1
MUSCLEBLIND-LIKE 1 REGULATION OF THE THYMIC TRANSCRIPTOME AND T
CELL DEVELOPMENT
By
MARINA MARLENE SCOTTI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
2
© 2016 Marina Marlene Scotti
3
To my family, in loving memory of my father and grandmother
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ACKNOWLEDGMENTS
I would like to thank my family. Firstly, I would like to thank my mother and father who
have cared for me tremendously since the moment I entered their lives. They have fostered my
inquisitiveness and encouraged my trek into the medical sciences beginning at a young age. My
mother, Susan E. Scotti, has been a rock in my scientific journeys, always encouraging me to
keep going when I was tired and to stay strong. My father, John A. Scotti, brought me with him
to observe veterinary surgical procedures and indulged my desire to watch surgeries at home
over dinner. He always pushed for me to take note of what the world needs and encouraged my
love of math and science. I would also like to thank my sister, Carmen C. Castellanos, for
having been a supporter and a friend to me throughout my PhD process. She has listened to my
gripes and to my successes over the years and has helped to distract me from myself when I have
been too caught up in my science life and needed someone to bring me back to reality. I owe a
debt of gratitude that I will spend my life repaying to my loving husband, Dr. David R. Knop.
He has truly been an inspiration and confidant throughout the graduate process. I strongly
believe I could not have “out-stubborned” these past 6 years if it had not been for his support.
I would like to thank my mentor, Dr. Maurice S. Swanson, for giving me the opportunity
to explore a research project slightly outside our realm of expertise but so thoroughly in-line with
my scientific aspirations. His guidance and support has helped me to persevere throughout this
process and hold fast to my research project. He has encouraged me and helped me to believe in
myself and begin to see the “bigger picture.” Dr. Swanson once told me that he looks for three
things in a potential student: good hands, a good head and creativity and he believed I exhibited
all three, even when I was unsure of myself. It has been an honor and a pleasure to work under
him and cultivate my scientific skills.
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I would also like to thank my committee members: Drs. Michael Clare-Salzler, Laura
Ranum and Edward Scott. They have served as tremendous resources for me and pushed me to
expand my scientific knowledge and thinking. Without their help and guidance, many of the
experiments I present here would not have been possible. I would especially like to thank Dr.
Laura Ranum, for lending me a helping hand, an open door and so many words of wisdom when
I needed them most.
I would like to send a very special thank you to Dr. James Resnick. Dr. Resnick has
always provided me with words of support and stories full of his wisdom and life experiences
any time I have needed them. He has always had an open door and a friendly ear for me. I can
never repay him enough for all his scientific support as well as moral support and, sometimes, a
respite away from the lab.
I especially appreciate my lab mentors and colleagues: Drs. Jihae Shin, Michael G.
Poulos, Konstantinos Charizanis and Mini Manchanda. They were my teachers, my lifelines and
my friends. I could not have completed my thesis work without them. Additionally, I would like
to thank my very eager, bright and enthusiastic undergraduate students, Lindsey Woody and
Kiril Poukalov. They have helped me grow as a mentor and teacher and put up with all the
growing pains that come with learning, but more importantly they contributed to the body of
work presented here. And finally, I would like to thank Catherine E Marten, Myrna Stenberg,
Tammy Reid and Dr. John Cleary. They have made my graduate experience unforgettable and
offered praise and help at every turn I have taken.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ...............................................................................................................4
LIST OF TABLES ...........................................................................................................................8
LIST OF FIGURES .........................................................................................................................9
LIST OF ABBREVIATIONS ........................................................................................................11
ABSTRACT ...................................................................................................................................16
CHAPTER
1 INTRODUCTION ..................................................................................................................18
RNA Splicing and Disease .....................................................................................................18 RNA Splicing Regulation ................................................................................................18
Alternative Splicing Regulation ......................................................................................19 Myotonic Dystrophy ...............................................................................................................20
Molecular Mechanism of DM .........................................................................................20
CELF Proteins in DM ......................................................................................................21 DM Immune System ........................................................................................................22
Immune System Development ................................................................................................23
T Cell Development ........................................................................................................23
Mbnl1-/-
Mice Display an Immune Phenotype.................................................................25
2 MBNL1 LOSS DISRUPTS THYMOCYTE DEVELOPMENT ...........................................36
Thymic Hyperplasia in 129-Mbnl1-/-
Males ...........................................................................36 MBNL1 Loss Specifically Dysregulates Thymocytes ...........................................................38
Loss of MBNL1 Alters Splicing Patterns and Differential Expression ..................................40 Splicing Alterations Occur in All Mbnl1
-/- Backgrounds .......................................................41
Discussion ...............................................................................................................................42
3 RNA ALTERATIONS AFFECT BOTH THYMOCYTE FUNCTION AND
PERIPHERAL T CELL POPULATIONS .............................................................................59
RNA Alterations Lead to Apoptosis Dysregulation in Thymocytes ......................................59 Mbnl1
-/- Mice Have Mature T Cell Deficits ............................................................................60
Peripheral T Cells Maintain Splicing Dysregulation Observed in Thymus ...........................62 Altered Splicing in DM Patients .............................................................................................62 Discussion ...............................................................................................................................63
4 MUSCLEBLIND 2 AND 3 INVOVLEMENT IN T CELL DEVELOPMENT ....................76
7
Mbnl2 and 3 Mouse Models ...................................................................................................76 Mbnl2
-/- Mouse Generation .............................................................................................76
Mbnl3 Whole Locus Knockout Mouse Generation .........................................................76 Mbnl2
-/- and Mbnl3
-/Y Aged Mice Have Limited T Cell Defects ............................................77
Discussion ...............................................................................................................................78
5 CONCLUDING REMARKS..................................................................................................89
6 MATERIALS AND METHODS ...........................................................................................92
Mbnl1 Animal Use for Thymic Analysis ................................................................................92 Thymocyte and Splenocyte Harvest .......................................................................................92
Flow Cytometry ......................................................................................................................92
Adoptive Transfer ...................................................................................................................93
Protein Lysate Generation and Immunoblot ...........................................................................94 Apoptosis Induction and Detection ........................................................................................95 Testosterone ELISA ................................................................................................................95 RNA-seq .................................................................................................................................96
RT-PCR Splicing Validation ..................................................................................................97 Human Peripheral Blood Lymphocyte Isolation and RNA Extraction ..................................97
Mbnl3 mESC Targeting ..........................................................................................................99 mESC Genomic DNA Isolation Southern Blot Analysis Mbnl3 condWL ...........................101 Mbnl3 condWL and Mbnl3 WLKO Genotyping ..................................................................103
APPENDIX
A HIGH CONFIDENCE MBNL1-DEPENDENT CASSETTE EXONS IDENTIFIED
FROM RNA-SEQ .................................................................................................................106
B GENE ONTOLOGY ANALYSIS 384 MBNL1 SPLICING-REGULATED TARGET
TRANSCRIPTS BENJAMINI FDR≤0.1 .............................................................................120
C MBNL1-DEPENDENT DIFFERENTIAL THYMUS EXPRESSION ANALYSIS (WT-
KO) .......................................................................................................................................122
D GENE ONTOLOGY ANALYSIS 808 DIFFERENTIALLY-REGULATED
TRANSCRIPTS IN MBNL1-/-
THYMUS BENJAMINI FDR≤0.1 ......................................137
LIST OF REFERENCES .............................................................................................................141
BIOGRAPHICAL SKETCH .......................................................................................................153
8
LIST OF TABLES
Table page
2-1 Paired end RNA-seq read mapping statistics. ....................................................................58
6-1 PCR primers. ....................................................................................................................105
9
LIST OF FIGURES
Figure page
1-1 RNA splicing regulation. ...................................................................................................27
1-2 Alternative splicing. ...........................................................................................................28
1-3 Myotonic dystrophy is a multisystemic disease.................................................................29
1-4 Molecular mechanism of myotonic dystrophy (DM). .......................................................30
1-5 MBNL family.....................................................................................................................31
1-6 Immune System. ................................................................................................................32
1-7 Receptors of the immune system. ......................................................................................33
1-8 T cell development. ............................................................................................................34
1-9 Mbnl1-/-
mouse generation schematic. ...............................................................................35
2-1 Decreased lifespan in Mbnl1-/-
knockout mice. .................................................................46
2-2 Mbnl1-/-
male mice show thymic hyperplasia. ...................................................................47
2-3 Mbnl1-/-
enlarged thymic phenotype has variable penetrance. ...........................................48
2-4 Mbnl1-/-
thymocyte development is dysregulated. .............................................................49
2-5 Adoptive transfer assay validation. ....................................................................................51
2-6 Adoptive transfer demonstrates Mbnl1-/-
thymocytes are dysregulated. ............................52
2-7 RNA-seq in Mbnl1-/-
and WT thymus. ...............................................................................53
2-9 Differential expression analysis of the Mbnl1-/-
thymic transcriptome. .............................55
2-10 Both 129 and BL/6 Mbnl1-/-
congenic lines show thymic splicing defects. .......................56
2-11 Testosterone ELISA. ..........................................................................................................57
3-1 Thymocyte TCR signaling model. .....................................................................................68
3-2 Mbnl1-/-
thymocytes are resistant to dexamethasone-induced apoptosis. ..........................69
3-3 Mbnl1-/-
CD4CD8 double positive thymocyte populations are most resistant to
apoptosis. ...........................................................................................................................70
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3-4 Mbnl1-/-
splenocytes are decreased. ....................................................................................71
3-5 CD3 positive Mbnl1-/-
T cells populations are perturbed in the periphery. ........................72
3-6 Mbnl1-/-
T cells functional phenotypes are altered. ............................................................73
3-7 Alternative splicing defects found in Mbnl1-/-
thymuses are recapitulated in Mbnl1-/-
splenocytes. ........................................................................................................................74
3-8 DM peripheral blood monocytes have similar splicing defects found in Mbnl1-/-
thymus. ...............................................................................................................................75
4-1 RNA-seq Mbnl expression levels.......................................................................................81
4-2 Mbnl2 targeting scheme and Mbnl2 knockout generation. ................................................82
4-3 Mbnl3 knockout mouse targeting scheme, germline transmission and knockout
generation. ..........................................................................................................................83
4-4 Mbnl2-/-
and Mbnl3-/Y
mice >20 weeks old have limited thymocyte defects. ....................85
4-5 Alternative splicing defects found in Mbnl1-/-
thymuses are not altered in Mbnl2-/-
and Mbnl3-/Y
thymuses. ......................................................................................................86
4-6 Mbnl2-/-
and Mbnl3-/Y
thymocytes are sensitive to apoptosis induction by
dexamethasone. ..................................................................................................................87
4-7 Mbnl2-/-
and Mbnl3-/Y
peripheral T cell populations are unaltered. ...................................88
6-1 Mbnl3 condWL and WLKO genotyping Schematic. .......................................................104
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LIST OF ABBREVIATIONS
Ab Antibody
ACS Animal Care Services
AFE Alternative first exon
Ag Antigen
ALE Alternative last exon
AOH Arm of homology
APC Antigen presenting cell
BCH BNIP2 and Cdc42GAP homology
BCR B cell receptor
Bnip2 BCL associated
BP Branch point
bp Base pair
BSA Bovine serum albumin
BSC Biological safety cabinet
Ca2+
Calcium
CD Cluster of differentiation
CD62L Cluster of differentiation 62 ligand
cDNA Complementary DNA
CNBP Cellular nucleic acid binding protein
CN Catalog number
CRAC Calcium release activated channel
CRD Context-dependent regulatory domain
CELF2 CUGBP, Elav-like family 2
CFSE Carboxyfluoroscein succinimidyl ester
Clcn1 Chloride channel 1
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condWL Conditional whole locus
CTD C-terminal domain
cTEC Cortical thymic epithelial cell
Dex Dexamethasone
DM Myotonic Dystrophy
DMEM Dubelco’s modified eagle medium
DMPK Dystrophia myotonica protein kinase
DMSO Dimethyl sulfoxide
DN Double negative
Dnm2 Dynamin 2
DP Double positive
dsRNA Double stranded RNA
EDTA Ethylenediaminetetraacetic acid
ENCODE Encyclopedia of DNA Elements
ELISA Enzyme linked immunosorbent assay
FACS Fluorescent assisted cell sorting
FBS Fetal bovine serum
Fc Fragment, crystallizable
FDR False discovery rate
FIAU Fialuridine
FITC Fluorescein isothiocyanate
Gapdh Glyceraldehyde-3-phosphate dehydrogenase
gDNA Genomic DNA
GO Gene ontology
HITS-CLIP High throughput sequencing coupled with crosslinking and immunoprecipitation
hnRNP Heterogeneous nuclear ribonucleoproteins
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HRP Horse radish peroxidase
HSC Hematopoietic stem cell
IACUC Institutional animal care and use committee
IgG Immunoglobulin gamma
IL Interleukin
Ion Ionomycin
IP Intraperitoneal
IP3 Inositol triphosphate
IP3R Inositol triphosphate receptor
IRB Institutional review board
ISP Induced single positive
kb kilobase
KO Knockout
Lef1 Lymphoid-enhancer binding factor 1
LIF Leukemia inhibitory factor
lncRNA Long non-coding RNA
Mbnl Muscleblind-like
MEF Murine embryonic fibroblast
mESC Murine embryonic stem cell
MHC Major histone compatibility complex
mTEC Medullary thymic epithelial cell
mRNA Messenger RNA
PBMC Peripheral blood mononucleocyte
PBS Phosphate buffered saline
PE Phycoerythrin
PI Propidium iodide
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PKC Protein Kinase C
PLC gamma Phospholipase C gamma
PMA Phorbol myristate acetate
Pol II RNA polymerase II
PSI Percent spliced in
Ptk2b Protein tyrosine kinase 2 beta
RADIL Research Animal Diagnostic Laboratory
RBP RNA binding protein
RNA Ribonucleic acid
RNA-seq High throughput RNA sequencing
Rnf130 Ring finger protein 130
RNP Ribonuclear protein
RPKM Reads per kilobase transcript per million mapped reads
RT Room temperature
RT-PCR Reverse transcriptase polymerase chain reaction
Scrib Scribbled planar ell polarity protein
SDS Sodium dodecyl sulfate
Serca Sarco/endoplasmic reticulum Ca2+
ATPase
snRNP Small nuclear ribonucleoproteins
SP Single positive
SPF Specific pathogen free
SR Ser/Arginine-rich
SS Splice site
SSC Saline Sodium Citrate
Tcf7 Transcription factor 7
TCR T cell receptor
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TLR Toll-like receptor
Trx Treatment
UF University of Florida
UTR Untranslated region
WLKO Whole locus knockout
WT Wild-type
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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MUSCLEBLIND-LIKE 1 REGULATION OF THE THYMIC TRANSCRIPTOME AND T
CELL DEVELOPMENT
By
Marina Marlene Scotti
May 2016
Chair: Maurice S. Swanson
Major: Medical Sciences -- Genetics
While transcriptional regulation plays a vital role in thymocyte development, co-/post-
transcriptional modifications are also important for T cell development. Here, we demonstrate
the loss of MBNL1, a developmentally regulated alternative RNA processing factor, leads to
dysregulation of thymocyte development and function. Mbnl1-/-
mice develop thymic
hyperplasia due to accumulation of T cells within the thymus and Mbnl1-/-
thymocytes are
resistant to steroid-induced apoptosis in vitro. As a consequence of thymocyte dysregulation,
peripheral T cell populations are perturbed in Mbnl1-/-
mice. Whole thymic transcriptome
comparisons between Mbnl1-/-
and wild-type mice indicate 474 mis-splicing events in >300
genes, including many involved in T cell receptor (TCR) assembly and signaling. TCR signaling
is a critical pathway involved in both thymocyte development and T cell activation and
maintenance. Additionally, >1600 RNA transcripts display altered expression (FDR≤0.05), with
half of the transcripts (808) altered having high confidence (FDR≤0.01) changes between wild-
type and Mbnl1-/-
thymuses. Enriched biological functions include immune processes, such as
lymphocyte activation and stimulus response. The T cell deficits and thymocyte development
blockages were specific to MBNL1 loss and were not recapitulated in either Mbnl2 or Mbnl3
knockout mouse models. Since MBNL1 regulates alternative splicing and polyadenylation
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during the postnatal period, this study suggests that fetal to adult RNA processing switches are
critical for normal thymocyte development and MBNL1 regulates alternative splicing of genes
critical for thymocyte development.
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CHAPTER 1
INTRODUCTION
RNA Splicing and Disease
The human genome consists of approximately 60,000 genes with transcription occurring
from ~20,000 protein-coding genes, according to the Encyclopedia of DNA Elements
(ENCODE) project (GrCH31, Ensembl79) with the remaining genes accounting for long non-
coding RNAs (lncRNAs), small non-coding RNAs and pseudogenes (1). Although the majority
of human genes currently identified are not protein encoding genes, the complexity of proteins
present in any given tissue, and the developmental changes that occur in every tissue, requires a
much larger proteome. This proteomic diversity is generated from a relatively small number of
genes by alternative splicing, which allows the synthesis of multiple isoforms from >90% of the
protein-coding genes (2-4).
RNA Splicing Regulation
RNA splicing – the removal of introns followed by exon ligation – is a two-step
biochemical process. Sequential transesterifcation reactions are initiated by a nucleophilic attack
of the 5’ splice site (5’ss) by the branch point adenosine (BP) in the downstream intron. This
first step results in the formation of an intron lariat intermediate with a 2’, 5’-phosphodiester
linkage. The intron lariat is resolved by a 5’ss-mediated attack on the 3’ss creating the spliced
RNA product (Figure 1-1a). Splice site identification and regulation is accomplished by the
major (U2-dependent) and minor (U12-dependent) spliceosomes. Both spliceosomes contain
five small nuclear ribonucleoproteins (snRNPs): U1, U2, U4, U5 and U6 for the major
spliceosome and U11, U12, U4atac, U5 and U6atac for the minor spliceosome. The major
spliceosome processes ~95.5% of all introns by recognition of consensus sequence elements at
the 5’ and 3’ ss and BP (5). Similarly, the minor spliceosome recognizes sequence consensus
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elements, however with a different sequence (Figure 1-1b) (6, 7). Additionally, the splicing
pathway is modulated by a number of cis-acting elements, such as exonic splicing enhancers,
exonic splicing silencers, intronic splicing enhancers and intronic splicing silencers. These
elements are recognized by auxiliary splicing factors, including a major group of splicing factors,
the Ser/Arg-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). These
auxiliary proteins and their RNA interactions result in the recruitment of the spliceosomal
constituents followed by the snRNP remodeling required for spliceosomal activation,
culminating in the generation of spliced RNA products.
Alternative Splicing Regulation
Co-/post-transcriptional processes such as splicing are also important for developmental
shifts (8-10). Many genes in the developmental repertoire are regulated by alternative splicing,
with high-throughput RNA sequencing (RNA-seq) studies suggesting that alternative splicing is
a routine activity in human cells with 90-95% of human multi-exon genes producing
alternatively spliced transcripts (3, 4). Alternative splicing adds another layer of complexity and
diversity for developmentally and tissue restricted splicing patterns through alternative first and
last exons (AFEs and ALEs), cassette exons, mutually exclusive cassette exons, alternative 5’
and 3’ ss usage and intron retention (Figure 1-2). Because of the complexity of the precursor
RNA sequence elements (cis-elements) and trans-acting splicing factors that control splicing, it
is fairly obvious that RNA processing steps could be particularly susceptible to mutations
implicated in disease (11). Further, RNA splicing patterns of particular subsets of genes have
been shown to shift during development and aging in multiple tissues (8-10, 12, 13), and aberrant
RNA splicing of these subsets has been found in human disease (14-17). One particular
regulatory protein, Muscleblind-like 1 (MBNL1) has been shown to be alternative splicing factor
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that is essential for proper development of muscle. Dysregulation of the MBNL1 protein plays a
major role in the disease pathology observed in myotonic dystrophy (DM) (9, 15, 18, 19).
Myotonic Dystrophy
Myotonic dystrophy (DM) is a multi-systemic disease affecting the musculoskeletal,
central nervous, gastric, endocrine and immune systems as well as others (Figure 1-3). DM is
the most common form of adult onset muscular dystrophy, affecting 1 in ~8000 adults (20). DM
is an autosomal dominant disease and an example of an RNA gain-of-function disease with
symptomology caused by missplicing of RNAs due to sequestration MBNL proteins by
expanded microsatellite expansion RNAs. These mutant expansion RNAs are encoded in either
the 3’ untranslated region (UTR) of DMPK (DM1) or within an intronic sequence of CNBP
(DM2) (Figure 1-4a) (20). Typically, CTG repeat sizes within the DMPK 3’ UTR range from 5-
37, whereas in DM1, disease-associated repeats can range from 37 to 3500 CTGs. Similarly, the
tetranucleotide repeat, CCTG found in intron 1 of CNBP normally contains less than 65 repeats,
but expansions >75 and up to 11,000 cause the disease symptomology seen in DM2 (21-23).
While the expansion mutations are similar, they occur in two very different regions of two
different genes. Yet, both of these mutations cause similar disease symptomology, even though
DM2 patients are generally considered to present with a milder clinical manifestation compared
to DM1 patients.
Molecular Mechanism of DM
Both forms of DM are caused by C(CTG) DNA microsatellite repeat expansions that
cause unusual DNA structures which lend themselves to expansions via slippage during DNA
replication (mitotic cells) or DNA repair (post-mitotic cells) (24, 25). C(CUG) RNA repeats
have been observed to create stable double-stranded (ds) RNA hairpins that accumulate in
nuclear foci in tissue sections from DM patient biopsies, as well as in cell culture systems, but
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not in control samples (26-29). The formation of these RNA foci created by the expanded
repeats result in sequestration of important RNA binding proteins (RBPs) necessary for
developmentally regulated splicing, including MBNL (Figure 1-4b) (28, 30-33).
The MBNL protein family consists of three paralogs; MBNL1, MBNL2 and MBNL3.
These proteins share sequence similarity and high sequence conservation in the C3H zinc finger
RNA binding motifs and, when ectopically expressed in cell culture, similar splicing activities
are observed (Figure 1-5) (34, 35). MBNL proteins are expressed in spatially and temporally
regulated patterns in mouse tissues, with MBNL1 highly expressed in muscle and MBNL2
highly expressed in brain (36). In contrast, MBNL3 is mainly expressed in embryonic and
regenerating tissues, while there is some expression in very few adult tissues (37).
MBNL proteins are RBPs involved in splicing, specifically, promoting a switch from
fetal to adult alternative splicing programs. An Mbnl1 knockout (KO) mouse model, Mbnl1-/-
that was developed to better elucidate DM molecular mechanisms, recapitulates several DM
symptoms (18). Additionally, these mice mis-splice several pre-messenger RNAs (pre-mRNA)
that have been shown to follow developmental splicing programs (Figure 1-4c), suggesting
MBNL1 protein loss is directly related to DM disease pathogenesis (38, 39).
CELF Proteins in DM
While MBNL proteins have been implicated to play a large role in DM pathogenesis,
other RBPs have been implicated as well. One such protein, CUG-binding proteins and Elav-
like family 1 (CELF1) protein is upregulated in DM patients due to stabilization and
hyperphosphorylation mediated by Protein Kinase C (PKC) (40, 41). MBNL and CELF proteins
act in an antagonistic fashion, with MBNL proteins promoting adult while CELF1 promotes fetal
splicing programs (32, 42, 43). Due to the combination of increased CELF1 activity and MBNL
sequestration DM patient tissues express fetal transcripts of gene transcripts that are specific
22
targets of these proteins (32, 38, 39, 41, 42, 44). The inappropriate expression of fetal isoforms
within an adult tissue is thought to ultimately lead to the functional failure of many genes and the
conclusion that DM is a development disease. Less is known about MBNL3 due to its primary
expression during embryogenesis, but it may also regulate developmental splicing decisions prior
to birth.
DM Immune System
To date, the immune system of DM patients has not been a focus of study although DM is
characterized by hypogammaglobulinemia, or low blood immunoglobulin gamma (IgG) levels
possibly due to increased turnover rate (45). IgG catabolism in DM has been attributed to an
increased expression of Fc receptor expression on DM monocytes (46). Additionally,
correlations have been drawn between repeat expansion size and IgG concentration as well as
circulating T cell numbers (47, 48). DM patients (~8%) have been reported to exhibit thymoma,
with a few cases characterized as a lymphocytic-rich thymoma (49-56). More recently, DM has
been associated with an increased risk for specific cancers including thyroid cancer, choroidal
melanoma and testicular cancer. The cancer risks in DM patients have not been correlated with
any other common cancer risk factors (57, 58). Recent work has demonstrated that the loss of
MBNL1 leads to increased metastasis in breast cancer cells (59). MBNL1 acts to stabilize
TACC1 and DBNL RNA transcripts in breast cancer cells. The stabilization of these transcripts
decreases the colonization capacity of tumorigenic cells, thus leading to a decrease in tumor
metastasis. Additionally, several DM patients also present with autoimmune disease (49, 51, 60-
63). The appearance of these symptoms highlights the importance of an intact, functioning
immune system.
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Immune System Development
The immune system is comprised of two branches: innate and adaptive (Figure 1-6).
While the innate branch is fast-acting and serves as the first line of defense against foreign
antigens, life-long immunity and cancer surveillance lies within the realm of the adaptive
immune response. The adaptive immune response is comprised of two types of immune cells: B
and T cells. Unlike the surface receptors of the innate immune cells, which are specific for
individual ligand recognition, B cell receptors (BCRs) and T cell receptors (TCRs) undergo
DNA splicing to generate a diverse repertoire of antigen recognition (Figure 1-7). Through the
education and maturation process of these two types of white blood cells, life-long immunity
against foreign antigens, as well as tolerance for self-antigens, is obtained. The ability of these
cells to discriminate self from non-self begins with education in the bone marrow (B cells) and
thymus (T cells) during pre- and post-natal development (64-66).
T Cell Development
Because the development of a competent immune system is critical for life, it is of great
importance to understand the regulatory mechanisms behind the development of a healthy,
functional immune system. T cell development is intricately intertwined with thymic
development. In mice and humans the thymic anlage differentiates from the 3rd
pharyngeal
pouch (67, 68). As the thymus develops, it signals for early thymic progenitors to enter for
further differentiation and development. As early thymic progenitors enter the thymus and begin
the differentiation process (Figure 1-8), the thymic environment continues to fully develop and
differentiate (68, 69). As T cells develop within the thymus as thymocytes, approximately 90-
95% are selectively destroyed throughout the maturation process to ablate high affinity binding
of self-antigens as well as promote recognition of non-self on antigen presenting cells (APCs)
(70, 71). These thymic-based selection processes are highly regulated events (72). Thymocytes
24
that are incorrectly selected, and/or loss of mature T cell populations, can lead to a compromised
immune state where T cells are unable to interact with the innate immune system or activate the
adaptive immune response. Dysfunctional immune system development can lead to autoimmune
disease and cancer susceptibility.
The selection processes that shape the T cell repertoire within the thymus are positive and
negative selection. In positive selection, developing T cells begin successive rearrangements of
the T cell receptor (TCR) beta chain. The TCR beta chain is paired with a pre-TCR alpha chain
for testing of a successful rearrangement that recognizes antigen in the context of the major
histone compatibility complex (MHC) on the surface of the cells of the thymic stroma or thymic
epithelial cells (TECs). Cells incapable of obtaining a survival signal through the TCR beta/pre-
TCR alpha complex are signaled to induce death. Positive selection is often referred to as death
by neglect. Once a successful beta chain rearrangement occurs, thymocytes undergo clonal
expansion and successive rearrangement of the TCR alpha chain for testing. Thymocytes
undergo another round of positive selection to obtain survival signaling through the complete
TCR. Negative selection also follows after a successful TCR alpha and beta chain
rearrangement has occurred. A strong TCR response to self-antigen presented in the context of
MHC elicits a death signal. This process eliminates T cells with too high an affinity to self-
antigens, thus reducing the potential for autoimmune disease development.
These selection processes are highly regulated events that take place in ordered and
compartmentalized environments within the thymus. The outer compartment (cortex) is
comprised primarily of cortical thymic epithelial cells (cTECs) and the inner compartment
(medulla) similarly comprised of medullary thymic epithelial cells (mTECs) (72). Within the
cortical region of the thymus the cTECs are mainly responsible for transitioning double negative
25
(DN) immature thymocytes to double positive (DP) thymocytes that have successfully
rearranged both the alpha and beta TCR chains. DP thymocytes migrate to the medulla for
further differentiation to single positive (SP) thymocytes, expressing either CD4 or CD8
dependent upon MHC class I or II restriction of the TCR and auto-antigen testing by AIRE
expressing mTECs (Figure 1-8) (70, 73, 74).
Transcriptional regulation has previously been shown to play a vital role in both thymic
organogenesis as well as thymocyte development (75-77). Expression of cytokines, cytokine
receptors, TCRs and other surface and secreted proteins are hallmarks of thymus and thymocyte
development and are all transcriptionally regulated. For example, the negative selection process
of mTECs is characterized by the promiscuous expression of tissue-specific proteins regulated by
the AIRE gene and other transcriptional factors to promote self-tolerance (78, 79). While AIRE
can open regions of the chromosome for protein synthesis and self-antigen presentation, co-
/post-transcriptional modifications of these proteins during thymocyte education would also be
crucial to create the diverse repertoire of self-epitopes that T cells would encounter in the
periphery post thymic emigration. Additionally, the importance of co-/post-transcriptional
modifications, such as RNA splicing, has also been implicated in thymocyte differentiation and
development (8, 80, 81).
Mbnl1-/-
Mice Display an Immune Phenotype
Interestingly, a constitutive Mbnl1-/-
mouse created to model DM exhibits an
immunologically-relevant phenotype: male knockout mice develop thymic hyperplasia during a
period in which the wild-type murine thymus undergoes involution (82). A disproportionate
number of thymocytes within the Mbnl1-/-
hyperplastic thymus are CD4CD8 DP with few single
positive thymocytes compared to wild type mice of similar age, indicating a disruption in
thymocyte development and selection pathways. This project is designed to test the hypothesis
26
that MBNL1 is necessary for RNA regulation during immune system development.
Understanding the regulatory events that control thymocyte selection and development and its
dysregulation in DM may shed light on the events regulating immune system aging and
autoimmune disease.
27
Figure 1-1. RNA splicing regulation. (a) Schematic diagram depicting the two-step biochemical
reactions resulting in intron lariat removal and RNA splice products. (b) Diagram
depicting U2-type and U12-type consensus sequences as well as the cis- and trans-
acting factors involved in spliceosomal recruitment and regulation. U2 and U12
consensus sequence frequencies correspond to residue height of each nucleotide in
each given position (83).
28
Figure 1-2. Alternative splicing. Depiction of the different alternative-splicing events
coordinated through cis- and trans-acting splicing factors and the spliceosome.
29
Figure 1-3. Myotonic dystrophy is a multisystemic disease. Cartoon highlighting the different
systems of the human body disrupted in myotonic dystrophy.
30
Figure 1-4. Molecular mechanism of myotonic dystrophy (DM). (a) DM1 is caused by a CTG
repeat expansion located in the 3’ UTR of DMPK and DM2 is caused by a CCTG
repeat expansion located in intron 1 of CNBP. (b) As RNA polymerase II (Pol II)
transcribes nascent RNA, RNA binding proteins (RBPs) associate with the Pol II C-
terminal domain (CTD) and are recruited to the nascent transcript. The binding of
RBPs MBNL1, 2 and 3 to the DM mutant RNA transcripts results in remodeling to a
double-stranded RNA (dsRNA) hairpin. (c) MBNL proteins promote adult exon
splicing patterns. The MBNL proteins sequestered by the dsRNA hairpin lead to loss
of adult splicing and promotion of fetal splicing.
31
Figure 1-5. MBNL family. The MBNL proteins are highly homologous. MBNL1, 2 and 3
proteins share zinc finger pairs for binding RNA and acting as splicing proteins.
MBNL2 shares 70% sequence homology with MBNL1. MBNL3 shares 60%
sequence homology with MBNL1. MBNL 2 and 3 isoforms with most homology to
most common MBNL1 isoform displayed.
32
Figure 1-6. Immune System. Cartoon of the two branches of the immune system with innate
immune organs are highlighted in red and adaptive immune organs are highlighted in
green.
33
Figure 1-7. Receptors of the immune system. Example receptors of the innate immune system
are ligand-specific and recognize either pathogen-associated molecular patterns or
damage-associated molecular patterns. The B cell receptor (BCR) and T cell receptor
(TCR) recognize a multitude of antigens.
34
Figure 1-8. T cell development. Early thymic progenitors originating from the bone marrow
enter the thymic cortex. Double negative (DN) thymocytes begin beta chain and T
cell receptor (TCR) rearrangements and positive testing through interactions with
cortical thymic epithelial cells (cTECs). Thymocytes that have made a successful
TCR beta chain rearrangement begin TCR alpha chain rearrangements and transition
to double positive thymocytes before differentiation to mature single positive CD4 or
CD8 thymocytes that undergo negative selection within the medulla and testing by
medullary thymic epithelial cells (mTECs).
35
Figure 1-9. Mbnl1-/-
mouse generation schematic. 129 murine embryonic stem cells (mESCs)
were targeted with plasmid DNA containing 5’ and 3’ arms of homology (AOH)
flanking a floxed neomycin resistance gene. The targeting construct (plasmid DNA
donor) recombines with the homologous genomic sequence replacing exon 3,
containing the Mbnl1 start codon, with the floxed neomycin resistance gene. The
DNA can then be recombined with Cre recombinase to remove the antibiotic
resistance gene and creating a gene specific knockout.
36
CHAPTER 2
MBNL1 LOSS DISRUPTS THYMOCYTE DEVELOPMENT
Thymic Hyperplasia in 129-Mbnl1-/-
Males
Mbnl1-/-
mice derived from 129 Sv/ImJ murine embryonic stem cells (18) were
backcrossed ten generations to the 129 Sv/ImJ congenic mouse strain to generate a 129 congenic
Mbnl1-/-
mouse strain. 129-Mbnl1-/-
mice, hereafter referred to as Mbnl1-/-
, exhibited a decreased
survival rate of 50% by 22 weeks (Log Rank ***p<0.001), as compared to wild-typ (WT)
littermates. When separated by sex, male and female mice both demonstrate similar lifespans,
21.6 weeks and 22.1 weeks, respectively (Figure 2-1). Necropsies of Mbnl1-/-
male mice
revealed abnormally large thymuses (Figure 2-2a). While females survive <1 week longer at
50% than males, survival rates decline more steeply for males than females. The occurrence of
an enlarged thymus is not the only factor leading to pre-mature death. Our lab and others have
already established Mbnl1-/-
mice suffer cardiac abnormalities (84, 85) that are most likely
responsible for the decreased survival rate of the mice. It is likely the combined cardiac
conduction defects and the stress upon the cardio-pulmonary system caused by the increased
thymic mass leads to the steeper decline in the male Mbnl1-/-
mice survival compared to female
mice.
MBNL1 is a known RNA binding protein involved in developmental regulation of RNA
splicing in other tissues (19, 82). Further characterization of gross thymic morphology and wet
weight in male Mbnl1-/-
mice at 4, 12 and >20 weeks of age was initiated to determine if the
thymus is also developmentally affected by MBNL1 loss. Four-week-old mice demonstrate no
observable gross morphological changes or differences in wet thymus/body weight ratios
compared to WT littermates (Figure 2-2a & b). At 12 weeks of age, thymic enlargement is
variable (Figure 2-3) with 23% of mice demonstrating enlargement, (data not shown), but no
37
significant changes occur in wet thymus/body weight ratios. By >20 weeks of age 76% of mice
demonstrate thymic enlargement (Pearson, ***p<0.001) with significant differences in wet
weight/body weight ratios, ~40-fold change (**p<0.01) compared to WT mice (Figure 2-2b &
c). The observed thymic enlargement could represent a thymoma, which is classically defined as
a benign overgrowth of thymic epithelial cells (TECs). Mbnl1-/-
mice demonstrating thymic
hyperplasia/enlargement also have significantly more thymocytes with an increase in thymic
cellularity greater than 7-fold over WT thymuses. In an attempt to determine if the enlargement
was a true thymoma or an overgrowth of the thymocyte population, viable cell counts were taken
at each of the developmental time points (Figure 2-3). No significant difference was noted at
each of the time points, however a large differential was observed at thymuses collected >20
weeks of age. The lack of significance at this time point is most likely due to the variable thymic
phenotype penetrance (Figure 2-3).
To determine if a particular thymocyte subpopulation was preferentially expanding
within the thymus, which might account for the increased cellularity, thymocytes were examined
for CD4 and CD8 surface markers by flow cytometry. The subpopulations of thymocyte
development can be characterized by the presence or absence of these two markers. Double
negative thymocytes (DN) do not express either CD4 or CD8 on the cell surface, double positive
(DP) thymocytes express both markers and single positive (SP) thymocytes express either CD4
or CD8. Further characterization of the developing thymocytes at 4 weeks, 12 weeks and >20
weeks of age showed alterations in the CD4 single positive (SP), CD8 SP and CD4CD8 double
positive (DP) populations in Mbnl1-/-
mice (Figure 2-4a-c). At 4 weeks of age Mbnl1-/-
mice
have decreased CD4 SP and CD8 SP populations, suggesting decreased differentiation potential
of the thymocytes. However, by 12 weeks of age, a shift occurs in Mbnl1-/-
mice that results in
38
intermittent accumulation of CD8 SP cells (Figure 2-4b). This shift in thymocyte populations
may correlate to modestly enlarged thymus size at 12 weeks of age (Figure 2-3). At ages >20
weeks the CD8 SP population remains increased compare to WT and flow cytometry dot plots
suggest a shift towards CD4CD8 DP populations. However, these shifts are not significant and
are most likely due to the mixed phenotype populations used in this analysis. The increasing
CD8 SP population could comprise either mature CD8 SP cells trapped within the thymus or a
transiently CD8 SP population that arises in the thymus between DN and DP developmental time
points, known as induced-single positive CD8 cells (ISP). As developing thymocytes transition
from the DN stage to DP, they pass through a brief period of CD8 SP expression on the cell
surface before CD4 expression is detectable (Figure 2-4d) (86).
MBNL1 is one of three members of the muscleblind-like family. It has been previously
demonstrated that other MBNL family members may compensate for loss of one protein in
tissues (84, 87). MBNL1 protein is undetectable in Mbnl1-/-
mouse lysates and unlike other
tissue systems; within the thymus MBNL1 loss does not result in upregulation of either MBNL2
or MBNL3 proteins (Figure 2-2d). This suggests the thymic changes observed in Mbnl1-/-
are a
sole result of MBNL1 loss.
MBNL1 Loss Specifically Dysregulates Thymocytes
Because the thymus is mainly composed of two cell types whose development are
intricately intertwined (Chapter 1), the cause of the dysregulated thymocyte development and
accumulation of thymocytes could be due to MBNL loss in either the developing cells
themselves (thymocyte intrinsic) or because of a defect in the educational cells (TEC intrinsic)
(67, 72, 86). Adoptive transfer was used to determine the cell type responsible for the impaired
development. Wilde-type mice expressing a different cell surface antigen (CD45.1) from the
Mbnl1-/-
mice (CD45.2) were used to create bone marrow chimeras (Figure 2-5a). Because the
39
thymic phenotype is most faithfully observed at 24 weeks of age, the bone marrow chimeras
would be allowed to repopulate white cells for 24 weeks post-transfer. Nine weeks post-sub-
lethal irradiation and hematopoietic stem cell transplant (HSC), peripheral blood was collected to
assay the engraftment success (Figure 2-5b & c). Mbnl1-/-
thymocytes successfully produced
white blood cells in the periphery of WT mice at proportions significantly greater than residual
endogenous HSCs (*p<0.05). Additionally, Mbnl1-/-
mice were able to educate and release WT
white cells into the periphery at proportions significantly greater than endogenous Mbnl1-/-
HSCs
(*p<0.05). These data indicate the adoptive transfer was successful and thymuses could be
collected for further analysis post the 24-week repopulation period.
Three of five WT recipients died either before or immediately at the 24-week endpoint.
Thymuses were extracted from these mice within 4-12 hours post-mortem and subjected to
thymocyte characterization. One of five Mbnl1-/-
mice died shortly after engraftment. This
mouse’s immune system failed to repopulate most likely due to a surgical failure at time of
transplantation of the HSCs or the extreme nature of the transplant procedure. Mice that
survived to the 24-week time point were sacrificed, thymuses removed and thymocytes extracted
for analysis by flow cytometry. An age matched non-irradiated mouse was also sacrificed for
each genotype to aid in control gating of flow cytometry.
Thymocytes collected from WT mice were comprised of mainly Mbnl1-/-
derived
progenitors although a non- negligible presence of endogenous thymocytes was present (Figure
2-6a). Mbnl1-/-
mice showed the vast majority of repopulated thymocytes were of WT lineage.
When CD4 and CD8 surface receptors were characterized on the engrafted populations only
Mbnl1-/-
thymocytes within the WT thymic environment had significantly decreased CD4 SP and
CD8 SP populations and significantly increased CD4CD8 DP populations (*p<0.05). These
40
population profiles were similar to observed profiles in hyperplastic thymuses of Mbnl1-/-
mice.
The conclusion is that MBNL1 loss dysregulated thymocyte development in a thymocyte
intrinsic manner.
Loss of MBNL1 Alters Splicing Patterns and Differential Expression
To gain further insight into the disruption observed in Mbnl1-/-
T cell development in
light of the fact that MBNL1 is an RNA binding protein that regulates the developmental
splicing program in muscle and other tissues (9, 18, 38, 87, 88), the thymic transcriptome was
compared between WT and Mbnl1-/-
mice. Thymuses 9 weeks of age were collected for RNA-
seq analysis (Table 2-1, Figure 2-7a). Since Mbnl1-/-
thymocytes demonstrate varied
developmental programs and size at 12 weeks of age (Figure 2-3 & Figure 2-4b), mice were
chosen from an age when peak T cell development would occur (adolescence) and well before
any phenotypic changes are observed. Paired end sequencing of thymic RNA yielded 474 high
confidence (coverage ≥ 20, |percent spliced in, or PSI| ≥ 0.1 and false discover rates (FDR) ≤
0.05) alternatively spliced targets within 386 unique genes. The vast majority (81%) of the
splicing events were alternative cassette exons as opposed to other splicing events, such as
mutually exclusive exon usage, alternative 3’ or 5’ ss and intron retention (Figure 2-3b). Some
of the top alternatively spliced targets included lymphocyte specific genes, including Tcf7 (also
known as Tcf1), Lef1 and Ptk2b (also known as Pyk2). Gene ontology (GO) analysis was
performed on the alternatively spliced cassette exons to determine if the splicing alterations were
functionally enriched. The most enriched biological processes were all processes that could be
linked with developing thymocytes undergoing differentiation (Figure 2-7c, Appendix B).
To validate the splicing data obtained from RNA-seq (Appendix A), reverse transcriptase
polymerase chain reaction (RT-PCR) was performed in WT and Mbnl1-/-
thymus (n=3, each
genotype) and PSI calculated for the top thymocyte specific RNA targets (Figure 2-8a). RT-
41
PCR validated alternative splicing for Lef1, Tcf7 and Ptk2b alterations was significant,
(***0.001, **p<0.01, ***p<0.001, respectively, Figure 2-8b).
In addition to alternative splicing data, RNA-seq was used to look at global alterations in
transcript expression between WT and Mbnl1-/-
thymus. Mbnl1-/-
thymus had slightly more
genes (58.5%, padj≤0.01) downregulated as compared to WT (Figure 2-9a, Appendix C). The
differential expression profile of all the significantly altered transcripts was analyzed by GO
(Figure 2-9b, Appendix D). Top biological functions included immune response, immune
system processes, defense response and antigen presentation and processing. These enriched
processes do not appear thymocyte specific but more in line with antigen presenting cell
processes. However, these process categories are not wholly surprising given the intricate
crosstalk between developing thymocytes and the thymic environment. Our conclusion is that
MBNL1 primarily regulates alternative cassette exon usage of transcripts involved in thymocyte
differentiation and the entirety of the thymic transcriptome is altered as a response to the defects
intrinsic to the Mbnl1-/-
thymocytes.
Splicing Alterations Occur in All Mbnl1-/-
Backgrounds
The original Mbnl1-/-
mouse line was created in a mixed background as previously noted.
However, neither the mixed background nor Mbnl1-/-
mice maintained on a C57 BL/6J
background demonstrate an enlarged thymic phenotype. Splicing of the top
thymocyte/lymphocyte specific RNA transcripts altered in the 129 Sv/ImJ strain Mbnl1-/-
thymus
were tested in the other Mbnl1-/-
backgrounds. Lef1, Tcf7 and Ptk2b splicing were found to be as
dysregulated as those transcripts in 129 Mbnl1-/-
mice (Figure 2-10). This suggests that it is not a
modifier of splicing in these mice that gives rise to the thymic phenotype, but another unknown,
modifier event. One possibility is a difference in testosterone levels in the 129 Sv/ImJ strain
Mbnl1-/-
compared to other Mbnl1-/-
strains. Androgens are potent inducers of apoptosis in
42
lymphocytes and reduced testosterone levels via castration models lead to thymic enlargement
(89, 90). To determine if 129 Mbnl1-/-
mice have decreased testosterone levels leading to thymic
enlargement, a testosterone ELISA was performed using Mbnl1-/-
and WT sera from both 129
Sv/ImJ sera and C57 BL/6J backgrounds at 12 weeks of age (n=3, each). Interestingly, Mbnl1-/-
sera from 129 Sv/ImJ background mice had more testosterone present in circulation than the
WT. The Mbnl1-/-
C57 BL/6J strain, also showed a slight increase in circulating testosterone
over WT (Figure 2-11). While the increases observed in both strains were not significant, the
129 Sv/ImJ Mbnl1-/-
males demonstrated a much larger increase than the C57 BL/6 strain.
Together, these data suggest that a loss of testosterone in Mbnl1-/-
mice is not the cause of the
observed enlarged thymus and the thymic phenotype is independent of the observed splicing
alterations of Lef1, Tcf7 and Ptk2b.
Discussion
Mbnl1-/-
mice die prematurely as compared to WT littermates due in part to a
substantially enlarged thymus. The hyperplastic thymus is characterized by a marked increase in
thymocyte accumulation with alterations in CD4 SP, CD8 SP and CD4CD8 DP populations.
Bone marrow chimeras revealed that the loss of MBNL1 dysregulates thymocyte development in
a thymocyte intrinsic manner and the Mbnl1-/-
thymus is capable of repopulating thymocytes
from WT donors. RNA-seq analysis uncovered several thymocyte/lymphocyte specific
transcripts alternatively spliced in Mbnl1-/-
thymus among the hundreds of alternatively spliced
transcripts. The top splicing targets Lef1, Tcf7 and Ptk2b had near zero false discovery rates,
suggesting inordinately high probabilities of the validity of the splicing events. These events
were validated by RT-PCR analysis. Consequently, both Tcf7 and Lef1 have been studied
extensively in thymocyte development (91-93). Tcf7-/-
mice also have disrupted thymocyte
development and decreased thymic cellularity however the deficit appears at the DN stage of T
43
cell development (94). Tcf7-/-
mice also show an increase (3-5 fold) in the ISP stage, but overall
decreased DP and SP thymocytes (94, 95). However, these observations were made in relatively
young mice, 6-8 weeks of age. When Tcf7-/-
mice are aged, 50% of mice develop enlarged
thymuses similar to Mbnl1-/-
mice (96). Lef1-/-
mice have no detectable disruptions with
thymocyte development but they die shortly after birth (97). There is a possible redundancy
between TCF7 and LEF1 that leads to an increased T cell development disruption in Tcf7/Lef1
compound knockouts (98). Tcf7-/-
Lef1-/-
compound knockout thymocytes may demonstrate an
increase in CD8 SP thymocyte development, but the increase in CD8 SP cells could result from a
block at the ISP stage as no TCRb transcript is detectable (98). Since Mbnl1-/-
splicing is
disrupted in both Tcf7 and Lef1, this may explain the similarities seen in both the Tcf7-/-
Lef1-/-
compound knockout thymocyte developmental block as well as the increase in thymus size that
phenocopies aged Tcf7-/-
mice.
The alternative exon excluded from Lef1 is located in the context dependent regulatory
domain (CRD). Exclusion of this exon has been demonstrated to reduce Lef1 binding affinity to
the enhancer region of TCRa resulting in decreased transcription and therefore reduced
production of TCR alpha chain (80). Additionally, Tcf7 regulates transcription of beta catenin
dependent transcripts and can serve as a negative regulator of Lef1 (99, 100). The alternative
exon included in Mbnl1-/-
mice encodes a c-clamp domain containing the amino acid sequence
“CRARF” that stabilizes the binding between TCF7 and DNA (101, 102). TCF7 has been
shown to negatively regulate expression of Lef1 transcription in a beta catenin dependent fashion
(100). The combined alternative splicing of both Tcf7 and Lef1 RNAs with the increased
expression of Lef1 in Mbnl1-/-
thymus suggests that TCR alpha chain production may be
compromised.
44
Developing thymocytes rely on receiving signals through the TCR during the selection
processes. PTK2B is a protein tyrosine kinase that is activated downstream of TCR signaling.
While PTK2B is a ubiquitously expressed protein, hematopoietic cells, such as T cells, express a
hematopoietic specific isoform (103, 104). TCR and calcium signaling activate PTK2B in T
cells (105, 106). However, the role of PTK2B in mature T cells and developing thymocytes is
less clear. PTK2B is involved in migration and adhesion in T cells, as well as apoptosis and
proliferation in other cell types (106-110). The alternatively spliced exon of Ptk2b included in
Mbnl1-/-
thymus switches the PTK2B protein away from the hematopoietic specific isoform to
the more ubiquitously expressed isoform. While the functional differences between these two
isoforms are still unknown, the exclusion of this exon shortens the proline-rich domain of the
protein and does not affect the protein kinase domain, focal adhesion domain or phosphorylation
sites (103, 107). How these splicing alterations affect thymocyte development requires further
exploration.
The development of thymic hyperplasia in the 129 Sv/ImJ Mbnl1-/-
mice is still
unexplained. One possible explanation is an unknown modifier present within the 129 Sv/ImJ
background strain that increases the splicing severity in these Mbnl1-/-
mice. Scnm1 is an
example of one such modifier that has been shown to produce more severe phenotypes in
different mouse strains (111). BL/6 mice were shown to carry an Scnm1 variant that produces a
truncated C-terminal domain. Ultimately, this mutation leads to decreased splicing of the non-
consensus splice donor site of Scn8a. The exclusion of these alternative exons produces more
severe neurologic phenotypes in BL/6 than other strains.
The thymic hyperplasia found in Mbnl1-/-
mice reflects an accumulation of thymocytes
however the observed splicing changes alone cannot account for the defect. Splicing occurs to
45
similar extents in 129 Sv/ImJ Mbnl1-/-
mice and C57 Bl/6J Mbnl1-/-
mice, as well as 129.BL/6
Mbnl1-/-
mice for the transcripts tested. These results suggest that the unknown modifier(s) is not
affecting splicing but some other pathway leading to thymocyte accumulation.
Genetic modifiers within differing mouse background strains is an important concern
being recognized in literature when utilizing genetically modified mouse strains for disease
modeling (112-114). However, the combination of current technology and affordability of high
throughput sequencing may permit the identification of potential modifiers and allow better
understanding of the corresponding phenotypes.
One possibility is that the modifier for the 129 Sv/ImJ strain lies somewhere in the
testosterone production pathway (89, 90). Surprisingly, Mbnl1-/-
mice assayed for circulating
testosterone concentration displayed increased testosterone levels compared to WT mice.
However, the mice assayed were 12 weeks in age. Mbnl1-/-
mice at 12 weeks of age only
demonstrate an intermittent phenotype, substantially lower than >20 weeks Mbnl1-/-
mice. At 12
weeks of age, Mbnl1-/-
mice have decreased viable thymocyte amounts compared to WT mice
(Figure 2-3). It would be more appropriate to assay testosterone concentration in animals at >20
weeks of age, when thymic enlargement is most prevalent. Another possibility to account for
observed increase of testosterone in the Mbnl1-/-
mice is a response to the increasing thymocyte
population in an attempt to induce apoptosis.
46
Figure 2-1. Decreased lifespan in Mbnl1-/-
knockout mice.
47
Figure 2-2. Mbnl1-/-
male mice show thymic hyperplasia. (a) Mbnl1-/-
male mice have an
enlarged thymus at ages >20 weeks of age. (b) Thymic wet weight is significantly
increased in Mbnl1-/-
mice when compared to WT mice, normalized to body weight
(c) Thymic phenotype is approximately 80% penetrant in male mice >20 weeks of
age. (d) MBNL1 protein is not detectable in Mbnl1-/-
mice. MBNL2 and MBNL3
proteins are not up regulated at the protein level in the absence of MBNL1.
48
Figure 2-3. Mbnl1-/-
enlarged thymic phenotype has variable penetrance. Mbnl1-/-
mice begin to
display changes in thymic morphology as early as 12 weeks of age. Note right-hand
12 week Mbnl1-/-
thymus and decreasing viable thymocytes. By >20 weeks of age,
Mbnl1-/-
mice have an increased amount of viable T cells within the thymus, however
not all >20 week old mice have an enlarged thymus (note left-hand >20 week Mbnl1-/-
thymus).
49
Figure 2-4. Mbnl1-/-
thymocyte development is dysregulated. Mbnl1-/-
thymocytes characterized
by flow cytometry for CD4 and CD8 expression at (a) 4 weeks (b) 12 weeks and (c)
>20 weeks of age; (d) schematic of thymocyte development.
50
Figure 2-4. Continued. Mbnl1-/-
thymocytes characterized by flow cytometry for CD4 and CD8
expression at (a) 4 weeks (b) 12 weeks and (c) >20 weeks of age; (d) schematic of
thymocyte development.
51
Figure 2-5. Adoptive transfer assay validation. (a) Cartoon diagram outlining adoptive transfer
process. Mbnl1-/-
mouse derived hematopoietic stem cells (HSCs) are in orange and
distinguished by CD45.2 cell surface receptors, WT HSCs are in blue and are
distinguished by CD45.1 cell surface receptors. HSCs were transplanted through
intravenous (IV) transplantation. (b) Flow cytometry dot plots of peripheral blood
lymphocytes for CD45.1 and CD45.2 to determine engraftment success. (c) Bar graph
representation of engrafted percentages.
52
Figure 2-6. Adoptive transfer demonstrates Mbnl1-/-
thymocytes are dysregulated. (a) Flow
cytometry dot plots showing gating strategy for CD4/CD8 analysis. Mbnl1-/-
derived
thymocytes are gated as CD45.2 positive and colored in orange. WT derived
thymocytes gated as CD45.1 positive and colored in blue. (b) Bar graphs
representing flow cytometry data of CD4 single positive (SP), CD8 SP and CD4CD8
double positive (DP) thymocyte percentages.
53
Figure 2-7. RNA-seq in Mbnl1-/-
and WT thymus. (a) Schematic of RNA-seq. (b) Alternative
splicing event distribution of high confidence, p<0.05, percent spliced in (PSI) ≥ 10%
and false discovery rates (FDR) ≤ 0.05. (c) Gene ontology (GO) analysis of top
alternative splicing biological processes p<0.05, FDR ≤ 0.1.
54
Figure 2-8. Key thymocyte developmental transcripts are mis-spliced in Mbnl1-/-
thymus. (a)
RT-PCR alternative splicing analysis of Lef1, Tcf7 and Ptk2b in 9-week thymic tissue. (b) RNA-
seq wiggle plots generated on the UCSC genome browser indicating the alternative splicing
events. (c) Bar graphs quantitating the percent spliced in (PSI) of each alternative splicing event
tested by RT-PCR.
55
Figure 2-9. Differential expression analysis of the Mbnl1-/-
thymic transcriptome. (a) Percent
thymic transcript up-, and down-, regulation in Mbnl1-/-
mice compared WT. (b) Gene
ontology (GO) analysis of top differentially expressed biological processes p<0.05,
FDR ≤ 0.1.
56
Figure 2-10. Both 129 and BL/6 Mbnl1-/-
congenic lines show thymic splicing defects.
57
Figure 2-11. Testosterone ELISA. Testosterone concentration in 12-week 129 Sv/ImJ and C57
BL/6J Mbnl1-/-
mice are not significantly increased over age matched WT controls.
58
Table 2-1. Paired end RNA-seq read mapping statistics.
Statistic AJAX14
WT
AJAX15
WT
AJAX16
WT
A01241
Mbnl-/-
A01242
Mbnl1-/-
A01245
Mbnl1-/-
Total Read Pairs 57900367 59035224 53239280 54489206 60767810 57836485 rRNA rate (%) 2.26 1.37 1.40 1.18 1.12 1.16 Mapping rate (%) 85.05 84.63 88.62 89.24 83.52 85.16 Uniquely mapping rate
(%) 78.72 77.16 82.34 83.36 77.91 79.46
Assignment to genes
rate (%) 71.90 75.62 72.30 72.32 72.97 72.46
Splicing rate (%) 13.05 14.74 13.16 12.94 13.02 12.87 Forward strand bias (%) 49.32 49.35 49.70 49.59 49.77 49.74 Reverse strand bias (%) 50.68 50.64 50.30 50.41 50.23 50.26 5prime vs. total (%) 15.60 16.07 15.71 15.76 15.66 15.49 3prime vs. total (%) 13.85 13.12 13.86 13.83 13.90 14.08 Mean GC content 48.37 48.50 48.27 48.39 48.48 48.32 Duplication rate (%) 11.53 13.40 10.65 10.09 10.47 10.26 Median insert size 200.00 205.00 197.00 197.00 198.00 197.00 Mean insert size 221.31 234.75 217.80 217.08 219.46 217.08 SD insert size 141.76 167.47 139.25 138.27 141.42 138.21
59
CHAPTER 3
RNA ALTERATIONS AFFECT BOTH THYMOCYTE FUNCTION AND PERIPHERAL T
CELL POPULATIONS
RNA Alterations Lead to Apoptosis Dysregulation in Thymocytes
TCR signaling in thymocytes and signal strength determine differentiation and selection
fates (115). The TCR receives signals when peptide antigens (Ag) are presented in the context
of major histone compatibility complexes (MHCs). Downstream signaling can either activate
PTK2B via FYN/LCK activation or Phospholipase C gamma (PLCgamma) activation generates
inositol triphosphate (IP3) that in turn interacts with inositol triphosphate receptors (IP3R) located
on the endoplasmic reticulum. This results in release of calcium into the cytosol, which can
activate PTK2B signaling. Because TCF7 can regulate LEF1 expression, which in turn affects
TCR alpha transcription leading to productive TCR assembly and expression and thereby
affecting PTK2B activation, cellular functions dependent on PTK2B are candidate pathways for
thymocyte dysregulation (Figure 3-1).
Apoptosis is a critical function of thymocyte development. Cells that respond
inappropriately, either too sensitive to apoptosis or insensitive to apoptotic signals, can cause a
block in thymocyte development. Given the disruptions to TCR signaling caused by changes in
alternative splicing in Mbnl1-/-
thymocytes, I hypothesized that Mbnl1-/-
thymocytes are less
sensitive to apoptosis.
To test for apoptotic sensitivity, thymocytes were collected from freshly dissected 4-, 12-
and >20-week thymuses and treated overnight with varying concentrations of dexamethasone
(Dex) , 10-6
M to 10-9
M or vehicle only control (0 M). Post-overnight incubation, cells were
collected and stained with Annexin V-FITC (Annexin V) and propidium iodide (PI) for apoptotic
analysis as well as CD4 and CD8 to determine if any particular sub-population is more or less
sensitive to apoptotic induction (Figure 3-2a). Annexin V labels phosphotidylserine residues that
60
are displayed on the extracellular surface on the plasma membrane in early stages of apoptosis.
PI is a DNA fluorescent exclusion dye that can only enter the cell when the plasma membranes
are structurally compromised and bind DNA (116).
Treatment (Trx) groups were normalized to no treatment controls. (n≥ 3, each group).
Total apoptosis was assayed first (Figure 3-2 b & c). Thymocytes collected at 4 weeks of age
did not show a decreased sensitivity to Dex induced apoptosis compared to WT. However, in
both 12- and >20-week thymocytes, Mbnl1-/-
thymocytes consistently demonstrate less apoptosis
(early or late) when normalized to vehicle only control as compared to WT thymocytes (Figure
3-2c).
CD4 and CD8 populations were then analyzed for apoptosis in the 4-, 12- and >20-week
samples. Thymocytes were first gated to CD4 SP, CD8 SP or CD4CD8 DP and then analyzed
for PI and Annexin V staining (Figure 3-3a). Only the strongest treatment condition, 10-6
M, in
CD4 SP populations showed insensitivity to Dex-induced apoptosis, in the late stage apoptosis of
4-week Mbnl1-/-
thymocytes. While Mbnl1-/-
thymocytes analyzed from 12- and >20-week old
animals consistently show reduced sensitivity to Dex induced apoptosis in the CD4CD8 DP
population (*p<0.05, **p<0.01) across multiple concentrations in early apoptosis and some
decreased populations in late apoptosis compared to WT thymocytes. Both CD4 SP and CD8 SP
Mbnl1-/-
thymocytes also show trends towards increased apoptotic resistance, but not as
pronounced as CD4CD8 DP populations. Cumulatively, these data suggest aging Mbnl1-/-
thymocytes are more resistant to apoptotic signaling than age-matched WT controls.
Mbnl1-/-
Mice Have Mature T Cell Deficits
Mbnl1-/-
mice have demonstrated perturbed thymocyte development caused by splicing
defects, however white blood cells were repopulated in the periphery of bone marrow chimeras.
To further evaluate how educational defects can affect the mature T cell population, lymphocytes
61
harvested from spleens (splenocytes), a major secondary lymphoid organ, were characterized.
Viable splenocytes collected from Mbnl1-/-
and WT mice at 4- and 12-weeks of age were
enumerated (Figure 3-4). At 12 weeks of age, Mbnl1-/-
mice had significantly less viable
splenocytes (***p<0.001). Because splenocytes encompass both B cell and T cell populations,
flow cytometry analysis using the B cell surface marker, B220, and the pan-T cell marker, CD3,
was used to identify differences in T cell populations. No significant changes were observed in
either B cell or T cell populations at 4 and 12 weeks (Figure 3-4b). CD4 SP, CD8 SP and
CD4CD8 DP subpopulations of T cells were evaluated by flow cytometry. CD3 positive cells
were first gated prior to gating CD4 and CD8 (Figure 3-5a). CD3 positive T cells assayed at 4
weeks of age showed no significant alterations to CD4/CD8 subpopulations. By 12 weeks of
age, CD3 positive T cells showed a significant increase in CD4 SP percentage (*p<0.05) and
marked decrease in the CD8 SP population (**p<0.01). No alterations were seen in CD4CD8
DP populations at either time point (Figure 3-5b).
To gain further insight into the functional status of Mbnl1-/-
T cells, CD62 ligand
(CD62L) and CD44 surface markers were analyzed to differentiate between naïve (CD62L SP),
memory (CD62LCD44 DP) and activated (CD44 SP) T cell populations. Splenocytes from 4-
and 12- week animals were first gated to CD3 positive T cell populations and then analyzed for
CD62L and CD44 expression (n=3, each group) (Figure 3.6a). As early as 4 weeks of age,
peripheral T cell populations demonstrated a significantly increased activated-phenotype
(**p<0.01) and significantly smaller proportions of memory T cell populations (*p<0.05) as
compared to WT T cells. At 12 weeks of age, naïve peripheral T cell populations were
significantly reduced in Mbnl1-/-
mice (*p<0.05) and activated T cells populations remained
62
increased (*p<0.05) (Figure3-6b). Early dysregulation of T cell development occurring in the
Mbnl1-/-
thymus had downstream effects on the mature T cell populations in the periphery.
Peripheral T Cells Maintain Splicing Dysregulation Observed in Thymus
While TCR signaling plays a critical role in thymocyte development, mature T cell
activation is also dependent on TCR signaling responses to antigen presentation. Apoptosis also
plays a critical role in the maintenance of mature T cells and reduction of Ag activated T cells
post immunologic response (117). Additionally, T cells activation involves endocytosis of the
TCR as well as endocytosis of other important signaling molecules (118, 119). Dysregulated
alternative splicing of transcripts associated with these pathways could affect T cell homeostasis
and function. In addition to the previously validated developmental genes (Lef1 and Ptk2b),
alternatively spliced events in both apoptosis (Bnip2 and Rnf130) and endocytosis (Dnm2 and
CD47) pathways were validated in splenocytes (Figure 3-7 a-c). Mbnl1-/-
splenocytes showed
similar dysregulation of alternatively spliced exons. However, the PSI of the alternative exons is
reduced in splenocytes.
Altered Splicing in DM Patients
Dysregulation of splicing in Mbnl knockout tissues has been faithfully recapitulated in
myotonic dystrophy (DM) patients (38, 39, 87, 120). Mbnl1-/-
mice demonstrated striking
splicing alterations in several genes implicated in thymocyte development as well genes in
pathways critical for maintenance of healthy peripheral T cells. For this reason, PTK2B, LEF1
and BNIP2 RNAs were assayed in DM patient peripheral blood. Additionally, the alternative
exons altered in these genes are highly conserved between mouse and humans. Briefly, at least
2.5 mL of DM patient (DM1 – 2 males, 1 female, aged 30-40 years; DM2 – 3 females, aged 45-
55 years) and control (2 males, 1 female aged 35-55 years) blood was collected into potassium
EDTA coated vials to prevent clotting. The blood was separated on a Ficoll-Paque density
63
gradient to isolate peripheral blood mononuclear cells (PBMCs) (Figure 3-8a). RNA was
extracted from the isolated PBMCs and RT-PCR analysis performed on control, DM1 and DM2
patient samples. While neither DM1 nor DM2 showed significant splicing alterations over
control samples in any of the genes tested, a clear trend was observed in the splicing changes of
DM samples. For all genes tested, DM2 samples exhibited altered splicing in the same direction
predicted by the Mbnl1-/-
mouse model (Figure 3-8b). In the case of LEF1 and PTK2B
expression, DM2 patient samples demonstrated increased alternative splicing changes over DM1
samples.
Discussion
Mbnl1-/-
thymocytes are less sensitive to the potent glucocorticoid apoptosis inducer, Dex
(121). However, this approach to apoptosis induction is limited because it is not an apoptotic
event mediated through TCR signaling but through glucocorticoid receptors (122).
Glucocorticoid receptors are expressed on the cell surface and upon ligand binding translocate to
the nucleus acting as a transcriptional regulator of a multitude of genes that lead to apoptosis.
The reduction in apoptosis observed in Mbnl1-/-
thymocytes may reflect an inherent dysfunction
in the apoptotic machinery. Another possible complication of the data interpretation is the
normalization of the Dex treatment conditions to an untreated control. If the control sample of
Mbnl1-/-
thymocytes had large apoptotic populations at the start of the experiment, then the
apoptotic change post-Dex treatment may be somewhat negated by a large starting value.
Additionally, this assay is an in vitro assay where thymocytes are removed from their normal
environment and processed through several buffer treatments and incubations. It is possible that
Mbnl1-/-
thymocytes are more fragile than WT thymocytes and begin to undergo programmed
cell death during the assay preparation. However, it seems unlikely that Mbnl1-/-
thymocytes
exhibit increased cell death over WT thymocytes based on the viable cell counts. Viable
64
thymocytes were processed quickly and total cell counts were taken with note of dead and viable
cell populations by Trypan blue exclusion dye. The Mbnl1-/-
thymocyte percent viability was not
significantly altered from WT ratios.
Additionally, the consistently observed apoptotic defects in older CD4CD8 DP
populations treated with Dex (Figure 3-3c & d) was in line with perturbations seen in the bone
marrow chimeras. Mbnl1-/-
-derived thymocytes had increased CD4CD8 DP populations in a
WT thymic environment (Figure 2-6b). It would be of interest to determine if directed signaling
through the TCR would fail to apoptose Mbnl1-/-
thymocytes by stimulating TCR with anti-CD3
Ab. Other known mutations in the TCR apoptosis pathways are not affected in the Mbnl1-/-
thymocytes. Alterations in caspases 3 and 8, previously identified as active in CD4CD8 DP
thymocyte apoptosis, are unaffected in the Mbnl1-/-
thymus (123) but splicing alterations are
found in other genes in the apoptosis pathway, such as Bnip2 and Rnf130 (Goliath).
Interestingly, BNIP2 cleavage promotes apoptosis and it is cleaved by caspases 3, 7, 8
and 9 (124). Because exon 10 lies within the BNIP2 and Cdc42GAP homology (BCH) domain,
it is possible exon 10 exclusion in Mbnl1-/-
thymocytes alters the BCH domain interactions with
Rho, preventing apoptosis post cleavage by caspase 3 and 8 in CD4CD8 DP thymocytes (125).
Rnf130 (Goliath) is another transcript implicated in apoptosis (126). The smaller isoform
of Rnf130, created by alternative exon 8 exclusion, is more ubiquitously expressed whereas
Rnf130 proteins including exon 8 are tissue specific for liver, lung, stomach, heart and thymus
(127, 128). The exact mechanism of Rnf130 involvement in apoptosis is still unclear although it
has been demonstrated to increase upon interleukin-3 (IL-3) mediated apoptosis (126).
Unfortunately, this is not an apoptosis pathway typically stimulated in developing thymocytes.
IL-3 is epigenetically silent in CD4CD8 DP thymocytes and is does not reach maximal
65
transcription potential until after TCR activation (129). RNF130 would, therefore, be important
for peripheral T cell homeostasis.
Splenocytes were also characterized in Mbnl1-/-
and WT mice at 4 and 12 weeks of age.
While 12-week animals showed decreased viable cell numbers, the B cell and T cell populations
were not significantly different from WT. The cumulative decrease in peripheral lymphocytes
might create the difference observed at enumeration or other cell populations, such as monocytes
and macrophages, could be reduced in Mbnl1-/-
spleens. CD3 positive peripheral T cells have
increased CD4 SP populations and correspondingly decreased CD8 SP populations. Given the
previously noted accumulation of CD8 SP thymocytes within the Mbnl1-/-
thymus (Figure 2-4b &
c), the decreased peripheral population could be due to blockage of CD8 SP cells in the thymus
either by blocked thymocyte development at the ISP stage or a failure of thymocytes to emigrate
into the periphery. Further flow cytometry characterization of the CD8 SP population within the
thymus should differentiate between ISP CD8 cells and mature CD8 SP cells failing to leave the
thymus by looking at several cell surface proteins. ISP populations express CD8, CD24 and
TCR beta but do not express CD3 or TCR alpha like CD8 SP cells (130, 131).
The increase in CD4 SP T cell population Mbnl1-/-
mice could reflect a proliferation of
this population to compensate for CD8 SP loss to maintain homeostasis. This idea is further
supported by the observed phenotype of Mbnl1-/-
peripheral T cells. As early as 4 weeks of age,
T cell populations have a more activated phenotype. This kind of phenotype is also observed in
Tcf7-/-
mice. As mentioned in Chapter 2, Tcf7-/-
mice demonstrate blockages in thymocyte
development and fail to pass the ISP stage. This loss of thymocyte development leads to a
decrease of peripheral T cells by 2-3 fold compared to control mice (94). Recent thymic
66
emigrants do not express CD44, whereas peripherally expanded T cells do, thus demonstrating
an activated phenotype similar to what is observed in the Mbnl1-/-
mice.
In addition to perturbations in peripheral T cell populations and phenotype, Mbnl1-/-
splenocytes recapitulate the altered splicing program observed in thymus. However, Mbnl1-/-
splenocytes to do not show splicing defects to the same extent as those observed within the
thymus. Decreased observed splicing alteration is most likely due to splenocytes not being
completely comprised of T cells, since they are a mixture of T cells, B cells, monocytes and
macrophages. Interestingly, splicing targets in the endocytosis pathway, Dnm2 and Cd47, are
faithfully mis-regulated in the splenocyte population as well. Dnm2 is an exceptionally
interesting target because Dnm2-/-
T cells have reduced TCR signaling, impaired clonal
expansion growth potential as well as compromised homeostatic proliferation (132). Peripheral
T cells of Dnm2-/-
mice underwent fewer divisions than control mice. Additionally, Dnm2-/-
mice
have impaired thymocyte egress. DNM2 regulates S1PR1 signaling which is critical for T cell
egress (133). This suggests the possibility CD8 SP loss in the periphery could be associated with
failure to emigrate. Also, CD8 SP T cell populations may not be able to proliferate to maintain
homeostasis in the periphery.
Cd47 alternative splicing modifies the C-terminal domain of this integrin-associated
protein. CD47 is another protein where splice isoforms are expressed in a tissue-restricted
pattern. CD47 form 2, which is the major isoform expressed in thymus and spleen, splices exon
8, 9 and 12 to create the C-terminus (134). CD47 signaling will depend on the CD47 ligand
presented by APCs during TCR ligation and activation. CD47 can activate apoptosis
independent of TCR-mediated induced cell death, but CD47 also enhances apoptosis signals
transmitted through the TCR (135). Mbnl1-/-
mice show increased usage of exons 10 and 11
67
creating CD47 forms 3 and 4, the isoforms typically expressed in testis, gut and brain (134). The
inappropriate expression of CD47 isoforms not normally present on T cells may not match
ligands presented by APCs and thus prevent efficient apoptosis in peripheral T cells and
developing thymocytes.
Mbnl1-/-
mice have shown defects in thymocyte development and function as well as
mature T cell populations. These defects may be attributed to dysregulation of specific splicing
targets due to loss of MBNL1 protein. DM1 and DM2 patient PBMCs surveyed for splicing
dysregulation did not show significant differences in splicing shifts. Although a clear trend was
observed in patient samples compared to control samples, DM transcripts were altered in the
same direction predicted by Mbnl1-/-
thymus splicing. Similar to mouse splenocytes, patient
PBMCs are a mixed population of white cells. It is possible splicing changes can be more
accurately surveyed by using purified T cell populations or patient thymic biopsy, noting that
purified T cell populations are more readily obtained than thymic biopsies. Another approach to
obtain significance, and verify the alternative splicing, would be to increase the sample number.
Three samples were assayed for each group and given that MBNL loss is incomplete in patients,
unlike knockout mice, observed splicing alterations tend to not be as severe as those in knockout
mice. Additionally, human samples are not on genetically homogenous backgrounds, unlike
Mbnl1-/-
mice. We have already explored the difference in strain background on observed
disease phenotype through genetic modifiers and humans as an out-bred population are an
exaggerated example of these effects.
68
Figure 3-1. Thymocyte TCR signaling model. T cell receptor (TCR) activation by peptide
antigen (Ag):major histone compatibility complex (MHC) begins intrathymocyte-
signaling cascades that can lead to activation of PTK2B, which potentially directs
apoptosis, proliferation and/or cellular migration. Additionally, TCR activation can
activate phospholipase C gamma (PLC) generating inositol triphosphate (IP3) which
is bound by inositol triphosphate receptors (IP3R) in the endoplasmic reticulum
leading to increased cytosolic calcium (CA2+
) levels that can activate PTK2B. Ca2+
can also enter the cytosol from the extracellular matrix via calcium release activated
channels, which can replenish the endoplasmic reticulum calcium stores or activate
PTK2B. In the nucleus, TCF7 can regulate genes responsible for differentiation,
survival and proliferation via beta catenin binding and act as a repressor LEF1. LEF1
increases TCR alpha transcription. Dysregulation of alternative splicing of Tcf7 or
Lef1 can either indirectly or directly affect TCR production, thereby affecting
downstream activities associated with PTK2B. Additionally, Ptk2b, alternative
splicing can potentially dysregulate apoptosis, proliferation and/or cellular migration
signaling.
69
Figure 3-2. Mbnl1-/-
thymocytes are resistant to dexamethasone-induced apoptosis. (a)
Schematic of apoptosis assay and flow cytometry analysis. (b) Early and late
apoptosis normalized to no treatment (Trx) controls for 4-, 12- and >20 week.
*p<0.05, **p<0.01
70
Figure 3-3. Mbnl1-/-
CD4CD8 double positive thymocyte populations are most resistant to
apoptosis. (a) Flow cytometry dot plots demonstrating thymocyte population specific
apoptosis analysis gating. (b) Four-week thymocytes separated by CD4 SP, CD4CD8
DP and CD8 SP populations analyzed for dexamethasone-induced apoptosis. (c)
Twelve-week thymocytes separated by CD4 SP, CD4CD8 DP and CD8 SP
populations analyzed for dexamethasone-induced apoptosis. (d) Greater than 20-week
thymocytes separated by CD4 SP, CD4CD8 DP and CD8 SP populations analyzed
for dexamethasone-induced apoptosis. *p<0.05, **p<0.01
71
Figure 3-4. Mbnl1-/-
splenocytes are decreased. (a) Viable cell counts taken from 4- and 12-
week splenocytes. (b) The 4- and 12- week analysis of B cell and T cell populations
in collected splenocytes. ***p<0.001
72
Figure 3-5. CD3 positive Mbnl1-/-
T cells populations are perturbed in the periphery. (a) Flow
cytometry dot plot demonstrating the T cell gating strategy. (b) 4- and 12- week T
cells were analyzed for CD4 SP, CD8 SP and CD4CD8 DP populations. *p<0.05,
**p<0.01
73
Figure 3-6. Mbnl1-/-
T cells functional phenotypes are altered. (a) Flow cytometry dot plots
showing the T cell gating strategy for CD62L and CD44 characterization. (b) The 4-
and 12-week T cells have an increased proportion of T cells with an activated
phenotype. Naïve and memory subsets are decreased in Mbnl1-/-
T cell, but were not
significant at both ages. *p<0.05, **p<0.01
74
Figure 3-7. Alternative splicing defects found in Mbnl1-/-
thymuses are recapitulated in Mbnl1-/-
splenocytes. (a) Developmental targets. (b) Apoptosis targets. (c) Endocytosis
targets.
75
Figure 3-8. DM peripheral blood monocytes have similar splicing defects found in Mbnl1-/-
thymus. (a) Peripheral blood monocytes (PBMCs) isolation schematic. (b) RT-PCR
analysis of dysregulated Mbnl1-/-
transcripts in DM1 and DM2 patient samples
compared to control.
76
CHAPTER 4
MUSCLEBLIND 2 AND 3 INVOVLEMENT IN T CELL DEVELOPMENT
Mbnl2 and 3 Mouse Models
Mbnl1 is the Mbnl family transcript that is most abundantly expressed in WT thymus
(Figure 4-1a). While MBNL2 and MBNL3 expression do not appear to up-regulate in the
absence of MBNL1 at the protein level (Figure 2-2d), RNA levels of both Mbnl2 and Mbnl3
increase in the Mbnl1-/-
thymus although only Mbnl3 upregulation is significant (*p<0.05)
(Figure 4-1b). MBNL3 expression in the thymus is particularly interesting because it is an
MBNL protein mostly expressed in embryonic tissues and very few adult tissues (37). For these
reasons, MBNL2 and MBNL3 were explored in thymocyte development using Mbnl2 and Mbnl3
knockout models.
Mbnl2-/-
Mouse Generation
An Mbnl2-/-
mouse model was previously created by our lab through gene targeting in
murine embryonic stem cells (mESCs) by floxing exon 2 (containing the protein start codon) and
creating a conditional knockout mouse line (Figure 4-2). Conditional Mbnl2 mice were then
crossed with a CMV-Cre mouse strain creating the Mbnl2-/-
mouse line. This mouse has been
used to study MBNL2 involvement in muscle and brain, but not in the immune system (84, 87).
Mbnl3 Whole Locus Knockout Mouse Generation
Similar to the design of the Mbnl2 knockout mouse, the Mbnl3 mouse was designed as a
conditional knockout. MBNL3, however, unlike the other two MBNL proteins, has two
isoforms coded by two separate start codons (37 kDa and 27 kDa). The original Mbnl3 knockout
mouse reported by Poulos et al. (37) removed exon 2, abrogating production of the 37 kDa
MBNL3 protein. However, in the absence of the large isoform, the smaller 27 kDa isoform is
upregulated (37). For this reason, a whole locus knockout was designed to better ascertain
77
MBNL3 involvement in thymocyte development. The original knockout targeting, floxing exon
2, was utilized as the starting donor mESCs for the whole locus generation. A third LoxP
sequence was inserted with a Puromycin resistance cassette, to aid in mESC selection, into the
intron between exons 7c and 8 (containing stop codon) (Figure 4-3a). Recombination in mESCs
created the Mbnl3 condition whole locus line (Mbnl3 condWL). Chimeric mice created from
Mbnl3 condWL mESCs were mated to obtain germline transmission of the mutant allele (Figure
4-3b & c). Further details of target construction, mESC targeting and screening are included in
Materials and Methods (Chapter 6). To generate Mbnl3 WLKO mice for further study, Mbnl3
condWL mice were mated with CMV-Cre mice to obtain recombination, eliminating exons 2-7
(Figure 4-3a). Because Mbnl3 is an X-linked gene and only male mice were used for the
remainder of this study, Mbnl3 WLKO is hereafter annotated as Mbnl3-/Y
.
Mbnl2-/-
and Mbnl3-/Y
Aged Mice Have Limited T Cell Defects
Because the most pronounced thymic defects were observed in Mbnl1-/-
mice >20 weeks
of age, Mbnl2-/-
and Mbnl3-/Y
mice were analyzed at ages > 20 weeks of age. Upon initial
dissection, thymic morphology of both Mbnl2-/-
and Mbnl3-/Y
mice similar to WT as well as
thymic wet weight to body weight ratios (n=3, per group) (Figure 4-4a). Thymocytes were
isolated and enumerated, but again, no significant changes were observed (Figure 4-4b).
Interestingly, when thymocytes were characterized for CD4 SP, CD8 SP and CD4CD8 DP
proportions Mbnl2-/-
mice showed statistically significant changes in CD4 SP and CD4CD8 DP
populations (*p<0.05) while Mbnl3-/Y
mice did not demonstrate any alterations (Figure 4-4c).
Developmental splicing of targets of MBNL1 were tested in Mbnl2-/-
and Mbnl3-/Y
thymuses. Neither MBNL2 nor MBNL3 loss resulted in splicing alterations like those observed
in Mbnl1-/-
mice (Figure 4-5). Sensitivity to apoptosis induction of Mbnl2-/-
and Mbnl3-/Y
thymocytes was also surveyed to test functionality of the thymocytes. As anticipated, no
78
observable change to apoptotic sensitivity was observed in any of the Dex treatment conditions
(Figure 4-6).
While few defects were observed on developing thymocytes, peripheral T cells were
assayed to determine if MBNL2 or MBNL3 proteins were necessary for maintaining
differentiated T cell populations. Spleens were extracted from Mbnl2-/-
and Mbnl3-/Y
mice (n=3,
each group) and splenocytes extracted and enumerated. Mbnl2-/-
mice showed significantly
decreased splenocyte amounts compared to WT (*p<0.05), while and Mbnl3-/Y
mice had an
increased amount of splenocytes compared to WT (*p<0.05). When T cell and B cell
populations of the Mbnl2-/-
and Mbnl3-/Y
splenocytes were analyzed, as previously described, no
changes in either B or T cell proportions were observed (Figure 4-7a).
Further exploration of T cell specific populations was performed and CD4 SP, CD8 SP,
CD4CD8 DP were evaluated. Again, neither Mbnl2-/-
and Mbnl3-/Y
mice showed any significant
changes compared to WT mice (Figure 4-7b). Even though no changes were observed in
peripheral T cell populations of Mbnl2-/-
and Mbnl3-/Y
mice, there still remained a possibility that
the phenotype of the peripheral T cell populations could be altered. To further evaluate this
possibility, CD62L and CD44 surface markers were analyzed on CD3 positive T cells. Neither
Mbnl2-/-
nor Mbnl3-/Y
mice showed any alteration in naïve, activated or memory phenotype T
cells compared to WT controls (Figure 4-7c).
Discussion
To obtain a complete picture of Mbnl regulation on thymocyte development, Mbnl3
knockout mice were generated to supplement the pre-existing Mbnl1 and Mbnl2 lines.
Following successful generation of the Mbnl3 WLKO line, Mbnl2-/-
and Mbnl3-/Y
thymuses and
thymocytes were analyzed for thymic defects observed in the Mbnl1-/-
mouse. Although Mbnl3
and Mbnl2 RNA are upregulated in the absence of MBNL1, neither Mbnl2-/-
nor Mbnl3-/Y
79
thymuses display an enlarged thymic phenotype. Viable thymocyte levels were unchanged in
Mbnl2-/-
and Mbnl3-/Y
compared to WT controls in animals >20 weeks of age. Unexpectedly,
Mbnl2-/-
thymocytes did demonstrate an increased proportion of CD4 SP populations and a
decrease in CD4CD8 DP populations. Alternative splicing analysis revealed thymocyte
developmental genes Lef1, Tcf7 and Ptk2b maintained WT splicing patterns in both Mbnl2-/-
and
Mbnl3-/Y
mice. This suggests that the splicing of Lef1, Tcf7 and Ptk2b RNAs is dependent on
MBNL1 only. Unsurprisingly, Mbnl2-/-
and Mbnl3-/Y
thymocyte apoptosis testing by Dex showed
no defects in either knockout compared to WT. Splenocyte analysis revealed both Mbnl2-/-
and
Mbnl3-/Y
mice were altered compared to WT with Mbnl2-/-
viable splenocyte counts were
decreased while Mbnl3-/Y
counts were increased. However, these observed changes in viable
splenocyte count were not reflected in either T cell or B cell peripheral populations. Peripheral T
cells were not altered in CD4 SP, CD8 SP or CD4CD8 DP populations, nor were T cell
phenotypes perturbed. The changes in both Mbnl2-/-
and Mbnl3-/Y
splenocyte populations could
be the product of other white cells, such as monocytes or macrophages.
Taken together, these data suggest that while MBNL3 loss does not appear to affect
thymocyte development or maintenance of peripheral T cell populations there may be MBNL2
specific transcripts that are altered to encourage CD4 SP thymocyte differentiation. CD4 SP and
CD8 SP selection choices within the thymus occur via instruction through TCR signaling
strength. Strong and prolonged TCR signals promote CD4 SP differentiation, while short,
repeated TCR signals promote CD8 SP differentiation (72). Loss of MBNL2 proteins may
promote either a more stabilized TCR signal or an increased signal intensity sending more
thymocytes towards CD4 SP differentiation. Perhaps this change in thymocyte populations
suggests a role for MBNL2 in thymocyte development, albeit, at a level below MBNL1
80
involvement. High-throughput crosslinking and immunoprecipitation assay (HITS-CLIP) has
revealed similar RNA binding motifs for both MBNL1 and MBNL2 proteins (136). Although
the loss of MBNL2 or MBNL3 does not alter splicing in the presence of MBNL1, it is possible
that a compound loss of MBNL proteins exacerbates thymic splicing changes as evidenced in
other tissue systems, such as muscle and brain (84, 137).
81
Figure 4-1. RNA-seq Mbnl expression levels. (a) Mbnl RNA expression in WT thymus in WT
and Mbnl1-/-
thymus. (b)Mbnl2 and Mbnl3 expression changes in WT vs. Mbnl1-/-
thymus. RPKM = Reads per kilobase transcript per million mapped reads
82
Figure 4-2. Mbnl2 targeting scheme and Mbnl2 knockout generation.
83
Figure 4-3. Mbnl3 knockout mouse targeting scheme, germline transmission and knockout
generation. (a) mESC targeting design utilizing previously targeted Mbnl3 mESCs
and Mbnl3 WLKO mouse construction. (b) Approximately 40% coat chimeric male
mouse received from University of Michigan Transgenic Mouse Models Core. (c)
Mbnl3 condWL germline transmission is evidenced by the appearance of a black
female mouse born to an Mbnl3 condWL chimeric male and albino female. Pup is
pictured with mother and albino littermates.
84
Figure 4-3. Continued. Mbnl3 knockout mouse targeting scheme, germline transmission and
knockout generation. (a) mESC targeting design utilizing previously targeted Mbnl3
mESCs and Mbnl3 WLKO mouse construction. (b) Approximately 40% coat
chimeric male mouse received from University of Michigan Transgenic Mouse
Models Core. (c) Mbnl3 condWL germline transmission is evidenced by the
appearance of a black female mouse born to an Mbnl3 condWL chimeric male and
albino female. Pup is pictured with mother and albino littermates.
85
Figure 4-4. Mbnl2-/-
and Mbnl3-/Y
mice >20 weeks old have limited thymocyte defects. (a)
Thymic morphology and thymic weight body to body weight ratios in Mbnl2-/-
and
Mbnl3-/Y
mice. (b) Mbnl2-/-
and Mbnl3-/Y
total viable thymocytes extracted from >20-
week thymuses. (c) CD4 SP, CD8 SP and CD4CD8 DP populations of Mbnl2-/-
and
Mbnl3-/Y
thymocytes.
86
Figure 4-5. Alternative splicing defects found in Mbnl1-/-
thymuses are not altered in Mbnl2-/-
and Mbnl3-/Y
thymuses.
87
Figure 4-6. Mbnl2-/-
and Mbnl3-/Y
thymocytes are sensitive to apoptosis induction by
dexamethasone. (a) The >20-week old thymocytes from Mbnl2-/-
mice are just as
sensitive to dexamethasone induced apoptosis as WT. (b) The >20-week old
thymocytes from Mbnl3-/Y
mice are just as sensitive to dexamethasone-induced
apoptosis as WT.
88
Figure 4-7. Mbnl2-/-
and Mbnl3-/Y
peripheral T cell populations are unaltered. (a) Mbnl2-/-
and
Mbnl3-/Y
viable splenocyte cell counts. (b) CD3 positive T cell populations analyzed
for CD4 and CD8 expression. (c) Mbnl2-/-
and Mbnl3-/Y
peripheral T cell phenotype
percentage.
89
CHAPTER 5
CONCLUDING REMARKS
This study explored the involvement of the RNA binding protein family, Muscleblind-
like (MBNL), on T cell development and function. Loss of MBNL proteins result in mis-
splicing of developmentally regulated genes (18, 37, 87). Based on RNA expression, MBNL1 is
the most highly expressed MBNL family member in the thymus. This knowledge, coupled with
the finding that Mbnl1-/-
mice develop a remarkably large thymus late in life, led the focus of the
analyses to be directed specifically at MBNL1 regulation of thymocyte development and
function.
We found Mbnl1-/-
thymocytes demonstrated dysregulation of CD4 SP, CD8 SP and
CD4CD8 DP populations, suggesting a defect in thymocyte differentiation. We also showed,
through use of bone marrow chimeras, that the defect associated with MBNL1 loss is thymocyte
intrinsic and not a result of dysregulation in the educational thymic epithelial cells (TECs).
Dysregulation of thymocyte development by MBNL1 is most likely due to RNA mis-splicing of
key thymocyte specific genes such as Lef1, Tcf7 and, possibly, Ptk2b.
The mis-splicing of these gene transcripts plays a role, directly or indirectly, in T cell
receptor (TCR) signaling (Figure 3-1). TCR signaling is one critical pathway in thymocyte
development and central tolerance as well as peripheral activation and recognition of foreign
antigen. Mbnl1-/-
mice present with multiple symptoms consistent with TCR signaling defects
such as impaired development beyond CD4CD8 DP, abnormal T cell survival, decreased
circulating CD8 SP T cells and normal proliferation when stimulated with Phorbol myristate
acetate (PMA) and Ionomycin (Ion) (data no shown). To more definitely provide evidence for
TCR signaling defects in Mbnl1-/-
thymocytes and T cells, stimulation assays crosslinking the
TCR would need to be performed.
90
Alternatively, MBNL1 also affects splicing of several calcium channels (39, 88).
Because PMA and Ion bypass the need for TCR signaling events to induce calcium flux,
thymocytes behave normally when treated with these two drugs but this highlights the
importance of calcium fluctuations in thymocyte development and activation. Further inquiry
into calcium regulation in Mbnl1-/-
thymus would be interesting.
Mbnl1-/-
defects on developing thymocytes lead to perturbations of peripheral T cell
populations as well. It is unclear if Mbnl1-/-
T cells fail to emigrate from the thymus or if loss in
CD8 SP peripheral populations are due to blockages in development. Tcf7 deficient mice also
share several similarities with our Mbnl1-/-
mice. Tcf7-/-
mice have decreased CD8 SP
populations due to a developmental defect of thymocytes causing blockages at the induced single
positive (ISP) stage. The CD4 SP population of Tcf7-/-
T cells, however, is also non-functional.
We have not yet tested the function of the mature T cells in the periphery of our Mbnl1-/-
mice. It
would be intriguing to determine if the splicing defects functionally affect Mbnl1-/-
ability to
mount a T cell-mediated response to antigen. As our animals are housed in specific pathogen
free (SPF) housing, there is a decreased probability of animals encountering pathogens that
would stimulate an immune response. The animals would have to be subjected to an antigenic
challenge to ascertain the ability of the peripheral T cells to mount an appropriate immune
response and coordinate with other cells of the immune system.
The observed thymic enlargement of Mbnl1-/-
mice was of initial interest because there
have been multiple case reports of DM patients presenting with a thymoma. Of these cases,
several have been classified as a lymphocytic thymoma similar to the Mbnl1-/-
mice. However,
the thymic enlargement phenotype was a separable event from alternative splicing dysregulation.
It would be interesting to further characterize the thymocytes of the thymoma for TCR clonality.
91
Our mice phenocopy a Tcf7-/-
Lef1-/-
double knockout mouse model that produce T cell leukemia
with age causing thymic enlargement owing to dysregulation of the Notch signaling pathway.
While DM patients have exhibited enlarged thymuses, thymoma is most often associated
with the autoimmune disease myasthenia gravis. The cause for thymoma in these patients is still
unknown even though 30% of patients develop thymoma (138). Altered B cell to T cell ratios
within the thymus also hallmarks myasthenia gravis thymoma (139). Analysis of thymic B cell
populations was not performed in our Mbnl1-/-
mice, but the 129 Sv/ImJ model may provide
insight into the understanding of thymoma development.
Importantly, we have started to show here that DM PBMCs share similar splicing
patterns as our Mbnl1-/-
mice. To date, the immune system of DM patients has not been
thoroughly investigated. Some correlative studies have shown decreased T cell populations in
DM patients inversely proportional to repeat size and other studies have associated DM with
autoimmune disease. The research presented here, lays the foundation for testing if altered
splicing shifts caused by MBNL sequestration leads to deficits in T cell immunology.
Several RNA binding proteins have important regulatory roles in both T cell development
and activation (140) so it is important to extend our knowledge of co-/post-transcriptional
regulation of immune specific splicing into other RNA-mediated diseases. Testing other
microsatellite repeat expansion diseases that lead to loss of RBP functions may uncover immune
deficits previously hidden or simply overlooked.
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CHAPTER 6
MATERIALS AND METHODS
Mbnl1 Animal Use for Thymic Analysis
129 Sv/ImJ mice were housed under specific pathogen free conditions. Mice were used
at ages 4 weeks, 12 weeks and 20-24 weeks (>20 weeks) for thymocyte and splenocyte
characterization. Animals 9 weeks of age were used for RNA-seq and splicing validation
studies. All animal procedures and endpoints were in accordance with University of Florida
approved Institutional Animal Care and Use Committee (IACUC) guidelines.
Thymocyte and Splenocyte Harvest
Animals were sacrificed in accordance to IACUC approved protocols. Thymus and
spleen were dissected into ice cold 1XPBS and connective tissue and fat removed. Single cell
suspensions were made by mincing thymic tissue through 100 um cell strainers (BD
Biosciences) into 1XPBS, pH 7.4. Cells were pelleted 400 × g, 10 minutes, room temperature
(RT) and resuspended in 150 mM ammonium chloride, 1 mM potassium bicarbonate, 0.01 mM
EDTA, pH 7.2 (Thermo Fisher) for red blood cell lysis. Cells were pelleted again, as above, and
resuspended in RPMI-1640 (Mediatech) supplemented with 10% fetal bovine serum (FBS)
(cRPMI; Hyclone). Viable cells were enumerated using Trypan Blue exclusion dye (Sigma).
Flow Cytometry
Anti-CD4-PE-Cy7 or anti-CD8-APC, anti-CD8-Pacific Blue or anti-CD8-PE-Cy5, anti-
CD3-AlexaFluor700, anti-CD45R-Pacific Blue, anti-L-selective-PE and anti-CD44-V500 were
purchased from BD Biosciences and were used for all flow cytometry analysis. 2 × 106 cells
were stained in 1XPBS, 0.5% Bovine Serum Albumin (BSA) (FACS Buffer, Sigma) or Brilliant
Violet Buffer for anti-CD44-V500 as provided by manufacturer’s protocol. Cells were analyzed
using a BD LSR II (BD Biosciences). Cells were gated away from debris based on forward
93
angle light scatter (FSC) and side angle light scatter (SSC) profiles. Data were analyzed using
FlowJo Data Analysis Software (Tree Star). For adoptive transfer experiments, CD45.1-FITC
and CD45.2 PE were used to determine engraftment success. CD4-PE-Cy7 and CD4-PE-Cy5
were used in conjunction with the aforementioned CD45 labels.
Adoptive Transfer
The adoptive transfer assay procedures and endpoints were in accordance with University
of Florida approved Institutional Animal Care and Use Committee (IACUC) guidelines. Gamma
irradiation and transplantation was performed with help and guidance of the laboratory of Dr.
Edward Scott.
Six-week old Mbnl1-/-
mice (CD45.2) and B6.SJL-Ptprca Pepc
b/BoyJ (CD45.1, WT)
(The Jackson Laboratory, CN: 002014) were utilized as both the sources of hematopoietic stem
cell (HSC) donors and transplant recipients. Five animals of each genotype were subjected to
total body irradiation in a cesium gamma irradiator at a single radiation dose of 750 rads. Two
animals of each genotype were sacrificed and bone marrow collected from the long bones
(bilateral femurs and tibias) with a 28-gauge needle attached to 3 mL syringe filled with RPMI-
1640 media (Mediatech). Bones were gently flushed of marrow and collected into a 15 mL
conical. Cells were pelleted 400 × g, 10 minutes, room temperature (RT) and resuspended in
150 mM ammonium chloride, 1 mM potassium bicarbonate, 0.01 mM EDTA, pH 7.2 (Thermo
Fisher) for red blood cell lysis. Cells were pelleted 400 × g, 10 minutes, room temperature (RT)
again and resuspended in 1 mL RPMI-1640 (Mediatech) and enumerated by hemacytometer
counting with Trypan blue exclusion dye (Sigma). Syringes (1 mL) fitted with a 30-gauge
needle were prepared with 107 viable HSCs in 100 uL RPMI-1640 for each animal to be injected.
Transplant recipients were anesthetized with Avertin/TBE anesthesia (220-280 mg/kg) delivered
via intraperitoneal (IP) injection. Once anesthetized, HSCs were delivered in 100 uL volumes
94
via retro-orbital sinus cavity injection. With the beveled side of the needle pointing towards the
eye, the needle is introduced gently between the eyeball and eyelid of the mouse in the corner
closest to the nose. Gently the needle is maneuvered to the back of the orbit while remaining
within the plane of eye. Once the needle is approximately two-thirds around the orbit, the needle
is lowered approximately 45° allowing the needle point to naturally and gently perforate the soft
tissue overlying the sinus cavity. The HSC suspension is slowly and gently delivered into this
space while monitoring the eyeball position for indicators of pressure build up. Once delivery is
complete, the needle is carefully removed and the anesthetized animal placed under warming
lights to allow recovery. For 14 days post transfer, mice are maintained on enrofloxacin (Bayril,
Bayer) supplemented in drinking water at 70-80 mg/kg/day, with fresh bottle changes each week.
Four to nine weeks post transfer, animals were bled via submandibular facial vein
puncture, collecting no more 200 uL blood into potassium EDTA vacutainer tubes (BD
Biosciences). Flow analysis was performed as indicated in flow cytometry section. Animals
were sacrificed 24 weeks post transfer for thymocyte analysis. In the event of early death,
animals were collected within 6-12 hours or less for necropsy and post-mortem analysis.
Protein Lysate Generation and Immunoblot
Dissected tissues were homogenized in lysis buffer (20 mM HEPES-KOH, pH 8.0, 100
mM KCl, 0.1% Igepal CA-630 (Sigma), 0.5 mM phenylmethylsulphonyl fluoride, 5 ug/mL
pepstatin A, 1 ug/mL chymostatin, 1 mM -aminocaproic acid, 1 mM p-aminobenzamidine, 1
ug/mL leupeptin, 2 ug/mL aprotinin) by disposable pestle followed by sonication on ice and
centrifugation (16,100 × g, 15 min, 4°C) (84, 87). Protein lysates were quantitated using the DC
Protein Assay (Bio-Rad) according to manufacturer’s instructions. Total proteins (50 ug/lane)
were resolved on 12.5% SDS-acrylamide and transferred to nitrocellulose membranes. Blots
95
were blocked for 45 minutes in 5% non-fat dry milk in 1XPBS, 0.1% Tween-20 (Sigma) 45
minutes. Primary antibodies, anti-Mbnl1 A2764 (a gift from Dr. Charles Thornton), anti-Mbnl2
3B5 (Santa Cruz Biotechnology), anti-MBNL3 M3g (37) and anti-Gapdh 6c5 (Abcam) were
used for immunoblotting either overnight, 4°C or 2 hours, RT. Blots were washed 3 × 1XPBS,
0.1% Tween-20 (Sigma), 10 minutes, RT prior to secondary antibody detection with anti-rabbit
IgG or anti-mouse-IgG, conjugated to horseradish peroxidase (HRP) (GE Healthcare) in wash
buffer. Secondary antibodies were incubated 1 hour, RT in wash buffer. Membranes were
washed, as described above, and developed in Western Lightning Plus ECL detection reagents
(Perkin Elmer) and exposed to Biomax Film (Kodak).
Apoptosis Induction and Detection
Thymocytes were plated in 24-well culture dishes (Corning) at 1.5 × 106 cells per well in
cRPMI medium. Dexamethasone solubilized in DMSO (Sigma) was added to each well at final
concentrations of 10-6
, 10-7
, 10-8
M or 0 M (vehicle only control) for each test sample. Cells
were incubated 12 hours at 37°C, 5% CO2 in a humidified chamber. Post treatment, cells were
collected into 5 mL round bottom tubes and centrifuged 400 × g, 5 minutes, RT. Cells were
resuspended in FACS Buffer and stained with anti-CD4 and anti-CD8 antibodies. Post
incubation 30 minutes, 4°C, light safe, cells were washed in FACS Buffer and centrifuged as
previously described. Apoptotic markers Annexin V and propidium iodide staining were
completed using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to
manufacturer’s protocol.
Testosterone ELISA
129 SvIm/J and C57 BL6/J WT and Mbnl1-/-
mice 12-13 weeks of age mice were bled via
facial vein puncture and approximately 200 uL of whole blood collected in untreated microfuge
tubes (n = 3, each group). Blood was allowed to clot on ice for 30 minutes. Post coagulation,
96
the blood was centrifuged 1000 × g, 10 minutes, RT. Separated sera was then collected and
frozen at -20ºC until assayed.
Frozen sera samples were thawed on ice and diluted 1:15 for the Parameter Testosterone
Assay (RND Systems). The assay was performed according to the manufacturer’s provided
protocol. Briefly, the Parameter Testosterone Assay is based on a competitive binding enzyme
linked immunosorbent assay (ELISA). A testosterone specific monoclonal antibody is bound to
microplate wells. Serum is added to the coated wells with an HRP-conjugated Testosterone that
acts as a competitor. Post incubation HRP enzyme substrate is added and a colorimetric readout
is measured with the intensity of the color being inversely proportional to sample testosterone
concentration. Absorbance readings (450 nm and 540 nm) were read on a Biotek Cytation3 with
Gen 5.0 collection software (Biotek).
RNA-seq
Total RNA was isolated from 9 week WT and Mbnl1-/-
thymus using 2 mL Tri Reagent
and homogenization by PolyTron (Kinematica) according to manufacturer’s provided protocol.
RNA was further cleaned of any potential contaminating DNA with the RNeasy Kit (Qiagen)
following according to manufacturer’s instructions. RNA quality was tested using a Bioanalyzer
2100 (Agilent Technologies) and quantitated using Quant-iT RiboGreen RNA Assay Kit
(Thermo Fisher). RNA-seq 50 nt paired end reads were generated by The New York Genome
Center from WT and Mbnl1-/-
thymic RNA (n = 3 each). Reads were aligned to the mm10
reference and known exon junctions utilizing Olego and processed using Quantas (141, 142) to
identify differentially spliced alternative exons. Exons with coverage ≥ 20, |percent spliced in
(PSI)| ≥ 0.1 and false discover rates (FDR) ≤ 0.05 were analyzed.
97
RT-PCR Splicing Validation
Mouse RNA was isolated from 8-9 week old thymus from WT, Mbnl1-/-
, Mbnl2-/-
and
Mbnl3-/Y
mice using Tri Reagent (Sigma) according to manufacturer’s protocol, it is worthy to
note that 1 mL Tri Reagent was used per thymic lobe extracted. For splenocyte RNA extraction,
0.5 mL Tri Reagent was used for purification. RNA was quantitated on a Nanodrop (Thermo
Scientific) and 2.5 ug used to generate complementary DNA (cDNA) utilizing SuperScript II
Reverse Transcriptase (Invitrogen) according to manufacturer’s provided protocol. PCR reaction
amplification conditions were 95ºC for 2 minutes, 95ºC for 30 seconds, 55ºC for 30 seconds,
72ºC for 1 minute (28× amplification cycles), 68ºC for 10 minutes (final elongation) and a 10ºC
hold. PCR reactions used 100 ng of RNA reverse transcription reaction as template. Each PCR
was spiked with 2.5 uCi (1 Ci = 37 GBq) of alpha-32
P dCTP (Perkin Elmer) in a 25 uL reaction
containing 0.4 mM dNTPS, 30 pmol forward primer, 30 pmol reverse primer 1X High Fidelity
Buffer (5 Prime), 1.25 U Triple Master Taq polymerase (5 Prime) (See Table 6-1 for primer
sequences). PCR products were resolved on 10-15% non-denaturing acrylamide gels followed
by autoradiography with Biomax MS film (Kodak). Splicing shifts were measured using a
Typhoon 9200 Imager (GE Healthcare). Percentage of each band in each PCR reaction was
quantified with ImageQuant TL software (GE Healthcare).
Human Peripheral Blood Lymphocyte Isolation and RNA Extraction
Human patient blood was collected in accordance with approved UF Institutional Review
Board (IRB) protocols. Blood was collected into 5 mL Potassium EDTA coated vacutainer tubes
(BD Biosciences). In accordance with biosafety level 2 precautions, all blood was processed
wearing appropriate personal protective equipment and handled in a biological safety cabinet
(BSC) until inactivation of samples was completed.
98
Peripheral blood lymphocytes were enriched on a Ficoll-Paque density gradient
according to manufacturer’s provided protocol (GE Healthcare). Blood samples collected
yielded ~2.5 to 3.5 mL whole blood for processing. All samples purified were normalized to the
lowest sample volume. Blood was underlayed with Ficoll-Paque and centrifuged to separate
blood constituents. Plasma was collected and stored for each patient sample. White blood cells
were collected and enumerated post RBC lysis. Lymphocytes were suspended in
cryopreservation media (60% cRPMI, 30% FBS, 10% DMSO) and stored in liquid nitrogen until
further use.
To extract RNA from cryopreserved patient lymphocytes, cryovials were first quickly
thawed in a 37ºC water bath and immediately resuspended in 10 mL pre-warmed cRPMI to
prevent cell lysis. Cells were centrifuged and lysed with 1 mL TRI reagent (Sigma) in a BSC.
Post cell lysis, remaining purifications steps were performed at a benchtop.
RNA was quantitated on a Nanodrop (Thermo Scientific) and 1 ug used to generate
complementary DNA (cDNA) utilizing SuperScript II Reverse Transcriptase (Invitrogen)
according to manufacturer’s provided protocol. PCR reaction amplification conditions were
95ºC for 2 minutes, 95ºC for 30 seconds, 55ºC for 30 seconds, 72ºC for 1 minute (30×
amplification cycles), 68ºC for 10 minutes (final elongation) and a 10ºC hold. PCRs each used
40 ng of RNA reverse transcription reaction as template. Each PCR was spiked with 2.5 uCi (1
Ci = 37 GBq) of alpha-32
P dCTP (Perkin Elmer) in a 25 uL reaction containing 0.4 mM dNTPS,
30 pmol forward primer, 30 pmol reverse primer 1X High Fidelity Buffer (5 Prime), 1.25 U
Triple Master Taq polymerase (5 Prime) (See Table 6-1 for primer sequences). PCR products
were resolved on 10-15% non-denaturing acrylamide gels followed by autoradiography with
Biomax MS film (Kodak). Splicing shifts were measured using a Typhoon 9200 Imager (GE
99
Healthcare). Percentage of each band in each PCR reaction was quantified with ImageQuant TL
software (GE Healthcare).
Mbnl3 mESC Targeting
The Mbnl3 conditional whole locus knockout (WLKO) targeting vector was created using
standard recombineering bacterial strains and techniques using protocols 1-4
(http://web.ncifcrf.gov/research/brb/protocol.aspx). Briefly, an approximate 7.5 kb fragment
containing Mbnl3 intron 7 was retrieved from a C57 BL/6 bacterial artificial chromosome (BAC)
into a high copy plasmid backbone containing herpes simplex virus thymidine kinase (HSV-TK).
A Puromycin resistance cassette with a single downstream LoxP sequence was inserted into the
intron 7 sequence creating a 3350 bp 5’ arm of homology (AOH) and 4180 bp 3’ AOH (Figure
4-3) within the Mbnl3condWL
targeting construct. Approximately 100 ug of Mbnl3condWL
targeting
construct was linearized with NotI (NEB), phenol:chloroform extracted, ethanol precipitated and
resuspended in 1X PBS, pH 7.2.
Murine embryonic stem cells (mESCs) previously targeted for Mbnl3 conditional large
isoform knockout (Mbnl3condE2/Y
) clone 85 served as the mESCs to be targeted. mESCs were
grown on mitotically inactivated murine embryonic fibroblast cells (MEFs) using standard
mESC growth techniques in a 37ºC, 5% CO2 humidified chamber. ESC growth media consisted
of DMEM (Invitrogen) supplemented with 15% heat-inactivated defined, ESC grade fetal bovine
serum (FBS) (Hyclone), 1X nucleoside stock (standard mESC formulation, Millipore
recommendation), 1% Penicillin 5000 U/mL:Streptomycin 5000 ug/mL (Invitrogen), ~10-4
M
beta mercaptoethanol (Invitrogen), 1% MEM non-essential amino acids (Invitrogen) and 103 U
LIF (Millipore). Twenty micrograms of purified, linearized targeting vector was electroporated
into 107 mESCs using a Bio-Rad Genepulser (300 V, 250 uF, infinite Ohms, exponential decay
pulse, 4-5 msec time constant in a 0.4 cm electroporation cuvette). Four to five 20 ug/107
100
mESCs electroporation cuvettes were pulsed. mESCs were plated immediately on 20-25 10 cm
culture dishes containing MEFs. Twenty-four hours post electroporation and plating, positive
selection was performed using both 250 ug active G418/mL (Invitrogen) and 1 ug/mL
Puromycin (Invitrogen) supplemented to the regular mESC growth media. Forty-eight hours
post electroporation mESCs were continued on the aforementioned positive selection regime and
began simultaneous negative selection with 0.2 uM fialuridine (FIAU) (Moravek Biochemicals)
for the next 72 hours. Following the negative selection period, positive selective pressure was
maintained on the mESCs throughout the culture duration. Individual colonies were picked with
a 200 uL sterile pipette tip, trypsinized and plated in 24-well plates, 1 well/colony. Colonies
were expanded until confluency was reached. At confluency, colonies were trypsinized. Half of
the each targeted colony was passaged into one well of a 12-well plate for further expansion and
gDNA extraction, the remaining half was cryopreserved in 0.5 mL cryopreservation media (60%
growth media, 30% FBS, 10% DMSO) for re-expansion post southern blot confirmation of
targeted homologous recombination.
Positively identified clones were thawed, expanded and cryopreserved for IMPACT I
profile mouse pathogen testing by the Research Animal Diagnostic Laboratory (RADIL)
(University of Missouri) and subsequent karyotyping and blastocyst injection at the University of
Michigan Mouse Transgenic Core at passage 27.
Briefly, 60 B6(Cg)-Tyrc-2J/J
donor blastocysts were injected with BL/6- Mbnl3condWL
clone 40. All 60 injected blastocysts were transferred to pseudo pregnant females. The birth rate
was 13%, with 50% of live births resulting in chimeric animals (chimeric coat range: 5-40%, 3
male/1female). Chimeric mice were imported to the University of Florida (UF) through Animal
101
Care Services (ACS) approved methods and quarantined for pathogen testing before release to
UF specific pathogen free (SPF) mouse colony housing.
Germline transmission of the Mbnl3condWL
allele was obtained by mating Mbnl3condWL
chimeric males with B6(Cg)-Tyrc-2J/J
females. Because Mbnl3 is an X-linked gene, males can
only transmit the mutant allele to F1 females. All F1 female pups with a black coat color
(indicating BL/6 targeted mESC derived gametes) were genotyped for the mutant allele.
To generate the Mbnl3 whole locus knockout mouse line (Mbnl3 WLKO), F1
Mbnl3condWL/+
females were mated to B6.C-Tg(CMV-Cre)1Cgn/J males (The Jackson
Laboratory, CN: 006054, also X-linked) to recombine the LoxP site, removing exons 2-7c and
creating F1 Mbnl3 WLKO strain animals. F1 females carrying Mbnl3-/Cre
were mated to
Mbnl3cond/Y
males
mESC Genomic DNA Isolation Southern Blot Analysis Mbnl3 condWL
Targeted mESC genomic DNA was extracted from confluent colonies. First, cells were
washed with 1X PBS, pH 7.2 (Invitrogen) to remove debris and culture medium. Next, cells
were lysed in 0.5 mL Lysis Buffer (200 mM NaCl, 100 mM Tris, pH 8.5, 5 mM EDTA, 0.2%
(w/v) SDS, 200 ug/mL Proteinase K), overnight in a 37ºC, 5% CO2 humidified chamber.
Twelve to 18 hours later, lysates were collected and ethanol precipitated. gDNA was
resuspended in 100 uL 1XTE buffer, pH 8.0 with a wide bore tip to prevent shearing of DNA.
5’ and 3’ probes were generated by PCR using 100 ng BL/6 mESC gDNA as template
and containing 0.4 mM dNTPS, 30 pmol forward primer, 30 pmol reverse primer 1X High
Fidelity Buffer (5 Prime), 2.5 U Triple Master Taq polymerase (5 Prime). PCR amplification
conditions were 96ºC for 4 minutes, 96ºC for 30 seconds, 68ºC 30 seconds, 72ºC 30 seconds
(30× amplification cycles), 72ºC for 10 minutes (final elongation) and a 10ºC hold (see Table 6-1
for primer sequences). PCR products were agarose gel purified and extracted using Qiagen’s
102
QIAquick gel extraction kit according to manufacturer’s protocol; 5’ probe PCR amplicon
expected 887 bp size and 3’ probe PCR amplicon expected 778 bp.
Approximately 10-20 ug of gDNA was digested with either SacI or BmtI (NEB) for 5’ or
3’ probing, respectively, in a 30 uL reaction containing 50-100 U of respective restriction
enzyme. Digestion reactions incubated overnight (12-16 hours), 37ºC. Digestions were spiked
the next day with 10-50 U of enzyme and incubated for an additional 6 hours. Samples were
resolved on 0.8% agarose gels, 30 V, overnight (12-16 hours), denatured for 1 hour in 0.5 M
NaOH, 1.5 M NaCl, pH 12 and neutralized for 1 hour in 0.5 M Tris, 1.5 mM NaCl, pH 7.4.
DNA was transferred to Hybond-N+ nylon membranes (GE Healthcare) in 10X SSC using a
neutral capillary action transfer. Membranes were cross-linked with a UV Stratalinker 1800
(Stratagene) on the autocrosslink setting. Prior to probing, membranes were stained with
methylene blue to visualize the DNA and DNA ladder, then scanned. Membranes were pre-
hybridized in either ExpressHyb Hybridization Solution (Clontech) (5’ probe) or Church Buffer
(3’ probe) for 1 hour, 68ºC. Southern probes were generated via alpha-32
P dCTP (Perkin Elmer)
random prime labeling reaction using 50 ng of probe template and Ready-to-Go DNA Labeling
Beads (GE Healthcare). Radiolabeled probes were purified on Illustra ProbeQuant G-50 Micro
columns (GE Healthcare) according to manufacturer’s instructions. Prior to probe hybridization,
probes were boiled at 100ºC, 10 min and added to pre-warmed (68ºC), fresh hybridization buffer.
Hybridization occurred overnight (12-18 hours), 68ºC. Post hybridization, blots were washed
with 2X SSC, 0.1% SDS at 50ºC, 2X SSC, 0.1% SDS at RT, twice with 0.5X SSC, 0.1% SDS at
RT, and finally 0.05X SSC, 0.1% SDS at RT for 30 minutes. Each wash was performed with
gentle agitation. Blots were exposed to Biomax MS Film and X-Omatic Intensifying Screens
103
(Kodak) at -80ºC. 5’ WT allele fragment size was 9.4 kb and mutant allele 6.6 kb. 3’ WT allele
fragment size was 9.6 kb and mutant allele 7.2 kb.
Mbnl3 condWL and Mbnl3 WLKO Genotyping
Mbnl3 condWL mice were genotyped using 100 ng genomic DNA template in 50 uL
reaction containing 0.4 mM dNTPS, 30 pmol forward primer, 30 pmol reverse primer 1X High
Fidelity Buffer (5 Prime), 2.5 U Triple Master Taq polymerase (5 Prime). Mbnl3 condWL and
Mbnl3 WLKO PCR amplification conditions were 96ºC for 2 minutes, 96ºC for 30 seconds,
60ºC for 30 seconds, 68ºC for 1 minute (35× amplification cycles), 68ºC for 10 minutes (final
elongation) and a 10ºC hold, amplicon sizes: Mbnl3 condWL 708 WT allele, 524 bp mutant
allele and Mbnl3 WLKO 708 WT allele, 414 bp mutant allele. Cre PCR amplification
conditions were 96ºC for 2 minutes, 96ºC for 30 seconds, 68ºC for 1 minute (35× amplification
cycles), 68ºC for 5 minutes (final elongation) and a 10ºC hold, amplicon size 390 bp (see Table
6-1 for primer sequences, Figure 6-1). All PCR products are resolved on 1-1.5% agarose gels.
104
Figure 6-1. Mbnl3 condWL and WLKO genotyping Schematic.
E1 E2 E4-6E3 E7a E7b-c
E8
Mbnl3 WLKO
Exon 2
247 bp
3377
3366WT
36256 bp
E7a E7b E7c
E8
4776
4775
Exon 2
247 bp
3377
3366
condWL
36256 bp
E7a E7b E7c
E8
4776
477541533365
neo puro
4774
3377
WLKO
E8
4776
38861 bp
E1
1453 bp 708 bp
3379 bp
2236 bp
717 bp 524 bp
2390 bp
273 bp
105
Table 6-1. PCR primers.
Primer name Gene Sequence 5' - 3'
Lef1ex7F Lef1 ACC TTC TAC CCC CTG TCT CC
Lef1ex7R Lef1/LEF1 ATG AGG GAT GCC AGT TGT GT
Tcf7ex13F Tcf7 AGA AGA AGA GGC GGT CAA GG
Tcf7ex13R Tcf7 GGA GGG CAA CAG AAG ATA CG
Ptk2bF Ptk2b TGG AGA AGG ACA TTG CCA TAG
Ptk2bR Ptk2b CAC TGG GAA TCT TCC ACC AT
Dnm2 10aF Dnm2 GGT GGT CAA GCT GAA AGA GC
Dnm2 R Dnm2 ACC AAG ATC TCC CCC TGA TT
Dnm2 10bF Dnm2 GGA GCT AGC CAC GGT CAT TA
Cd47 F Cd47 TCA GGT TTG GGG ATC ATA GC
Cd47 R Cd47 CCG TCA CTT CCC TTC ACC TA
Bnip2 F Bnip2 TGT TCC CAT GGA GTA TGT CG
Bnip2 R Bnip2 TGA GCC ATG AGT GCA ATA GG
Rnf130 F Rnf130 GCC TTG AGC CAC TGA GAA CT
Rnf130 R Rnf130 ACG CTT GTG TGG CAT GAT TGG T
LEF1 F LEF1 ATG AGG GAT GCC AGT TGT GT
PTK2B F PTK2B TGG AGA AGG ACA TTG CCA TGG
PTK2B R PTK2B CAC TGG TAG TCC TCC ACC AT
BNIP2 BNIP2 TTG GCA GAA CTA GCA GAA CTT G
BNIP2 BNIP2 GCA TTA TGA ATG CAG AAA CAG C
MSS2684 Cre
TGC AAC GAG TGA TGA GGT TCG CAA GAA
CCT G
MSS2685 Cre
GAA CGA ACC TGG TCG AAA TCA GTG CGT
TCG
MSS4774 Mbnl3 condWL GAT CAA TTC TCT AGA GCT CGC TGA T
MSS4775 Mbnl3 condWL TTA TTT TAC TTC TTT TCC CAC TCT TAA TTC
MSS4776 Mbnl3 condWL
CGA TCC TAC ACC TTT GTA GAA ATA TCC
TGA GTA AGT C
MSS3377 Mbnl3 WLKO
CCG CCT TGC TTG TAG TTC ACT GTC AGC
AAC TGG
MSS4775 Mbnl3 WLKO TTA TTT TAC TTC TTT TCC CAC TCT TAA TTC
MSS4776 Mbnl3 WLKO
CGA TCC TAC ACC TTT GTA GAA ATA TCC
TGA GTA AGT C
Mbnl3 condWL 3' probe F Mbnl3 TAC CCT CTA CAA TCC ACA GTT CAA
Mbnl3 condWL 3' probe R Mbnl3 ATC AAT GTC GAG CTT GTT CTA CTG
MSS4205 Mbnl3 GGT GAC AAG ATG ATG TGA GTG AGT GGA C
MSS4206 Mbnl3 GGA ACA GCT AAT CCT TCA GTA ACA GG
106
APPENDIX A
HIGH CONFIDENCE MBNL1-DEPENDENT CASSETTE EXONS IDENTIFIED FROM RNA-SEQ
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
19229 Ptk2b chr14 66158530 66165328 - 735.5 0.837397 0.00E+00 0.00E+00 1
21414 Tcf7 chr11 52257683 52260620 - 19980.4 -0.224709 0.00E+00 0.00E+00 -1
21414 Tcf7 chr11 52253941 52256305 - 8598.3 0.438720 0.00E+00 0.00E+00 1
101706 Numa1 chr7 101998299 102007553 + 1326.7 -0.764334 0.00E+00 0.00E+00 -1
56758 Mbnl1 chr3 60613262 60615772 + 1958.3 0.836561 0.00E+00 0.00E+00 1
16842 Lef1 chr3 131189334 131193250 + 2412.3 -0.744823 0.00E+00 0.00E+00 -1
68339 Ccdc88c chr12 100923179 100929064 - 315.0 -0.862396 7.89E-201 6.24E-198 -1
56758 Mbnl1 chr3 60501264 60595763 + 812.8 -0.362098 1.48E-185 1.02E-182 -1
68682 Slc44a2 chr9 21352466 21355027 + 1363.6 0.228791 8.71E-139 5.36E-136 1
116870 Mta1 chr12 113133184 113135827 + 740.1 -0.597938 2.47E-134 1.37E-131 -1
68682 Slc44a2 chr9 21352466 21355027 + 935.5 0.177087 2.25E-128 1.13E-125 1
72475 Ssbp3 chr4 107009678 107028251 + 817.6 0.416578 2.37E-110 1.09E-107 1
223828 Pphln1 chr15 93441500 93455529 + 419.8 -0.700482 8.29E-108 3.53E-105 -1
104318 Csnk1d chr11 120961754 120968133 - 1281.6 -0.394667 1.17E-103 4.64E-101 -1
22379 Fmnl3 chr15 99328057 99332069 - 200.0 0.713130 4.34E-98 1.60E-95 1
16403 Itga6 chr2 71849380 71856757 + 376.4 -0.608850 4.78E-88 1.65E-85 -1
105559 Mbnl2 chr14 120396450 120429625 + 406.1 0.564791 8.69E-86 2.83E-83 1
16319 Incenp chr19 9878318 9883832 - 928.7 -0.331947 1.96E-85 6.03E-83 -1
26431 Git2 chr5 114733860 114739280 - 676.7 -0.285508 6.21E-85 1.81E-82 -1
26921 Map4k4 chr1 40000623 40004912 + 911.5 0.336610 1.11E-83 3.08E-81 1
26921 Map4k4 chr1 40000623 40004912 + 822.4 0.299187 1.21E-83 3.20E-81 1
232341 Wnk1 chr6 119951897 119956195 - 581.6 0.386135 7.21E-80 1.81E-77 1
99889 Arfip1 chr3 84527685 84534694 - 304.8 -0.647560 4.83E-73 1.16E-70 -1
17931 Ppp1r12a chr10 108252756 108259845 + 352.8 0.494743 1.20E-70 2.77E-68 1
105559 Mbnl2 chr14 120388899 120396603 + 560.3 0.493629 2.59E-68 5.74E-66 1
17245 Mdm1 chr10 118152174 118157369 + 361.6 -0.453589 6.59E-65 1.40E-62 -1
107
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
20637 Snrnp70 chr7 45380716 45383884 - 3511.0 -0.163545 1.08E-64 2.21E-62 -1
18541 Pcnt chr10 76401331 76403873 - 178.2 -0.783201 4.91E-62 9.71E-60 -1
72313 Fryl chr5 73112748 73118584 - 361.7 0.569316 1.73E-61 3.29E-59 1
76719 Kansl1 chr11 104342362 104343672 - 426.3 -0.366674 2.04E-56 3.76E-54 -1
217030 Synrg chr11 83982195 83987989 + 241.0 -0.554167 3.36E-54 5.99E-52 -1
20637 Snrnp70 chr7 45380716 45383884 - 3511.0 -0.161734 8.99E-54 1.56E-51 -1
56449 Csda chr6 131370322 131379479 - 751.8 0.314474 1.10E-53 1.85E-51 1
53313 Atp2a3 chr11 72988969 72993042 + 1321.8 -0.191260 3.89E-52 6.16E-50 -1
27360 Add3 chr19 53242503 53245217 + 217.0 0.480468 4.66E-52 7.16E-50 1
53313 Atp2a3 chr11 72988969 72993042 + 1493.5 -0.236496 7.73E-52 1.16E-49 -1
16801 Arhgef1 chr7 24917894 24919350 + 520.4 -0.401569 3.68E-49 5.23E-47 -1
18286 Odf2 chr2 29892185 29893568 + 231.1 -0.398587 5.05E-47 6.99E-45 -1
69710 Arap1 chr7 101404279 101408128 + 251.3 -0.402823 6.63E-47 8.95E-45 -1
64009 Syne1 chr10 5043610 5047857 - 195.6 -0.612985 9.65E-47 1.27E-44 -1
18286 Odf2 chr2 29892185 29893568 + 231.1 -0.462229 3.40E-46 4.38E-44 -1
18222 Numb chr12 83795438 83799656 - 135.7 0.718805 1.29E-45 1.62E-43 1
18483 Palm chr10 79815184 79820895 + 297.4 -0.417451 4.73E-45 5.81E-43 -1
53332 Mtmr1 chrX 71369798 71383274 + 185.0 -0.580151 2.05E-43 2.46E-41 -1
57757 Pglyrp2 chr17 32413369 32417087 - 123.1 0.383140 5.74E-43 6.76E-41 1
237542 Osbpl8 chr10 111204734 111233689 + 430.1 0.380799 2.69E-40 3.04E-38 1
105439 Slain1 chr14 103656932 103685817 + 240.2 0.241322 8.29E-39 9.18E-37 1
69940 Exoc1 chr5 76554099 76559166 + 174.5 0.645230 1.03E-38 1.12E-36 1
224829 Trerf1 chr17 47317767 47323661 + 90.9 0.770210 3.42E-38 3.64E-36 1
99412 Golga2 chr2 32292092 32296720 + 136.7 -0.373737 5.25E-38 5.49E-36 -1
224829 Trerf1 chr17 47317767 47323661 + 65.2 0.717490 5.93E-37 6.08E-35 1
16801 Arhgef1 chr7 24917894 24919775 + 1325.4 -0.169681 3.70E-36 3.73E-34 -1
12175 Bnip2 chr9 70003568 70007114 + 603.5 -0.187850 1.18E-34 1.14E-32 -1
16859 Lgals9 chr11 78967997 78971426 - 824.4 0.113518 2.72E-34 2.55E-32 1
13430 Dnm2 chr9 21489742 21494616 + 577.8 -0.344132 5.22E-33 4.74E-31 -1
108
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
217030 Synrg chr11 84039210 84042093 + 404.5 -0.387713 7.50E-33 6.69E-31 -1
99412 Golga2 chr2 32292092 32297007 + 147.0 0.489134 1.59E-32 1.40E-30 1
17931 Ppp1r12a chr10 108251839 108253494 + 337.1 -0.286992 1.72E-32 1.49E-30 -1
71268 Lrrfip2 chr9 111190167 111199834 + 184.3 -0.552051 2.58E-32 2.20E-30 -1
232341 Wnk1 chr6 119952684 119956195 - 353.7 0.182437 3.35E-31 2.77E-29 1
170644 Ubn1 chr16 5081417 5086284 + 940.0 -0.269885 2.16E-30 1.76E-28 -1
18717 Pip5k1c chr10 81314991 81318144 + 324.2 0.182030 1.63E-29 1.27E-27 1
64652 Nisch chr14 31180821 31185049 - 110.0 -0.102815 1.67E-29 1.28E-27 -1
15441 Hp1bp3 chr4 138216634 138222187 + 392.9 -0.205793 2.87E-29 2.18E-27 -1
14235 Foxm1 chr6 128370956 128372602 + 194.7 -0.310024 3.40E-29 2.54E-27 -1
407823 Baz2b chr2 59932104 59933714 - 198.6 -0.404003 6.30E-29 4.65E-27 -1
26407 Map3k4 chr17 12239969 12243595 - 102.3 0.297406 2.19E-28 1.57E-26 1
26400 Map2k7 chr8 4238828 4243444 + 306.9 -0.367652 2.38E-28 1.69E-26 -1
14056 Ezh2 chr6 47540676 47542405 - 1375.1 -0.168972 4.72E-28 3.31E-26 -1
70762 Dclk2 chr3 86786149 86792110 - 171.7 -0.757259 1.84E-27 1.26E-25 -1
16709 Ktn1 chr14 47726225 47730422 + 453.1 -0.326368 5.92E-27 3.95E-25 -1
12704 Cit chr5 115986893 115988235 + 82.2 -0.639713 7.55E-26 4.86E-24 -1
105782 Scrib chr15 76061133 76063046 - 143.7 0.243319 8.31E-26 5.29E-24 1
227723 Prrc2b chr2 32212074 32216232 + 541.3 -0.213543 3.22E-25 2.01E-23 -1
18044 Nfya chr17 48395649 48400579 - 228.8 -0.185625 7.43E-25 4.52E-23 -1
18044 Nfya chr17 48395649 48400579 - 261.0 -0.204292 7.43E-25 4.57E-23 -1
26931 Ppp2r5c chr12 110570695 110580440 + 464.6 -0.237977 9.05E-25 5.39E-23 -1
214290 Zcchc6 chr13 59778751 59782342 - 340.7 -0.349459 2.26E-24 1.33E-22 -1
16842 Lef1 chr3 131204255 131223328 + 1107.7 -0.138719 4.56E-24 2.66E-22 -1
20778 Scarb1 chr5 125277793 125284044 - 90.8 -0.383655 4.96E-24 2.86E-22 -1
70497 Arhgap17 chr7 123292035 123296565 - 178.8 0.439466 9.81E-24 5.60E-22 1
73683 Atg16l2 chr7 101293875 101296233 - 140.9 -0.446507 1.54E-23 8.69E-22 -1
56457 Clptm1 chr7 19655717 19665015 - 75.1 -0.126165 3.34E-22 1.87E-20 -1
26921 Map4k4 chr1 40001429 40004912 + 143.3 0.287193 4.81E-22 2.66E-20 1
109
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
80905 Polh chr17 46190606 46198767 - 81.0 -0.438057 7.09E-22 3.89E-20 -1
100169 Phactr4 chr4 132378404 132387064 - 208.9 -0.416790 8.75E-22 4.75E-20 -1
78798 Eml4 chr17 83427999 83440153 + 289.7 -0.380780 1.61E-21 8.67E-20 -1
76614 Immt chr6 71863140 71868802 + 462.0 0.223207 2.22E-21 1.18E-19 1
107173 Gpr137 chr19 6939260 6940400 - 152.8 0.441399 2.72E-20 1.43E-18 1
11308 Abi1 chr2 22962400 22971258 - 337.4 -0.101092 4.33E-20 2.26E-18 -1
70497 Arhgap17 chr7 123279148 123287012 - 181.0 -0.240997 8.65E-20 4.48E-18 -1
232341 Wnk1 chr6 119952684 119956195 - 236.6 0.120191 9.62E-19 4.89E-17 1
20740 Spna2 chr2 29999913 30002428 + 412.7 -0.209197 1.50E-18 7.57E-17 -1
70497 Arhgap17 chr7 123279148 123287012 - 121.9 -0.178336 1.45E-17 7.06E-16 -1
78798 Eml4 chr17 83421605 83427350 + 86.4 -0.276924 2.93E-17 1.40E-15 -1
66656 Eef1d chr15 75896819 75901254 - 740.5 0.154853 3.10E-17 1.47E-15 1
66147 Necap2 chr4 141070055 141071694 - 45.9 0.334320 4.04E-17 1.89E-15 1
17758 Mtap4 chr9 110068692 110078083 + 361.9 -0.172835 4.14E-17 1.93E-15 -1
18789 Papola chr12 105829056 105834783 + 705.5 -0.126126 6.32E-17 2.91E-15 -1
57439 Tmem183a chr1 134352354 134354840 - 443.1 -0.172192 1.32E-16 6.04E-15 -1
76707 Clasp1 chr1 118510205 118521933 + 73.8 -0.156019 4.58E-16 2.06E-14 -1
13682 Eif4a2 chr16 23108718 23110152 + 1232.0 -0.116418 4.65E-16 2.08E-14 -1
17918 Myo5a chr9 75193938 75197735 + 132.7 -0.381618 1.36E-15 5.99E-14 -1
70088 Meaf6 chr4 125102838 125108355 + 214.5 -0.295218 2.03E-15 8.83E-14 -1
18080 Nin chr12 70040754 70045636 - 75.8 -0.222277 5.29E-15 2.29E-13 -1
50926 Hnrpdl chr5 100034725 100036194 - 618.8 -0.160950 8.69E-15 3.73E-13 -1
66314 Tpd52l2 chr2 181508165 181513086 + 362.0 0.140648 9.67E-15 4.09E-13 1
227723 Prrc2b chr2 32226944 32230937 + 1059.2 -0.167168 1.98E-14 8.29E-13 -1
233833 Tnrc6a chr7 123174143 123176903 + 173.3 -0.216757 2.00E-14 8.32E-13 -1
56218 Patz1 chr11 3293279 3299204 + 1047.1 -0.107458 1.23E-13 5.03E-12 -1
19231 Ptma chr1 86529172 86529803 + 39.7 -0.471848 1.73E-13 7.04E-12 -1
224088 Atp13a3 chr16 30315872 30322951 - 514.7 -0.106409 1.85E-13 7.47E-12 -1
14218 Sh3pxd2a chr19 47278312 47286717 - 66.6 -0.461234 1.87E-13 7.50E-12 -1
110
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
26931 Ppp2r5c chr12 110574744 110580440 + 86.4 0.387024 2.35E-13 9.35E-12 1
59079 Erbb2ip chr13 103823313 103835027 - 464.6 -0.159863 4.38E-13 1.71E-11 -1
59079 Erbb2ip chr13 103823313 103835027 - 464.6 -0.151382 4.93E-13 1.91E-11 -1
21429 Ubtf chr11 102310442 102311499 - 1211.4 0.125180 5.13E-13 1.97E-11 1
75991 Slain2 chr5 72958148 72974845 + 197.8 -0.158098 7.06E-13 2.70E-11 -1
20411 Sorbs1 chr19 40318021 40324831 - 78.9 -0.488477 1.12E-12 4.22E-11 -1
382034 Gse1 chr8 120537487 120562824 + 70.0 0.110368 1.13E-12 4.24E-11 1
57438 6-Mar chr2 60243556 60247956 + 387.9 0.215401 1.22E-12 4.54E-11 1
13682 Eif4a2 chr16 23111816 23113947 + 810.8 0.104204 1.29E-12 4.76E-11 1
13682 Eif4a2 chr16 23111816 23113947 + 417.2 0.110821 1.63E-12 5.93E-11 1
23880 Fyb chr15 6643693 6646661 + 1200.1 0.122627 2.31E-12 8.37E-11 1
24086 Tlk2 chr11 105209793 105221352 + 141.9 -0.254016 2.54E-12 9.14E-11 -1
277854 Depdc5 chr5 32955852 32964782 + 87.4 0.272674 3.74E-12 1.33E-10 1
114674 Gtf2ird2 chr5 134203270 134208941 + 61.6 0.548474 3.74E-12 1.33E-10 1
241846 Lsm14b chr2 180031360 180032221 + 226.8 0.113799 7.14E-12 2.50E-10 1
75560 Ep400 chr5 110739295 110742059 - 334.1 0.241476 1.15E-11 3.97E-10 1
13383 Dlg1 chr16 31743105 31781834 + 334.6 -0.215279 1.34E-11 4.61E-10 -1
328365 Zmiz1 chr14 25658106 25664718 + 91.0 0.147662 1.58E-11 5.41E-10 1
11308 Abi1 chr2 22946575 22957117 - 1014.7 0.138376 1.66E-11 5.59E-10 1
11308 Abi1 chr2 22946575 22957117 - 1014.7 0.126597 1.66E-11 5.63E-10 1
68259 Ift80 chr3 68918489 68930984 - 305.1 0.139417 1.72E-11 5.77E-10 1
11877 Arvcf chr16 18404459 18407075 + 79.1 0.279544 2.32E-11 7.75E-10 1
17237 Mgrn1 chr16 4924422 4927900 + 277.2 -0.273273 2.81E-11 9.26E-10 -1
66923 Pbrm1 chr14 31107108 31114003 + 755.8 0.146939 2.83E-11 9.27E-10 1
270058 Mtap1s chr8 70910944 70911927 + 69.1 -0.112670 3.09E-11 1.01E-09 -1
14182 Fgfr1 chr8 25532249 25557819 + 27.1 0.619738 3.64E-11 1.17E-09 1
70834 Spag9 chr11 94112032 94114348 + 109.9 -0.124541 4.35E-11 1.39E-09 -1
270058 Mtap1s chr8 70910944 70911927 + 65.0 -0.105851 5.55E-11 1.76E-09 -1
53357 Pla2g6 chr15 79302618 79305810 - 188.2 0.348663 8.37E-11 2.62E-09 1
111
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
54645 Gripap1 chrX 7792275 7799427 + 237.0 -0.213666 8.72E-11 2.71E-09 -1
77040 Atg16l1 chr1 87774144 87775858 + 214.6 -0.218993 1.01E-10 3.11E-09 -1
68671 Pcyt2 chr11 120612462 120613422 - 333.2 0.225530 1.11E-10 3.41E-09 1
69232 Qrich1 chr9 108517106 108528917 + 326.3 -0.242047 1.62E-10 4.94E-09 -1
665113 Tnik chr3 28594021 28596259 + 42.1 0.103045 2.08E-10 6.26E-09 1
70762 Dclk2 chr3 86789408 86792110 - 34.1 0.408984 2.29E-10 6.87E-09 1
69232 Qrich1 chr9 108517106 108528917 + 326.3 -0.221678 2.36E-10 7.04E-09 -1
16975 Lrp8 chr4 107847443 107851426 + 88.9 -0.330393 3.13E-10 9.26E-09 -1
14235 Foxm1 chr6 128370956 128372602 + 82.5 -0.209760 4.82E-10 1.42E-08 -1
68379 Ciz1 chr2 32364218 32367531 + 68.0 -0.148306 5.88E-10 1.72E-08 -1
63959 Slc29a1 chr17 45592178 45595507 - 348.0 0.173371 6.85E-10 2.00E-08 1
381511 Pdp1 chr4 11958184 11966374 - 54.4 0.473654 1.01E-09 2.94E-08 1
74383 Ubap2l chr3 90028324 90034176 - 743.7 0.126299 1.15E-09 3.32E-08 1
77626 Smpd4 chr16 17631985 17638640 + 91.4 -0.114000 1.36E-09 3.87E-08 -1
19679 Pitpnm2 chr5 124123938 124125462 - 377.4 -0.113595 1.42E-09 4.02E-08 -1
74383 Ubap2l chr3 90028324 90034176 - 743.7 0.141530 1.57E-09 4.44E-08 1
20185 Ncor1 chr11 62345234 62351545 - 278.5 -0.109993 1.63E-09 4.57E-08 -1
72055 Slc38a10 chr11 120141599 120151237 - 76.1 -0.133232 2.29E-09 6.33E-08 -1
17925 Myo9b chr8 71334312 71339019 + 245.4 0.106423 2.37E-09 6.52E-08 1
66314 Tpd52l2 chr2 181501904 181508266 + 155.5 -0.120557 2.40E-09 6.58E-08 -1
68671 Pcyt2 chr11 120612462 120613422 - 333.2 0.209198 2.95E-09 8.01E-08 1
30957 Mapk8ip3 chr17 24918087 24923906 - 72.5 0.113678 3.04E-09 8.22E-08 1
63959 Slc29a1 chr17 45592178 45595507 - 348.0 0.197637 4.08E-09 1.10E-07 1
72552 Hsdl1 chr8 119567765 119575174 - 420.0 0.197422 4.49E-09 1.20E-07 1
107817 Jmjd6 chr11 116838989 116840544 - 349.6 -0.127024 4.93E-09 1.31E-07 -1
208638 Slc25a38 chr9 120117452 120120435 + 108.1 -0.143490 5.15E-09 1.36E-07 -1
19707 Reps1 chr10 18104153 18114485 + 433.6 0.143518 6.60E-09 1.73E-07 1
19707 Reps1 chr10 18104153 18114485 + 433.6 0.150818 6.60E-09 1.74E-07 1
77980 Sbf1 chr15 89295394 89299619 - 353.1 0.122069 7.96E-09 2.06E-07 1
112
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
78798 Eml4 chr17 83421605 83428024 + 27.9 -0.269466 1.02E-08 2.62E-07 -1
26921 Map4k4 chr1 40000623 40003986 + 254.0 -0.160539 1.10E-08 2.79E-07 -1
239667 Dip2b chr15 100142021 100154305 + 71.8 0.184906 1.34E-08 3.38E-07 1
69582 Plekhm2 chr4 141634252 141639861 - 152.0 -0.308234 1.42E-08 3.57E-07 -1
26364 Cd97 chr8 83729381 83733565 - 763.7 0.103197 1.46E-08 3.65E-07 1
59044 Rnf130 chr11 50095775 50104730 + 140.0 -0.226387 2.28E-08 5.62E-07 -1
56406 Ncoa6 chr2 155402671 155411645 - 32.0 -0.109865 2.31E-08 5.65E-07 -1
59044 Rnf130 chr11 50095775 50104730 + 140.0 -0.166627 2.28E-08 5.65E-07 -1
74198 Dtx2 chr5 136021754 136028571 + 151.0 -0.246570 3.36E-08 8.15E-07 -1
67706 Tmem179b chr19 8773027 8774452 - 111.9 0.187756 5.01E-08 1.21E-06 1
57783 Tnip1 chr11 54939597 54956103 - 125.8 -0.250277 5.67E-08 1.36E-06 -1
80285 Parp9 chr16 35941032 35948353 + 53.8 0.215421 8.73E-08 2.06E-06 1
67706 Tmem179b chr19 8773027 8774452 - 111.9 0.282623 8.86E-08 2.09E-06 1
80285 Parp9 chr16 35941032 35948353 + 53.8 0.168383 1.41E-07 3.25E-06 1
53357 Pla2g6 chr15 79302618 79304468 - 47.8 -0.225465 1.43E-07 3.29E-06 -1
59035 Carm1 chr9 21587507 21589466 + 140.9 0.222475 1.81E-07 4.12E-06 1
16201 Ilf3 chr9 21387699 21388752 + 715.6 0.138728 2.31E-07 5.21E-06 1
74100 Arpp21 chr9 112187589 112217350 - 1059.6 -0.109811 2.44E-07 5.50E-06 -1
78798 Eml4 chr17 83421605 83428024 + 27.9 -0.151339 2.84E-07 6.37E-06 -1
192119 Dicer1 chr12 104731799 104738847 - 77.5 -0.261961 3.44E-07 7.67E-06 -1
269338 Vps39 chr2 120346327 120350228 - 59.3 -0.124067 3.95E-07 8.68E-06 -1
71819 Kif23 chr9 61932074 61935549 - 343.5 -0.144334 3.94E-07 8.68E-06 -1
665775 Bod1l chr5 41787536 41794379 - 482.7 -0.144966 3.92E-07 8.69E-06 -1
13518 Dst chr1 34295461 34299837 + 129.8 -0.270800 4.18E-07 9.10E-06 -1
20441 St3gal3 chr4 118031839 118107692 - 54.5 -0.339113 4.26E-07 9.25E-06 -1
207165 Bptf chr11 107043632 107047342 - 734.1 -0.104224 5.39E-07 1.14E-05 -1
228880 Zmynd8 chr2 165785310 165791970 - 120.5 0.190445 5.37E-07 1.15E-05 1
228880 Zmynd8 chr2 165785310 165791970 - 120.5 0.224023 5.53E-07 1.17E-05 1
12908 Crat chr2 30412982 30415517 - 28.4 -0.159526 5.92E-07 1.23E-05 -1
113
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
65963 Tmem176b chr6 48838171 48841027 - 167.9 -0.164373 6.18E-07 1.28E-05 -1
75710 Rbm12 chr2 156079338 156103428 - 209.6 0.158098 8.62E-07 1.76E-05 1
234875 Ttc13 chr8 124695635 124709972 - 221.6 -0.215373 9.94E-07 2.02E-05 -1
546071 Mast3 chr8 70789205 70789919 - 310.9 -0.123650 1.04E-06 2.11E-05 -1
214895 Lman2l chr1 36428299 36439725 - 283.4 -0.140206 1.06E-06 2.13E-05 -1
70435 Inf2 chr12 112611409 112615556 + 37.5 -0.288910 1.11E-06 2.22E-05 -1
77090 Ocel1 chr8 71371770 71372801 + 164.2 -0.165922 1.13E-06 2.25E-05 -1
65963 Tmem176b chr6 48838171 48841027 - 430.6 -0.155847 1.15E-06 2.29E-05 -1
65963 Tmem176b chr6 48838171 48841027 - 440.7 -0.145836 1.16E-06 2.30E-05 -1
224619 Traf7 chr17 24514124 24516595 - 263.3 0.139758 1.20E-06 2.37E-05 1
224619 Traf7 chr17 24514124 24516595 - 263.3 0.163101 1.22E-06 2.40E-05 1
56215 Acin1 chr14 54651716 54653680 - 239.9 -0.160991 1.34E-06 2.63E-05 -1
20602 Ncor2 chr5 125018810 125022923 - 230.5 0.181844 1.46E-06 2.87E-05 1
100986 Akap9 chr5 3951689 3954961 + 95.7 -0.316361 1.51E-06 2.94E-05 -1
223828 Pphln1 chr15 93398349 93410782 + 127.2 -0.157501 1.55E-06 2.99E-05 -1
104457 0610010K14Rik chr11 70235207 70236210 - 733.3 -0.110231 1.58E-06 3.03E-05 -1
15441 Hp1bp3 chr4 138217261 138222187 + 117.6 -0.157878 1.82E-06 3.46E-05 -1
97820 4833439L19Rik chr13 54564184 54565355 - 22.5 0.156448 2.11E-06 3.96E-05 1
11426 Macf1 chr4 123440549 123444951 - 279.5 0.154785 2.29E-06 4.27E-05 1
56215 Acin1 chr14 54651716 54653680 - 239.9 -0.144365 2.43E-06 4.51E-05 -1
20975 Synj2 chr17 6028418 6032808 + 62.9 -0.368193 3.96E-06 7.29E-05 -1
20975 Synj2 chr17 6028418 6032808 + 62.9 -0.360951 4.04E-06 7.36E-05 -1
97820 4833439L19Rik chr13 54561692 54565355 - 21.0 0.128243 4.23E-06 7.55E-05 1
97820 4833439L19Rik chr13 54561692 54565355 - 21.0 0.120855 4.21E-06 7.56E-05 1
230861 Eif4g3 chr4 138105313 138126633 + 221.3 0.111899 4.20E-06 7.58E-05 1
230861 Eif4g3 chr4 138105313 138126633 + 221.3 0.102747 4.20E-06 7.60E-05 1
71752 Gtf3c2 chr5 31172781 31174088 - 477.5 -0.115014 4.19E-06 7.60E-05 -1
234875 Ttc13 chr8 124690276 124709972 - 254.6 -0.173323 5.47E-06 9.64E-05 -1
59079 Erbb2ip chr13 103824719 103835027 - 68.0 -0.174349 5.64E-06 9.91E-05 -1
114
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
74143 Opa1 chr16 29588251 29589747 + 259.1 -0.124841 5.97E-06 1.05E-04 -1
75710 Rbm12 chr2 156079338 156103428 - 209.6 0.158660 7.19E-06 1.25E-04 1
76789 Mzt1 chr14 99034547 99046084 - 230.8 -0.122089 7.33E-06 1.27E-04 -1
74168 Zdhhc16 chr19 41939599 41940734 + 180.3 -0.158422 7.48E-06 1.29E-04 -1
111173 Erc1 chr6 119773733 119779631 - 66.3 -0.317940 8.06E-06 1.39E-04 -1
23792 Adam23 chr1 63572886 63592911 + 23.9 -0.590996 9.26E-06 1.58E-04 -1
26931 Ppp2r5c chr12 110570695 110578167 + 24.5 -0.192112 9.39E-06 1.59E-04 -1
21374 Tbp chr17 15499917 15503127 + 34.2 0.379299 1.41E-05 2.36E-04 1
66960 Fam188a chr2 12400022 12404167 - 37.9 0.116631 1.47E-05 2.43E-04 1
67669 l7Rn6 chr7 89923563 89941049 - 159.9 -0.119002 1.47E-05 2.44E-04 -1
75710 Rbm12 chr2 156079338 156103428 - 209.6 0.154069 1.54E-05 2.51E-04 1
207165 Bptf chr11 107086711 107095924 - 79.3 -0.311103 1.55E-05 2.53E-04 -1
108100 Baiap2 chr11 120000551 120006778 + 82.6 -0.138320 1.81E-05 2.93E-04 -1
108100 Baiap2 chr11 120000551 120006778 + 148.6 -0.153267 1.81E-05 2.93E-04 -1
68750 Rreb1 chr13 37929557 37947662 + 315.3 -0.148429 1.87E-05 3.01E-04 -1
67956 Setd8 chr5 124445543 124447369 + 199.5 -0.188404 2.10E-05 3.34E-04 -1
208266 Dot1l chr10 80790319 80794346 + 40.2 0.523234 2.16E-05 3.41E-04 1
18771 Pknox1 chr17 31564772 31583802 + 276.6 -0.132126 2.19E-05 3.44E-04 -1
71720 Osbpl3 chr6 50328669 50336344 - 69.0 0.332536 2.34E-05 3.67E-04 1
14924 Magi1 chr6 93686781 93697486 - 56.2 0.192264 2.37E-05 3.68E-04 1
14924 Magi1 chr6 93686781 93697486 - 56.2 0.169955 2.37E-05 3.69E-04 1
70802 Pwwp2a chr11 43684144 43705476 + 118.7 -0.279984 2.66E-05 4.10E-04 -1
14268 Fn1 chr1 71602309 71605082 - 100.3 0.223913 2.94E-05 4.52E-04 1
76074 Gbp8 chr5 105031209 105051070 - 124.7 -0.135189 3.04E-05 4.64E-04 -1
66975 2410002O22Rik chr13 104150049 104154339 - 195.1 -0.149069 3.15E-05 4.79E-04 -1
30957 Mapk8ip3 chr17 24917994 24923906 - 47.7 0.245497 3.21E-05 4.87E-04 1
71720 Osbpl3 chr6 50344873 50346430 - 83.2 0.319323 3.34E-05 5.05E-04 1
22404 Wiz chr17 32358942 32361963 - 348.0 0.133273 3.61E-05 5.41E-04 1
14924 Magi1 chr6 93686781 93697486 - 56.2 0.135045 3.88E-05 5.78E-04 1
115
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
75210 Prr3 chr17 35978380 35979254 - 136.0 -0.245108 4.20E-05 6.22E-04 -1
233545 2210018M11Rik chr7 98641492 98646522 - 127.4 -0.240238 4.37E-05 6.46E-04 -1
71765 Klhdc3 chr17 46678280 46680800 - 94.7 -0.139336 4.68E-05 6.86E-04 -1
114604 Prdm15 chr16 97821767 97835829 - 39.1 -0.234107 5.27E-05 7.65E-04 -1
75785 Klhl24 chr16 20097573 20107642 + 163.2 -0.163894 5.95E-05 8.60E-04 -1
229877 Rap1gds1 chr3 138965867 139015670 - 246.2 -0.117310 6.77E-05 9.68E-04 -1
66209 1110054O05Rik chr4 59775446 59783851 - 454.6 -0.121364 6.93E-05 9.86E-04 -1
74213 Rbm26 chr14 105131881 105140446 - 611.8 -0.105020 7.53E-05 1.06E-03 -1
237860 Ssh2 chr11 77216424 77327495 + 29.9 -0.135247 7.76E-05 1.09E-03 -1
74741 5730419I09Rik chr6 143017882 143029245 - 285.2 -0.124249 9.36E-05 1.31E-03 -1
70508 Bbx chr16 50209084 50220592 - 122.0 -0.146150 9.98E-05 1.39E-03 -1
234839 Fam38a chr8 122489098 122490162 - 138.7 0.214471 1.06E-04 1.48E-03 1
75415 Arhgap12 chr18 6064340 6070090 - 220.3 -0.194260 1.15E-04 1.60E-03 -1
69710 Arap1 chr7 101348088 101367097 + 26.8 -0.171930 1.20E-04 1.66E-03 -1
68837 Foxk2 chr11 121298558 121307899 + 228.7 0.130275 1.22E-04 1.67E-03 1
16593 Klc1 chr12 111789348 111806073 + 151.4 -0.173291 1.34E-04 1.81E-03 -1
76594 Dnajc18 chr18 35680765 35683323 - 37.3 0.129302 1.35E-04 1.82E-03 1
13859 Eps15l1 chr8 72340998 72358444 - 77.4 -0.161056 1.36E-04 1.82E-03 -1
215351 Senp6 chr9 80067452 80092411 + 296.7 -0.119750 1.43E-04 1.91E-03 -1
20623 Snrk chr9 122131892 122137600 + 78.4 -0.230598 1.44E-04 1.91E-03 -1
232566 Amn1 chr6 149170832 149188672 - 25.6 -0.181081 1.73E-04 2.27E-03 -1
14000 Drosha chr15 12905598 12912554 + 81.7 0.125440 2.11E-04 2.74E-03 1
226412 R3hdm1 chr1 128181985 128186908 + 164.4 -0.131279 2.16E-04 2.80E-03 -1
213498 Arhgef11 chr3 87717420 87722482 + 128.0 -0.250111 2.19E-04 2.83E-03 -1
22129 Ttc3 chr16 94403313 94416008 + 38.4 -0.127841 2.23E-04 2.86E-03 -1
54135 Lsr chr7 30959152 30966087 - 35.4 0.316642 2.29E-04 2.92E-03 1
73724 Mcee chr7 64392774 64412120 + 90.7 -0.179239 2.29E-04 2.93E-03 -1
30954 Siva1 chr12 112644902 112647980 + 122.0 -0.146827 2.45E-04 3.12E-03 -1
114874 Ddhd1 chr14 45620711 45629752 - 108.6 -0.119343 2.57E-04 3.22E-03 -1
116
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
73724 Mcee chr7 64392774 64412120 + 90.7 -0.210850 2.71E-04 3.37E-03 -1
77031 Slc9a8 chr2 167443448 167451349 + 82.2 -0.189870 2.89E-04 3.56E-03 -1
73341 Arhgef6 chrX 57240593 57243514 - 123.4 -0.104025 3.06E-04 3.72E-03 -1
19725 Rfx2 chr17 56787578 56803738 - 49.4 0.246549 3.21E-04 3.87E-03 1
226412 R3hdm1 chr1 128153188 128162857 + 25.7 -0.450936 3.22E-04 3.87E-03 -1
19826 Rnps1 chr17 24415040 24418611 + 120.0 0.166912 3.24E-04 3.89E-03 1
19826 Rnps1 chr17 24415040 24418611 + 120.0 0.113769 3.29E-04 3.94E-03 1
211651 Fancd2 chr6 113585471 113587455 + 62.5 -0.181942 3.80E-04 4.46E-03 -1
232566 Amn1 chr6 149170832 149188672 - 25.6 -0.279153 3.80E-04 4.46E-03 -1
192897 Itgb4 chr11 116005530 116006609 + 93.8 -0.241306 3.85E-04 4.49E-03 -1
72046 Urgcp chr11 5726830 5741145 - 20.0 -0.290085 4.36E-04 5.03E-03 -1
12725 Clcn3 chr8 60912984 60919555 - 224.6 -0.122210 4.46E-04 5.13E-03 -1
72149 Strada chr11 106173678 106187179 - 78.9 -0.258074 4.62E-04 5.30E-03 -1
208266 Dot1l chr10 80790610 80792772 + 42.8 -0.280498 5.41E-04 6.11E-03 -1
13132 Dab2 chr15 6424634 6431106 + 23.6 -0.338136 6.16E-04 6.85E-03 -1
56430 Clip1 chr5 123630452 123640553 - 147.4 0.167949 7.06E-04 7.77E-03 1
12763 Cmah chr13 24435606 24439256 + 170.4 -0.144433 7.44E-04 8.15E-03 -1
227446 2310035C23Rik chr1 105735922 105741094 + 64.1 0.177481 7.79E-04 8.45E-03 1
246277 Csad chr15 102177318 102178831 - 33.0 -0.148494 8.30E-04 8.96E-03 -1
20411 Sorbs1 chr19 40364993 40373616 - 67.7 -0.233481 9.03E-04 9.72E-03 -1
27369 Dguok chr6 83480218 83481461 - 99.4 -0.105586 9.44E-04 1.01E-02 -1
668661 2410002F23Rik chr7 44246789 44248294 + 35.9 -0.251763 9.72E-04 1.03E-02 -1
668661 2410002F23Rik chr7 44246789 44248294 + 35.9 -0.272322 9.93E-04 1.05E-02 -1
14115 Fbln2 chr6 91257724 91263472 + 26.7 -0.221114 1.02E-03 1.08E-02 -1
319638 Nt5dc1 chr10 34324287 34399816 - 54.0 -0.159155 1.05E-03 1.09E-02 -1
319638 Nt5dc1 chr10 34324287 34399816 - 54.0 -0.201420 1.05E-03 1.09E-02 -1
72805 Zfp839 chr12 110854999 110864055 + 50.8 -0.284757 1.04E-03 1.10E-02 -1
224938 Pja2 chr17 64292838 64309665 - 242.1 -0.125678 1.05E-03 1.10E-02 -1
72836 Pot1b chr17 55706105 55710905 - 70.2 -0.171802 1.08E-03 1.12E-02 -1
117
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
54725 Cadm1 chr9 47818733 47848297 + 91.3 0.245956 1.28E-03 1.30E-02 1
72080 2010317E24Rik chr2 25372359 25375063 + 48.6 -0.292736 1.34E-03 1.36E-02 -1
68327 0610007P22Rik chr17 25240176 25241256 + 24.0 0.175754 1.36E-03 1.37E-02 1
73998 Herc3 chr6 58831524 58843857 + 43.7 -0.143002 1.41E-03 1.42E-02 -1
227446 2310035C23Rik chr1 105740434 105741359 + 126.1 -0.158891 1.47E-03 1.46E-02 -1
69934 Rg9mtd3 chr4 45297157 45300626 + 24.0 -0.242424 1.49E-03 1.47E-02 -1
68327 0610007P22Rik chr17 25240176 25241256 + 24.0 0.231653 1.50E-03 1.48E-02 1
13823 Epb4.1l3 chr17 69287256 69289984 + 95.5 -0.163004 1.51E-03 1.49E-02 -1
270669 Mbtps2 chrX 157591353 157598673 - 169.1 -0.170128 1.54E-03 1.51E-02 -1
18987 Pou2f2 chr7 25092649 25093685 - 20.0 0.580405 1.55E-03 1.52E-02 1
66151 Prr13 chr15 102459213 102460145 + 210.4 -0.133634 1.81E-03 1.74E-02 -1
207777 Bzrap1 chr11 87777046 87779327 + 33.0 -0.482432 1.81E-03 1.74E-02 -1
16764 Aff3 chr1 38536825 38664953 - 41.8 -0.144749 1.84E-03 1.75E-02 -1
109689 Arrb1 chr7 99596022 99598560 + 63.0 0.197610 1.96E-03 1.84E-02 1
16548 Khk chr5 30924738 30928514 + 77.2 0.155458 1.97E-03 1.84E-02 1
110197 Dgkg chr16 22571670 22575399 - 21.7 -0.139916 1.98E-03 1.84E-02 -1
51812 Mcrs1 chr15 99249875 99251941 - 66.5 -0.182091 2.02E-03 1.88E-02 -1
66960 Fam188a chr2 12397492 12405919 - 44.9 0.135090 2.03E-03 1.88E-02 1
207565 Camkk2 chr5 122737432 122743956 - 63.1 -0.134490 2.05E-03 1.89E-02 -1
66897 Naa16 chr14 79344894 79351500 - 225.6 -0.130440 2.07E-03 1.91E-02 -1
76589 Unc5cl chr17 48463361 48464541 + 79.1 0.167488 2.13E-03 1.94E-02 1
16423 Cd47 chr16 49908053 49911084 + 77.7 0.257788 2.21E-03 2.01E-02 1
75415 Arhgap12 chr18 6052861 6057590 - 71.3 -0.210317 2.26E-03 2.05E-02 -1
65945 Clstn1 chr4 149638232 149643351 + 327.0 -0.103928 2.44E-03 2.18E-02 -1
66990 Tmem134 chr19 4127549 4131281 + 68.9 0.102835 2.55E-03 2.27E-02 1
12419 Cbx5 chr15 103213071 103215351 - 23.4 0.123010 2.56E-03 2.27E-02 1
12419 Cbx5 chr15 103213071 103215351 - 23.4 0.138399 2.56E-03 2.27E-02 1
72754 Arhgef10l chr4 140568338 140570401 - 44.4 -0.287216 2.60E-03 2.29E-02 -1
77733 Rnf170 chr8 26120439 26125978 + 34.4 -0.234742 2.80E-03 2.45E-02 -1
118
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
69232 Qrich1 chr9 108517108 108528917 + 77.2 -0.136565 2.88E-03 2.50E-02 -1
53413 Exoc7 chr11 116295543 116297662 - 30.4 0.323944 2.88E-03 2.51E-02 1
15950 Ifi203 chr1 173926805 173935176 - 213.3 -0.129967 2.94E-03 2.54E-02 -1
57783 Tnip1 chr11 54939597 54962908 - 75.1 -0.131208 2.98E-03 2.57E-02 -1
69232 Qrich1 chr9 108517108 108528917 + 77.2 -0.148487 3.03E-03 2.60E-02 -1
208718 Dis3l2 chr1 86791415 86821449 + 30.5 -0.219323 3.07E-03 2.62E-02 -1
13436 Dnmt3b chr2 153649456 153661601 + 29.8 -0.388441 3.40E-03 2.87E-02 -1
227446 2310035C23Rik chr1 105691905 105693176 + 185.9 0.112057 3.58E-03 2.99E-02 1
20411 Sorbs1 chr19 40299462 40311862 - 25.1 0.117368 3.66E-03 3.05E-02 1
51801 Ramp1 chr1 91196770 91206789 + 40.6 -0.220874 3.75E-03 3.11E-02 -1
14130 Fcgr2b chr1 170961195 170965823 - 30.0 -0.133956 3.78E-03 3.12E-02 -1
76816 Sdccag8 chr1 176826198 176831181 + 37.5 -0.205754 3.83E-03 3.15E-02 -1
193796 Kdm4b chr17 56326090 56351796 + 105.3 -0.180975 3.90E-03 3.18E-02 -1
269639 Zfp512 chr5 31452489 31465632 + 68.2 0.132658 4.00E-03 3.25E-02 1
57815 Spata5 chr3 37528157 37579089 + 110.9 0.166273 4.14E-03 3.35E-02 1
78787 Usp54 chr14 20577454 20583489 - 28.9 -0.213810 4.23E-03 3.41E-02 -1
67501 Ccdc50 chr16 27435561 27436601 + 68.1 -0.172003 4.26E-03 3.43E-02 -1
26409 Map3k7 chr4 31992386 32002152 + 247.6 0.102756 4.46E-03 3.57E-02 1
67144 Lrrc40 chr3 158040440 158043689 + 72.9 0.120802 4.77E-03 3.78E-02 1
72053 Tmub2 chr11 102284961 102287870 + 210.2 -0.112269 4.86E-03 3.83E-02 -1
69706 Lrr1 chr12 69171861 69179007 + 132.5 -0.104726 4.90E-03 3.85E-02 -1
107986 Ddb2 chr2 91212449 91217229 - 72.5 -0.104911 4.89E-03 3.85E-02 -1
217430 Pqlc3 chr12 16992277 16995670 - 101.7 0.128799 5.21E-03 4.05E-02 1
225432 Rbm27 chr18 42305679 42317749 + 196.0 -0.119469 5.25E-03 4.06E-02 -1
224139 Golgb1 chr16 36885008 36887402 + 21.0 0.235337 5.24E-03 4.07E-02 1
224139 Golgb1 chr16 36885008 36887402 + 21.0 0.258838 5.24E-03 4.07E-02 1
68031 Rnf146 chr10 29354291 29362441 - 71.1 -0.154029 5.68E-03 4.35E-02 -1
15950 Ifi203 chr1 173928274 173935176 - 150.7 -0.154765 5.78E-03 4.39E-02 -1
13728 Mark2 chr19 7278041 7280011 - 37.5 0.240046 6.11E-03 4.59E-02 1
119
Gene
ID Gene Symbol Chromosome
Chromosome
Start
Chromosome
End Strand Coverage PSI P Value FDR Direction
69612 Kansl2 chr15 98529289 98531897 - 102.0 -0.128000 6.15E-03 4.60E-02 -1
208718 Dis3l2 chr1 86760287 86821449 + 117.9 0.146902 6.23E-03 4.64E-02 1
68556 Uckl1 chr2 181570499 181573134 - 176.1 0.120324 6.24E-03 4.64E-02 1
72775 Fance chr17 28320789 28326272 + 62.4 -0.159656 6.26E-03 4.65E-02 -1
70373 1700020O03Rik chr12 86268975 86291369 + 210.5 -0.122302 6.27E-03 4.65E-02 -1
108105 B3gnt5 chr16 19765107 19772752 + 49.1 -0.205822 6.57E-03 4.85E-02 -1
211660 Cspp1 chr1 10095865 10108480 + 208.2 -0.130756 6.60E-03 4.86E-02 -1
225912 Cybasc3 chr19 10577733 10580561 + 81.5 -0.129429 6.62E-03 4.87E-02 -1
74741 5730419I09Rik chr6 143017882 143020373 - 84.3 -0.178905 6.78E-03 4.96E-02 -1
120
APPENDIX B
GENE ONTOLOGY ANALYSIS 384 MBNL1 SPLICING-REGULATED TARGET
TRANSCRIPTS BENJAMINI FDR≤0.1
Term Count %
Fold
Enrichment Benjamini FDR p-Value Biological Process
GO:0016043~cellular component organization 68 23.1 2.423 2.60E-09 1.90E-12
GO:0009987~cellular process 177 60.2 1.289 6.30E-07 9.20E-10
GO:0006996~organelle organization 36 12.2 2.250 3.60E-03 7.80E-06
GO:0051276~chromosome organization 20 6.8 3.336 3.10E-03 8.90E-06
GO:0016568~chromatin modification 14 4.8 3.998 1.40E-02 4.90E-05
GO:0006325~chromatin organization 16 5.4 3.423 1.60E-02 7.00E-05
GO:0043687~post-translational protein
modification 32 10.9
2.044 3.40E-02 1.80E-04
GO:0006897~endocytosis 11 3.7 3.943 5.40E-02 4.90E-04
GO:0010324~membrane invagination 11 3.7 3.943 5.40E-02 4.90E-04
GO:0016044~membrane organization 13 4.4 3.221 7.60E-02 7.40E-04
GO:0006468~protein amino acid
phosphorylation 21 7.1
2.211 1.20E-01 1.30E-03
GO:0016192~vesicle-mediated transport 17 5.8 2.458 1.30E-01 1.50E-03
Cellular Component
GO:0005622~intracellular 200 68.0 1.368 5.78E-13 2.05E-15
GO:0044424~intracellular part 194 66.0 1.387 1.11E-12 7.69E-15
GO:0043229~intracellular organelle 164 55.8 1.375 1.64E-07 1.70E-09
GO:0043226~organelle 164 55.8 1.374 1.28E-07 1.78E-09
GO:0005634~nucleus 101 34.4 1.607 3.96E-06 6.86E-08
GO:0005623~cell 230 78.2 1.072 3.20E-05 6.65E-07
GO:0044464~cell part 230 78.2 1.072 3.20E-05 6.65E-07
GO:0043227~membrane-bounded organelle 143 48.6 1.352 2.97E-05 7.20E-07
GO:0005737~cytoplasm 131 44.6 1.390 3.32E-05 9.19E-07
GO:0043231~intracellular membrane-
bounded organelle 142 48.3 1.343 4.24E-05 1.32E-06
GO:0016323~basolateral plasma membrane 12 4.1 5.841 1.92E-04 6.63E-06
GO:0030054~cell junction 21 7.1 3.067 4.62E-04 1.76E-05
GO:0005911~cell-cell junction 12 4.1 4.761 1.08E-03 4.50E-05
GO:0005856~cytoskeleton 34 11.6 2.080 1.77E-03 7.98E-05
GO:0019898~extrinsic to membrane 19 6.5 2.763 3.86E-03 1.87E-04
GO:0005874~microtubule 13 4.4 3.718 3.95E-03 2.05E-04
GO:0015630~microtubule cytoskeleton 18 6.1 2.745 5.69E-03 3.16E-04
GO:0043228~non-membrane-bounded
organelle 47 16.0 1.681 6.53E-03 3.85E-04
GO:0043232~intracellular non-membrane-
bounded organelle 47 16.0 1.681 6.53E-03 3.85E-04
GO:0044422~organelle part 68 23.1 1.451 1.30E-02 8.15E-04
GO:0005912~adherens junction 8 2.7 5.180 1.35E-02 8.91E-04
GO:0005913~cell-cell adherens junction 5 1.7 11.070 1.43E-02 9.98E-04
GO:0044446~intracellular organelle part 67 22.8 1.440 1.54E-02 1.13E-03
GO:0070161~anchoring junction 8 2.7 4.464 2.74E-02 2.11E-03
GO:0030055~cell-substrate junction 6 2.0 6.240 3.31E-02 2.68E-03
GO:0044430~cytoskeletal part 22 7.5 1.951 5.17E-02 4.40E-03
GO:0044428~nuclear part 30 10.2 1.659 8.11E-02 7.29E-03
GO:0045178~basal part of cell 4 1.4 9.152 9.73E-02 9.16E-03
GO:0042995~cell projection 17 5.8 2.029 1.01E-01 9.86E-03
GO:0005938~cell cortex 7 2.4 3.813 1.00E-01 1.02E-02
121
Term Count %
Fold
Enrichment Benjamini FDR p-Value Cellular Component (continued)
GO:0000118~histone deacetylase complex 4 1.4 8.580 1.04E-01 1.10E-02
Molecular Function
GO:0005488~binding 209 71.1 1.228 2.46E-08 6.15E-11
GO:0005515~protein binding 124 42.2 1.426 1.20E-04 5.98E-07
GO:0008092~cytoskeletal protein binding 20 6.8 3.250 1.77E-03 1.33E-05
GO:0016772~transferase activity, transferring
phosphorus-containing groups 31 10.5 2.179 7.93E-03 7.97E-05
GO:0008017~microtubule binding 7 2.4 7.719 2.19E-02 2.77E-04
GO:0003676~nucleic acid binding 66 22.4 1.505 2.41E-02 3.65E-04
GO:0016301~kinase activity 26 8.8 2.130 2.82E-02 5.01E-04
GO:0005083~small GTPase regulator activity 12 4.1 3.620 2.47E-02 5.01E-04
GO:0015631~tubulin binding 7 2.4 6.196 3.96E-02 9.08E-04
GO:0046872~metal ion binding 79 26.9 1.380 4.22E-02 1.08E-03
GO:0003779~actin binding 13 4.4 3.036 4.41E-02 1.24E-03
GO:0043167~ion binding 80 27.2 1.368 4.20E-02 1.29E-03
GO:0043169~cation binding 79 26.9 1.368 4.27E-02 1.42E-03
GO:0016740~transferase activity 42 14.3 1.639 4.04E-02 1.44E-03
GO:0016773~phosphotransferase activity,
alcohol group as acceptor 22 7.5 2.123 4.23E-02 1.62E-03
GO:0003677~DNA binding 42 14.3 1.586 6.40E-02 2.64E-03
GO:0030695~GTPase regulator activity 14 4.8 2.609 6.54E-02 2.87E-03
GO:0000287~magnesium ion binding 15 5.1 2.467 6.77E-02 3.15E-03
GO:0060589~nucleoside-triphosphatase
regulator activity 14 4.8 2.566 6.73E-02 3.30E-03
GO:0000166~nucleotide binding 48 16.3 1.479 8.99E-02 4.70E-03
GO:0030234~enzyme regulator activity 21 7.1 1.959 9.58E-02 5.27E-03
122
APPENDIX C
MBNL1-DEPENDENT DIFFERENTIAL THYMUS EXPRESSION ANALYSIS (WT-KO)
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
20091 Rps3a -5.2007 5.8871 2.27E-110 5.22E-106 upregulated
16574 Kif5c -2.9220 5.1418 4.48E-57 5.16E-53 upregulated
233274 Siglech 2.7081 3.3112 3.47E-18 2.67E-14 downregulated
71768 Vwce -1.9977 3.6628 1.08E-16 6.23E-13 upregulated
54377 Cacng4 -1.9741 3.1722 8.37E-16 3.86E-12 upregulated
59095 Fxyd6 -1.8973 3.7759 5.10E-15 1.96E-11 upregulated
83674 Cnnm1 -3.1412 3.6301 7.22E-15 2.38E-11 upregulated
18142 Npas1 2.4039 1.2133 3.51E-14 1.01E-10 downregulated
70080 2210010C17Rik -1.5468 5.5279 8.64E-14 2.21E-10 upregulated
320472 Ppm1e -1.1807 5.5583 1.55E-13 3.58E-10 upregulated
20201 S100a8 -2.5915 5.6580 8.46E-13 1.77E-09 upregulated
12478 Cd19 2.4597 2.7489 1.01E-12 1.95E-09 downregulated
268451 Rab11fip4 -1.0758 6.3115 1.17E-12 2.07E-09 upregulated
14938 Gzma -3.1504 7.5269 2.01E-12 3.30E-09 upregulated
20202 S100a9 -2.7693 5.8354 2.41E-12 3.70E-09 upregulated
13417 Dnahc8 1.0578 9.3212 2.80E-12 4.03E-09 downregulated
12145 Cxcr5 2.6472 0.8262 3.33E-12 4.35E-09 downregulated
15404 Hoxa7 4.1110 -1.2688 3.40E-12 4.35E-09 downregulated
216881 Wscd1 -1.3427 4.5933 3.85E-12 4.66E-09 upregulated
240667 Sec31b 2.0505 2.2151 1.06E-11 1.22E-08 downregulated
18054 Ngp -2.7923 5.1431 1.43E-11 1.57E-08 upregulated
244234 5830411N06Rik -2.4767 5.2517 1.70E-11 1.78E-08 upregulated
26456 Sema4g -1.4494 3.7204 1.93E-11 1.93E-08 upregulated
67441 Isoc2b 1.9616 2.0632 2.38E-11 2.29E-08 downregulated
232414 Clec9a 1.9053 2.0893 3.38E-11 3.11E-08 downregulated
14221 Fjx1 -2.4520 1.2915 4.00E-11 3.54E-08 upregulated
51799 Rundc3a -1.8405 3.4041 7.68E-11 6.56E-08 upregulated
12816 Col12a1 1.1863 5.7608 8.51E-11 7.01E-08 downregulated
20558 Slfn4 -2.9805 3.3410 9.22E-11 7.32E-08 upregulated
384214 Ephx4 -2.1088 1.6642 1.17E-10 8.61E-08 upregulated
277360 Prex1 0.8549 8.0011 1.20E-10 8.61E-08 downregulated
12518 Cd79a 2.1886 2.0575 1.20E-10 8.61E-08 downregulated
22042 Tfrc -1.2692 7.7983 1.27E-10 8.86E-08 upregulated
12796 Camp -2.9330 4.1138 1.81E-10 1.23E-07 upregulated
116847 Prelp 1.5269 4.2053 1.88E-10 1.24E-07 downregulated
319582 6430573F11Rik -4.3350 3.5009 1.96E-10 1.25E-07 upregulated
69169 Faim3 3.6509 0.3624 2.13E-10 1.32E-07 downregulated
20271 Scn5a -1.4373 3.5690 3.42E-10 2.08E-07 upregulated
13797 Emx2 -4.0898 -1.3370 7.64E-10 4.51E-07 upregulated
93719 Ear6 -4.6700 1.4155 8.90E-10 5.13E-07 upregulated
16852 Lgals1 1.0201 6.3052 9.75E-10 5.48E-07 downregulated
243958 Siglecg 1.9786 1.9150 1.46E-09 7.99E-07 downregulated
231510 Agpat9 -1.5805 3.0060 1.54E-09 8.27E-07 upregulated
12482 Ms4a1 2.3565 2.0144 1.74E-09 9.10E-07 downregulated
19679 Pitpnm2 -0.7922 8.8291 1.85E-09 9.46E-07 upregulated
12458 Ccr6 2.1358 1.2192 1.90E-09 9.51E-07 downregulated
73368 Col20a1 1.2821 3.9526 1.94E-09 9.51E-07 downregulated
18548 Pcsk1 -3.8158 1.0456 2.06E-09 9.87E-07 upregulated
12944 Crp 2.2648 0.6840 2.11E-09 9.93E-07 downregulated
66395 Ahnak 0.9901 6.9870 2.52E-09 1.16E-06 downregulated
66953 Cdca7 -0.7685 8.4393 2.99E-09 1.35E-06 Upregulated
123
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
20716 Serpina3n 1.5141 5.1639 3.21E-09 1.42E-06 downregulated
19395 Rasgrp2 0.7913 7.0963 4.83E-09 2.09E-06 downregulated
17476 Mpeg1 0.7871 6.9538 4.96E-09 2.09E-06 downregulated
244233 Cd163l1 -4.3435 6.3132 4.99E-09 2.09E-06 upregulated
13390 Dlx1 2.7428 -0.7517 5.25E-09 2.16E-06 downregulated
18507 Pax5 3.0720 -0.6123 6.23E-09 2.52E-06 downregulated
170813 Ms4a3 -3.4740 0.4407 7.31E-09 2.86E-06 upregulated
11749 Anxa6 1.0394 7.6947 7.33E-09 2.86E-06 downregulated
13861 Epx -4.1015 3.0770 8.87E-09 3.41E-06 upregulated
100504362 Gm1987 1.1687 5.7540 1.19E-08 4.48E-06 downregulated
19074 Prg2 -3.8199 4.8238 1.32E-08 4.92E-06 upregulated
13404 Dmc1 2.4596 -0.5812 1.37E-08 4.99E-06 downregulated
240055 Neurl1b 1.4699 4.2550 1.42E-08 5.12E-06 downregulated
140488 Igf2bp3 -3.5172 4.3543 1.48E-08 5.26E-06 upregulated
14800 Gria2 -2.6171 2.2962 1.60E-08 5.59E-06 upregulated
78250 Iqch -3.3495 1.6702 1.63E-08 5.59E-06 upregulated
53374 Chst3 2.2056 1.1258 2.07E-08 7.01E-06 downregulated
382571 Kcnf1 -2.2312 3.6845 2.18E-08 7.28E-06 upregulated
223697 Sun2 0.7577 8.6976 3.04E-08 1.00E-05 downregulated
14264 Fmod 1.5143 2.5503 3.17E-08 1.02E-05 downregulated
15402 Hoxa5 2.9459 -1.2521 3.20E-08 1.02E-05 downregulated
13421 Dnase1l3 0.9858 5.6946 3.52E-08 1.11E-05 downregulated
223650 Eppk1 1.3190 3.6595 3.76E-08 1.17E-05 downregulated
11801 Cd5l -3.7421 2.7289 4.09E-08 1.26E-05 upregulated
58218 Trem3 -3.1601 -0.8038 4.73E-08 1.44E-05 upregulated
20129 Rptn 1.6982 3.9258 5.07E-08 1.52E-05 downregulated
19250 Ptpn14 -1.0147 6.7043 5.39E-08 1.59E-05 upregulated
19259 Ptpn5 2.2732 0.0002 5.96E-08 1.74E-05 downregulated
16415 Itgb2l -3.2084 -0.5197 6.49E-08 1.84E-05 upregulated
14658 Glrb 1.9132 1.9447 6.52E-08 1.84E-05 downregulated
20408 Sh3gl3 -3.6365 2.7897 6.55E-08 1.84E-05 upregulated
64095 Gpr35 1.5680 1.9409 6.88E-08 1.88E-05 downregulated
237256 Zc3h12d 1.7537 3.0264 6.89E-08 1.88E-05 downregulated
258055 Olfr524 -3.4730 0.8833 6.95E-08 1.88E-05 upregulated
17537 Meis3 -0.9660 5.2583 7.08E-08 1.90E-05 upregulated
56643 Slc15a1 -2.2590 2.8763 7.78E-08 2.06E-05 upregulated
12526 Cd8b1 0.7990 9.9082 9.46E-08 2.48E-05 downregulated
15403 Hoxa6 6.9508 -3.1699 9.71E-08 2.49E-05 downregulated
239017 Ogdhl -3.6982 1.5501 9.72E-08 2.49E-05 upregulated
17394 Mmp8 -2.4280 2.3140 1.01E-07 2.54E-05 upregulated
68355 2010204K13Rik -2.3297 0.7121 1.03E-07 2.54E-05 upregulated
15018 H2-Q7 1.1100 5.1838 1.03E-07 2.54E-05 downregulated
18601 Padi3 -2.8049 3.1834 1.04E-07 2.54E-05 upregulated
225912 Cybasc3 1.0135 5.5959 1.09E-07 2.64E-05 downregulated
81703 Jdp2 -1.9079 3.6211 1.13E-07 2.72E-05 upregulated
99899 Ifi44 1.2329 4.7883 1.18E-07 2.80E-05 downregulated
108897 Aif1l -1.6644 3.2646 1.32E-07 3.11E-05 upregulated
67111 Naaa 0.9860 5.6314 1.53E-07 3.57E-05 downregulated
50928 Klrg1 3.3561 -1.5777 1.62E-07 3.72E-05 downregulated
109272 Mybpc1 1.7966 1.7759 1.68E-07 3.83E-05 downregulated
110558 H2-Q9 1.1647 4.6396 1.80E-07 4.01E-05 downregulated
17869 Myc -1.5640 6.4964 1.81E-07 4.01E-05 upregulated
109828 C7 2.2192 2.5562 1.82E-07 4.01E-05 downregulated
76867 Rhbdd1 1.1929 3.7449 1.83E-07 4.01E-05 downregulated
98752 Fcrla 2.1795 1.5647 1.91E-07 4.14E-05 downregulated
124
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
14255 Flt3 1.3610 4.1174 1.98E-07 4.25E-05 downregulated
11492 Adam19 -1.7467 6.3511 2.18E-07 4.66E-05 upregulated
270109 Pcnxl2 2.3993 -0.9181 2.38E-07 5.03E-05 downregulated
100043335 Gm4371 -1.8303 2.5711 2.44E-07 5.11E-05 upregulated
381260 Gm973 -3.0810 1.3697 2.48E-07 5.11E-05 upregulated
30877 Gnl3 -1.3387 6.9621 2.49E-07 5.11E-05 upregulated
212073 4831426I19Rik 0.7924 5.9859 2.74E-07 5.59E-05 downregulated
12227 Btg2 0.8965 8.8233 2.84E-07 5.75E-05 downregulated
269629 Lhfpl3 -2.7753 1.1503 3.10E-07 6.22E-05 upregulated
53318 Pdlim3 1.4549 2.0133 3.16E-07 6.28E-05 downregulated
66912 Bzw2 -0.8009 7.5351 3.23E-07 6.36E-05 upregulated
100034684 BC100530 -4.3223 0.9740 3.41E-07 6.66E-05 upregulated
18606 Enpp2 1.0835 5.4389 3.55E-07 6.86E-05 downregulated
68501 Nsmce2 -0.8677 5.5815 3.75E-07 7.19E-05 upregulated
66892 Eif4e3 0.8420 6.7181 3.87E-07 7.37E-05 downregulated
235472 Prtg -2.4416 0.2676 4.36E-07 8.23E-05 upregulated
12274 C6 2.3402 -0.6376 4.46E-07 8.35E-05 downregulated
16664 Krt14 1.0727 5.7623 4.64E-07 8.63E-05 downregulated
245195 Retnlg -2.6943 2.0586 4.79E-07 8.82E-05 upregulated
20017 Polr1b -1.3160 5.5374 5.21E-07 9.53E-05 upregulated
268902 Robo2 1.5974 2.4364 5.41E-07 9.82E-05 downregulated
15245 Hhip 1.0637 4.2535 5.59E-07 1.01E-04 downregulated
12180 Smyd1 -1.5486 3.9611 5.66E-07 1.01E-04 upregulated
626058 E330020D12Rik 1.6190 1.4596 5.69E-07 1.01E-04 downregulated
100736249 Mira 2.6729 -1.7319 5.80E-07 1.02E-04 downregulated
20533 Slc4a1 -3.9691 1.0303 5.93E-07 1.03E-04 upregulated
258571 Olfr1033 2.2518 2.3925 5.96E-07 1.03E-04 downregulated
16149 Cd74 0.9609 10.4055 6.35E-07 1.09E-04 downregulated
54598 Calcrl 1.0846 4.7371 6.90E-07 1.18E-04 downregulated
13035 Ctsg -2.7237 0.6840 6.98E-07 1.18E-04 upregulated
12655 Chi3l3 -1.8034 3.4480 7.07E-07 1.18E-04 upregulated
100043314 Tigit 2.3427 1.0543 7.10E-07 1.18E-04 downregulated
14969 H2-Eb1 0.9497 8.5540 7.43E-07 1.23E-04 downregulated
12902 Cr2 1.8611 1.6380 7.55E-07 1.24E-04 downregulated
110557 H2-Q6 0.9534 5.4649 8.00E-07 1.31E-04 downregulated
215821 D10Bwg1379e -2.6894 3.3243 8.27E-07 1.34E-04 upregulated
18829 Ccl21a 1.1550 4.6304 8.66E-07 1.40E-04 downregulated
15360 Hmgcs2 1.0082 4.2804 8.84E-07 1.41E-04 downregulated
15900 Irf8 0.7869 6.4558 8.86E-07 1.41E-04 downregulated
54672 Gpr97 -1.3095 3.7439 9.27E-07 1.46E-04 upregulated
12759 Clu 1.1451 5.8337 9.37E-07 1.47E-04 downregulated
21664 Phlda1 -0.8875 5.4630 9.43E-07 1.47E-04 upregulated
20810 Srm -1.4773 5.4949 9.73E-07 1.50E-04 upregulated
18576 Pde3b -0.7784 6.3297 1.10E-06 1.69E-04 upregulated
328417 Parp4 0.8069 6.6459 1.12E-06 1.71E-04 downregulated
93730 Lztfl1 -0.7537 6.6566 1.19E-06 1.79E-04 upregulated
212108 Rln3 2.0941 -0.7385 1.19E-06 1.79E-04 Downregulated
20887 Sult1a1 1.2975 3.0778 1.20E-06 1.79E-04 downregulated
65972 Ifi30 0.8667 5.6621 1.26E-06 1.87E-04 downregulated
16792 Laptm5 0.7389 9.6248 1.28E-06 1.87E-04 downregulated
17313 Mgp 1.1243 4.2731 1.28E-06 1.87E-04 downregulated
170677 Cdhr1 1.3808 3.1489 1.29E-06 1.89E-04 downregulated
18534 Pck1 2.1068 2.3949 1.31E-06 1.90E-04 downregulated
214895 Lman2l 0.6876 6.4427 1.36E-06 1.95E-04 downregulated
66696 Snx31 -1.5422 3.1108 1.36E-06 1.95E-04 upregulated
125
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
78887 Sfi1 1.0170 6.8677 1.38E-06 1.97E-04 downregulated
17329 Cxcl9 1.5172 3.6261 1.43E-06 2.02E-04 downregulated
114332 Lyve1 1.7112 0.1473 1.53E-06 2.14E-04 downregulated
54445 Unc93b1 0.7872 5.5176 1.53E-06 2.14E-04 downregulated
111970 Dlx1as 2.9972 -1.8985 1.72E-06 2.39E-04 downregulated
17172 Ascl1 1.3438 3.2033 1.74E-06 2.40E-04 downregulated
56045 Samhd1 1.1736 6.8934 1.79E-06 2.45E-04 downregulated
11747 Anxa5 0.8185 7.6220 1.85E-06 2.53E-04 downregulated
13070 Cyp11a1 -1.7565 1.2616 1.95E-06 2.64E-04 upregulated
14805 Grik1 -4.2212 1.8525 1.98E-06 2.67E-04 upregulated
17312 Clec10a 1.5919 1.8752 2.06E-06 2.76E-04 downregulated
17523 Mpo -2.2976 3.6533 2.11E-06 2.81E-04 upregulated
13849 Ephx1 1.3537 5.3043 2.13E-06 2.83E-04 downregulated
20668 Sox13 -2.2127 4.6832 2.18E-06 2.87E-04 upregulated
19152 Prtn3 -2.3296 1.8349 2.22E-06 2.89E-04 upregulated
17231 Mcpt8 -4.8636 -0.1735 2.22E-06 2.89E-04 upregulated
18302 Oit3 -3.1522 -1.4947 2.36E-06 3.05E-04 upregulated
236312 Pyhin1 0.9602 4.2586 2.39E-06 3.08E-04 downregulated
67839 Gpsm1 0.9026 4.3293 2.43E-06 3.10E-04 downregulated
20425 Shmt1 -1.3395 5.7871 2.52E-06 3.21E-04 upregulated
272382 Spib 1.1840 4.2852 2.62E-06 3.32E-04 downregulated
12978 Csf1r 0.7285 5.7193 2.68E-06 3.36E-04 downregulated
19695 Reg3g 1.2546 4.3962 2.69E-06 3.36E-04 downregulated
72515 Wdr43 -0.8734 7.1563 2.70E-06 3.36E-04 upregulated
16069 Igj 2.2354 5.5326 2.76E-06 3.41E-04 downregulated
329877 Dennd4c 0.7045 7.4680 2.87E-06 3.53E-04 downregulated
93671 Cd163 1.7730 3.2290 2.94E-06 3.61E-04 downregulated
16145 Igtp 0.6925 5.9684 3.01E-06 3.67E-04 downregulated
74145 F13a1 1.3146 7.1592 3.03E-06 3.67E-04 downregulated
212190 Ubxn10 -1.9287 0.1460 3.04E-06 3.67E-04 upregulated
15201 Hells -0.8639 7.4728 3.13E-06 3.75E-04 upregulated
14114 Fbln1 -1.9966 5.2567 3.25E-06 3.88E-04 upregulated
21894 Tln1 0.6114 9.7779 3.29E-06 3.91E-04 downregulated
67313 5730559C18Rik -1.7491 3.9973 3.39E-06 4.00E-04 upregulated
16541 Napsa 1.2746 3.9923 3.43E-06 4.03E-04 downregulated
17005 Ltk -0.8622 4.4047 3.50E-06 4.09E-04 upregulated
50701 Elane -2.4189 1.7457 3.57E-06 4.15E-04 upregulated
29877 Hdgfrp3 -0.7442 6.6219 3.62E-06 4.16E-04 upregulated
329693 Fcrl5 2.7916 -0.7168 3.62E-06 4.16E-04 downregulated
13108 Cyp2g1 1.3975 2.3499 3.67E-06 4.16E-04 downregulated
81897 Tlr9 1.2547 2.5182 3.71E-06 4.16E-04 downregulated
72022 Slc35f2 -1.4312 3.8584 3.73E-06 4.16E-04 upregulated
328505 Skint7 1.3326 2.8645 3.74E-06 4.16E-04 downregulated
17127 Smad3 -0.6972 6.7089 3.74E-06 4.16E-04 upregulated
230613 Skint10 1.3207 3.7888 3.74E-06 4.16E-04 downregulated
75096 4930524O05Rik -1.8725 -0.4218 3.75E-06 4.16E-04 upregulated
58226 Cacna1h 1.4281 1.7798 3.76E-06 4.16E-04 downregulated
21652 Phf1 0.7887 6.1255 3.80E-06 4.19E-04 downregulated
73139 Cenpv -2.6259 3.9735 3.83E-06 4.20E-04 upregulated
15109 Hal 1.1622 3.2561 3.94E-06 4.29E-04 downregulated
69863 Ttc39b 0.8506 4.6413 3.95E-06 4.29E-04 downregulated
231296 Lrrc66 -4.0576 -1.1662 4.27E-06 4.61E-04 upregulated
16490 Kcna2 0.7609 5.0479 4.38E-06 4.71E-04 downregulated
100502619 AW046200 1.7743 -0.3678 4.46E-06 4.78E-04 downregulated
18766 Pkdrej -3.5744 -0.9858 4.56E-06 4.86E-04 upregulated
126
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
56213 Htra1 1.0551 4.9320 4.60E-06 4.89E-04 downregulated
268445 Ankrd13b -1.2251 5.1123 4.77E-06 5.04E-04 upregulated
17829 Muc1 1.3861 1.3929 4.81E-06 5.06E-04 downregulated
16842 Lef1 -0.7938 8.5693 4.88E-06 5.11E-04 upregulated
213002 Ifitm6 -2.8636 0.5994 4.94E-06 5.15E-04 upregulated
78416 Rnase6 1.5722 1.7335 5.15E-06 5.35E-04 downregulated
338365 Slc41a2 1.5452 0.8667 5.26E-06 5.42E-04 downregulated
17913 Myo1c 0.6526 7.0000 5.27E-06 5.42E-04 downregulated
66614 Gpatch4 -1.0701 4.9060 5.31E-06 5.44E-04 upregulated
237523 Ptprq 1.4434 3.3556 5.34E-06 5.44E-04 downregulated
14289 Fpr2 -2.2621 -0.0669 5.43E-06 5.51E-04 upregulated
16997 Ltbp2 1.2457 4.1754 5.54E-06 5.60E-04 downregulated
278507 Wfikkn2 0.8947 4.9430 5.58E-06 5.61E-04 downregulated
18647 Cdk14 1.3255 1.9094 5.64E-06 5.65E-04 downregulated
14585 Gfra1 -1.7203 -0.4439 5.79E-06 5.78E-04 upregulated
24050 2-Sep 1.2889 2.7382 6.14E-06 6.10E-04 downregulated
76459 Car12 -1.7844 3.3565 6.26E-06 6.19E-04 upregulated
380698 Obscn 1.4789 1.3245 6.31E-06 6.21E-04 downregulated
14219 Ctgf 1.3224 2.3810 6.54E-06 6.42E-04 downregulated
74467 Pus10 -0.6115 6.1590 6.77E-06 6.61E-04 upregulated
110308 Krt5 0.8323 7.9186 6.97E-06 6.75E-04 downregulated
14635 Galk1 -0.6646 5.9159 6.99E-06 6.75E-04 upregulated
110749 Chaf1b -0.6735 5.9058 7.00E-06 6.75E-04 upregulated
15002 H2-Ob 1.1607 3.9856 7.04E-06 6.76E-04 downregulated
244550 Podnl1 -1.0648 4.7569 7.08E-06 6.77E-04 upregulated
11820 App 0.8728 6.7559 7.16E-06 6.81E-04 downregulated
245474 Dkc1 -0.9144 7.1978 7.30E-06 6.89E-04 upregulated
68891 Cd177 -1.9409 2.8084 7.32E-06 6.89E-04 upregulated
12162 Bmp7 -0.9551 4.1463 7.33E-06 6.89E-04 upregulated
20371 Foxp3 0.9034 4.3208 7.45E-06 6.95E-04 downregulated
229898 Gbp5 1.0681 4.6428 7.45E-06 6.95E-04 downregulated
52897 Rbfox3 -3.3924 0.4123 7.68E-06 7.13E-04 upregulated
666926 Gm8369 1.5631 0.5240 8.05E-06 7.45E-04 downregulated
107094 Rrp12 -1.0681 5.3871 8.14E-06 7.49E-04 upregulated
72504 Taf4b -1.5768 4.2220 8.16E-06 7.49E-04 upregulated
14934 Gypa -6.5540 -1.2348 8.29E-06 7.58E-04 upregulated
13199 Ddn -1.6087 1.3824 9.21E-06 8.39E-04 upregulated
14708 Gng7 -1.5681 0.8595 9.70E-06 8.80E-04 upregulated
16531 Kcnma1 1.0157 3.6574 9.81E-06 8.86E-04 downregulated
18432 Mybbp1a -1.0534 8.0323 9.92E-06 8.93E-04 upregulated
73895 4930431P03Rik 2.3106 -1.6016 1.04E-05 9.33E-04 downregulated
116872 Serpinb7 1.4703 0.7345 1.04E-05 9.33E-04 downregulated
15016 H2-Q5 1.2392 2.7899 1.06E-05 9.42E-04 downregulated
269356 Slc4a11 1.3333 2.8576 1.06E-05 9.42E-04 downregulated
230796 Wdtc1 0.5766 7.4076 1.11E-05 9.83E-04 downregulated
56504 Srpk3 2.2300 -0.3416 1.12E-05 9.85E-04 downregulated
243369 Sspo 0.9573 4.9587 1.15E-05 1.01E-03 downregulated
14964 H2-D1 0.6051 9.0044 1.16E-05 1.01E-03 downregulated
94109 Csmd1 1.4430 2.1158 1.17E-05 1.02E-03 downregulated
20307 Ccl8 1.4166 2.7215 1.20E-05 1.04E-03 downregulated
269608 Plekhg5 -0.7637 4.8631 1.21E-05 1.04E-03 upregulated
56047 Msln 1.2120 2.9383 1.21E-05 1.04E-03 downregulated
12672 Chrm4 -3.4343 1.1559 1.23E-05 1.06E-03 upregulated
13087 Cyp2a5 1.2791 3.9430 1.25E-05 1.07E-03 downregulated
27966 Rrp9 -1.0665 5.4914 1.25E-05 1.07E-03 upregulated
127
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
13830 Stom 0.7859 4.7441 1.26E-05 1.07E-03 downregulated
16400 Itga3 0.9738 4.3246 1.31E-05 1.10E-03 downregulated
100043489 1300002E11Rik 0.9057 5.6490 1.34E-05 1.13E-03 downregulated
17996 Neb 1.4529 2.7569 1.35E-05 1.13E-03 downregulated
230971 Megf6 1.0766 3.6670 1.38E-05 1.15E-03 downregulated
12374 Casr 1.8663 0.9146 1.39E-05 1.15E-03 downregulated
544963 Iqgap2 -0.5816 6.7572 1.47E-05 1.21E-03 upregulated
56325 Abcb9 -0.9089 5.1081 1.48E-05 1.22E-03 upregulated
320092 E030003E18Rik 1.9909 -1.3836 1.50E-05 1.23E-03 downregulated
50908 C1s 1.0810 4.3724 1.50E-05 1.23E-03 downregulated
21847 Klf10 0.6672 6.0662 1.55E-05 1.26E-03 downregulated
214572 Prmt7 -0.8750 6.0076 1.56E-05 1.27E-03 upregulated
55989 Nop58 -1.1135 7.7348 1.60E-05 1.29E-03 upregulated
55990 Fmo2 1.4648 3.2012 1.60E-05 1.29E-03 downregulated
12268 C4b 1.3727 3.0499 1.60E-05 1.29E-03 downregulated
19273 Ptpru 1.4371 1.5298 1.61E-05 1.30E-03 downregulated
239081 Tlr11 1.3189 2.4884 1.64E-05 1.32E-03 downregulated
237988 Cdr2l -0.8477 4.2320 1.69E-05 1.35E-03 upregulated
74754 Dhcr24 -0.6869 7.1065 1.72E-05 1.37E-03 upregulated
15507 Hspb1 1.4481 1.7913 1.74E-05 1.38E-03 downregulated
17754 Mtap1a 1.4312 1.4392 1.75E-05 1.38E-03 downregulated
50927 Nasp -0.5744 7.8659 1.82E-05 1.43E-03 upregulated
104662 Tsr1 -0.9551 6.6466 1.87E-05 1.47E-03 upregulated
117229 Stk33 1.5498 1.3022 1.96E-05 1.53E-03 downregulated
16667 Krt17 0.6820 7.1931 1.96E-05 1.53E-03 downregulated
11567 Avil 1.3286 2.8831 1.97E-05 1.53E-03 downregulated
23832 Xcr1 1.0908 3.2433 1.98E-05 1.53E-03 downregulated
212483 Fam193b 0.7137 8.1737 2.01E-05 1.55E-03 downregulated
244418 D8Ertd82e -1.1958 5.5176 2.02E-05 1.55E-03 upregulated
380684 Nefh -1.2570 4.0462 2.03E-05 1.55E-03 upregulated
70661 Sik3 0.5544 6.9706 2.03E-05 1.55E-03 downregulated
16182 Il18r1 1.1888 4.2303 2.12E-05 1.61E-03 downregulated
21810 Tgfbi 1.0219 5.4751 2.15E-05 1.63E-03 downregulated
75580 Zbtb4 0.9169 5.4009 2.17E-05 1.64E-03 downregulated
110794 Cebpe -2.7938 -0.4338 2.22E-05 1.67E-03 upregulated
320790 Chd7 -1.6076 6.0218 2.26E-05 1.70E-03 upregulated
434341 Nlrc5 0.7663 6.6222 2.29E-05 1.71E-03 downregulated
239096 Cdh24 0.7209 6.6363 2.30E-05 1.72E-03 downregulated
19214 Ptgdr 1.9488 0.2022 2.37E-05 1.76E-03 downregulated
13090 Cyp2b19 1.3904 1.6803 2.39E-05 1.77E-03 downregulated
72373 Psca 1.2799 1.7402 2.41E-05 1.78E-03 downregulated
13511 Dsg2 0.8628 4.9965 2.46E-05 1.81E-03 downregulated
258216 Olfr1034 2.8574 -1.0383 2.47E-05 1.82E-03 downregulated
66526 2210012G02Rik 0.8157 4.6709 2.49E-05 1.82E-03 downregulated
76484 Kndc1 1.0062 3.7378 2.49E-05 1.82E-03 downregulated
74626 Tmem81 1.0582 2.7454 2.53E-05 1.84E-03 downregulated
27273 Pdk4 2.0357 2.4273 2.53E-05 1.84E-03 downregulated
320027 Fstl4 -2.2729 0.3317 2.54E-05 1.84E-03 upregulated
16188 Il3ra 0.7696 4.5901 2.55E-05 1.84E-03 downregulated
231452 Sdad1 -0.7585 6.7966 2.59E-05 1.86E-03 upregulated
56095 Ftsj3 -0.7604 7.1066 2.71E-05 1.94E-03 upregulated
70426 Tekt5 1.8217 -0.7830 2.75E-05 1.96E-03 downregulated
75590 Dusp9 -2.6333 0.9366 2.75E-05 1.96E-03 upregulated
258443 Olfr164 2.8887 -2.3999 2.76E-05 1.96E-03 downregulated
14134 Fcnb -2.5228 1.0977 2.78E-05 1.96E-03 upregulated
128
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
387314 Tmtc1 1.2483 2.7656 2.79E-05 1.96E-03 downregulated
387160 Mir142 1.4938 0.7955 2.88E-05 2.02E-03 downregulated
14945 Gzmk 4.3328 -2.5588 2.89E-05 2.02E-03 downregulated
97775 D930048N14Rik 1.0289 3.4108 2.92E-05 2.04E-03 downregulated
13195 Ddc -3.7401 2.4710 2.94E-05 2.04E-03 upregulated
110109 Nop2 -0.9944 6.3498 2.94E-05 2.04E-03 upregulated
13681 Eif4a1 -0.7826 6.9052 3.03E-05 2.10E-03 upregulated
98970 Fibcd1 -2.6662 1.5136 3.09E-05 2.13E-03 upregulated
67133 Gp2 1.0943 3.4673 3.15E-05 2.16E-03 downregulated
639774 Skint8 1.4738 2.5035 3.15E-05 2.16E-03 downregulated
212996 Wbscr17 1.3514 1.9923 3.18E-05 2.17E-03 downregulated
246278 Cd207 1.2404 3.4149 3.20E-05 2.18E-03 downregulated
20112 Rps6ka2 -0.9644 3.8294 3.31E-05 2.25E-03 upregulated
12480 Cd1d2 1.1934 4.4500 3.34E-05 2.26E-03 downregulated
14960 H2-Aa 0.8127 8.9029 3.34E-05 2.26E-03 downregulated
108075 Ltbp4 0.7405 5.3798 3.50E-05 2.36E-03 downregulated
319293 A530099J19Rik 1.3627 2.1149 3.52E-05 2.36E-03 downregulated
18392 Orc1 -0.9843 4.8138 3.56E-05 2.38E-03 upregulated
226169 Pprc1 -0.9245 6.2730 3.65E-05 2.44E-03 upregulated
52589 Ncald -1.5537 3.4689 3.67E-05 2.44E-03 upregulated
105670 Rcbtb2 -0.5552 8.4363 3.72E-05 2.47E-03 upregulated
13602 Sparcl1 0.7841 5.9017 3.73E-05 2.47E-03 downregulated
12348 Car11 -1.3383 1.9167 3.74E-05 2.47E-03 upregulated
69165 Cd209b 1.7940 0.2480 3.77E-05 2.48E-03 downregulated
12181 Bop1 -0.8953 6.7323 3.79E-05 2.49E-03 upregulated
11670 Aldh3a1 1.5794 1.0656 3.81E-05 2.49E-03 downregulated
22423 Wnt8b 2.1335 -1.4096 3.83E-05 2.50E-03 downregulated
100040736 9130206I24Rik 1.2011 3.0687 3.84E-05 2.50E-03 downregulated
67895 Ppa1 -0.9400 6.2954 3.96E-05 2.57E-03 upregulated
14115 Fbln2 -0.8939 4.6996 4.02E-05 2.60E-03 upregulated
73677 Psma8 -1.9544 2.3927 4.06E-05 2.62E-03 upregulated
13179 Dcn 0.9073 6.1839 4.18E-05 2.69E-03 downregulated
269615 Plch2 1.0294 3.1677 4.21E-05 2.70E-03 downregulated
13555 E2f1 -0.8403 5.2297 4.29E-05 2.74E-03 upregulated
108888 Atad3a -0.8675 4.9007 4.31E-05 2.75E-03 upregulated
66143 Eef1e1 -1.0097 4.9443 4.33E-05 2.75E-03 upregulated
108096 Slco1a5 1.5068 1.4015 4.38E-05 2.78E-03 downregulated
12765 Cxcr2 -1.9042 1.6864 4.41E-05 2.78E-03 upregulated
68723 Hrnr 1.5953 1.2005 4.41E-05 2.78E-03 downregulated
240168 Rasgrp3 1.3297 2.8305 4.43E-05 2.78E-03 downregulated
14262 Fmo3 1.6465 -0.3244 4.43E-05 2.78E-03 downregulated
100504399 C130021I20Rik -1.4996 0.7490 4.49E-05 2.81E-03 upregulated
383075 Enthd1 -3.0255 -0.1308 4.55E-05 2.84E-03 upregulated
110310 Krt7 0.6701 5.7631 4.66E-05 2.90E-03 downregulated
14696 Gnb4 0.6958 6.5558 4.69E-05 2.91E-03 downregulated
70113 Odf3b -1.1832 1.8204 4.75E-05 2.94E-03 upregulated
56200 Ddx21 -0.7722 7.9861 4.76E-05 2.94E-03 upregulated
15139 Hc -2.6166 1.8396 4.81E-05 2.97E-03 upregulated
14038 Expi 1.1833 3.6322 4.86E-05 2.99E-03 downregulated
238384 Slc24a4 1.1976 2.8912 4.90E-05 3.00E-03 downregulated
107766 Haao 1.3113 1.3241 4.95E-05 3.02E-03 downregulated
100504265 Gm20139 1.6571 0.2621 4.97E-05 3.02E-03 downregulated
15571 Elavl3 -1.5929 1.3773 4.97E-05 3.02E-03 upregulated
11792 Apex1 -0.9079 5.7665 4.99E-05 3.03E-03 upregulated
16612 Klk1 1.6467 -0.0740 5.13E-05 3.10E-03 downregulated
129
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
19073 Srgn 0.7618 6.3988 5.23E-05 3.15E-03 downregulated
268885 Stfa2l1 -5.0464 -1.3475 5.24E-05 3.15E-03 upregulated
52118 Pvr -0.7569 5.3174 5.25E-05 3.15E-03 upregulated
56863 Cldn9 1.9958 -1.6792 5.38E-05 3.22E-03 downregulated
241846 Lsm14b -0.5231 7.1321 5.55E-05 3.31E-03 upregulated
244895 C230081A13Rik 0.6642 5.4394 5.61E-05 3.34E-03 downregulated
53856 Prg3 -2.9470 1.0404 5.64E-05 3.35E-03 upregulated
19009 Pou6f1 0.6939 5.8077 5.78E-05 3.42E-03 downregulated
14525 Gcet2 1.3801 1.7296 5.79E-05 3.42E-03 downregulated
11426 Macf1 0.5377 9.5847 5.82E-05 3.42E-03 downregulated
68133 Gcsh -1.0829 4.4373 5.82E-05 3.42E-03 upregulated
16765 Stmn1 -0.6131 6.0824 5.83E-05 3.42E-03 upregulated
50783 Lsm4 -0.5924 6.3542 5.86E-05 3.43E-03 upregulated
50720 Sacs -1.6098 2.0499 5.89E-05 3.43E-03 upregulated
19027 Sypl 0.5919 6.2424 5.90E-05 3.43E-03 downregulated
77794 Adamtsl2 -1.3166 3.7675 5.93E-05 3.44E-03 upregulated
21886 Tle2 1.3653 4.0290 6.04E-05 3.49E-03 downregulated
69071 Tmem97 -1.2215 3.5748 6.06E-05 3.50E-03 upregulated
266632 Irak4 0.8612 3.9599 6.10E-05 3.52E-03 downregulated
259277 Klk8 -1.1698 4.3307 6.16E-05 3.53E-03 upregulated
13121 Cyp51 -0.6956 5.1093 6.17E-05 3.53E-03 upregulated
20019 Polr1a -0.8302 6.5919 6.18E-05 3.53E-03 upregulated
68718 Rnf166 0.6500 6.3962 6.19E-05 3.53E-03 downregulated
140743 Rem2 -1.7482 1.2780 6.24E-05 3.55E-03 upregulated
67937 Tmem59l -1.5929 0.4676 6.28E-05 3.56E-03 upregulated
60611 Foxj2 0.6398 5.8010 6.35E-05 3.60E-03 downregulated
103149 Upb1 1.4715 0.4255 6.41E-05 3.62E-03 downregulated
17988 Ndrg1 0.9505 5.6212 6.47E-05 3.64E-03 downregulated
13731 Emp2 1.2024 3.8933 6.52E-05 3.66E-03 downregulated
52552 Parp8 0.8890 5.4429 6.54E-05 3.67E-03 downregulated
64058 Perp 0.8181 5.6356 6.64E-05 3.72E-03 downregulated
227753 Gsn 0.7881 8.1616 6.73E-05 3.75E-03 downregulated
12010 B2m 0.5417 9.4791 6.80E-05 3.79E-03 downregulated
15401 Hoxa4 2.0722 -1.3140 6.91E-05 3.82E-03 downregulated
260297 Prrt1 0.9857 5.3853 6.92E-05 3.82E-03 downregulated
16790 Anpep 0.8152 4.3207 6.94E-05 3.82E-03 downregulated
212198 Wdr25 -2.0684 3.4696 6.94E-05 3.82E-03 upregulated
20190 Ryr1 -2.1276 2.0699 6.95E-05 3.82E-03 upregulated
229499 Fcrl1 1.4924 2.3355 6.96E-05 3.82E-03 downregulated
17219 Mcm6 -0.7592 8.7052 6.98E-05 3.82E-03 upregulated
229900 Gbp7 0.6147 6.6150 7.03E-05 3.84E-03 downregulated
52331 Stbd1 1.8844 -1.1563 7.05E-05 3.84E-03 downregulated
20449 St8sia1 -0.5941 7.3714 7.08E-05 3.84E-03 upregulated
171212 Galnt10 -0.6410 6.2645 7.12E-05 3.86E-03 upregulated
100040462 Mndal 0.6182 6.5187 7.16E-05 3.87E-03 downregulated
17215 Mcm3 -0.7127 8.2383 7.26E-05 3.92E-03 upregulated
171283 Havcr1 1.4500 0.5395 7.29E-05 3.93E-03 downregulated
106042 Prickle1 -0.9324 5.3022 7.35E-05 3.95E-03 upregulated
14357 Dtx1 -1.3772 5.7306 7.44E-05 3.99E-03 upregulated
16480 Jup 0.5756 7.0599 7.60E-05 4.06E-03 downregulated
224813 Gm88 1.2672 0.9143 7.75E-05 4.11E-03 downregulated
78697 Pus7 -1.1493 5.2001 7.77E-05 4.11E-03 upregulated
74104 Abcb6 -1.1478 3.1126 7.77E-05 4.11E-03 upregulated
72169 Trim29 0.8724 5.4871 7.78E-05 4.11E-03 downregulated
23887 Ggt5 0.9757 3.9542 7.78E-05 4.11E-03 downregulated
130
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
67564 Tmem35 -1.0997 2.2117 7.80E-05 4.11E-03 upregulated
11474 Actn3 1.0131 3.7032 7.81E-05 4.11E-03 downregulated
207819 4930539E08Rik 1.0590 3.4410 7.84E-05 4.11E-03 downregulated
319803 A430090L17Rik -1.5460 1.9427 7.89E-05 4.12E-03 upregulated
20356 Sema5a 1.4614 1.8778 7.89E-05 4.12E-03 downregulated
17191 Mbd2 0.5260 7.4933 8.01E-05 4.17E-03 downregulated
13436 Dnmt3b -0.9203 4.2175 8.01E-05 4.17E-03 upregulated
13007 Csrp1 0.5322 8.0777 8.05E-05 4.17E-03 downregulated
14586 Gfra2 1.0817 2.8463 8.07E-05 4.18E-03 downregulated
14061 F2 -2.4121 1.9350 8.10E-05 4.18E-03 upregulated
14972 H2-K1 0.6776 8.1795 8.30E-05 4.27E-03 downregulated
20775 Sqle -0.6031 5.7216 8.31E-05 4.27E-03 upregulated
16591 Kl 2.7128 0.8961 8.42E-05 4.32E-03 downregulated
66180 Leprel4 0.9194 3.7471 8.47E-05 4.33E-03 downregulated
329977 Fhad1 1.2834 1.7245 8.48E-05 4.33E-03 downregulated
546134 Gramd2 1.7409 -1.0700 8.54E-05 4.35E-03 downregulated
14256 Flt3l 0.9306 4.3570 8.69E-05 4.42E-03 downregulated
72462 Rrp1b -0.8809 5.4709 8.81E-05 4.47E-03 upregulated
70683 Utp20 -0.8816 6.7814 8.83E-05 4.47E-03 upregulated
110862 Kcnq3 1.0944 1.9845 8.84E-05 4.47E-03 downregulated
544791 Myh13 1.4384 0.4507 9.03E-05 4.54E-03 downregulated
18128 Notch1 -1.0328 8.9192 9.03E-05 4.54E-03 upregulated
382551 Cd300lh 1.4487 -0.1108 9.08E-05 4.55E-03 downregulated
241770 Rims4 -3.6574 -1.7410 9.09E-05 4.55E-03 upregulated
228071 Sestd1 0.7173 4.7818 9.10E-05 4.55E-03 downregulated
21771 Cirh1a -0.7590 6.1023 9.19E-05 4.57E-03 upregulated
16918 Mycl1 1.3295 2.3801 9.19E-05 4.57E-03 downregulated
217143 Gpr179 -1.5538 1.9344 9.25E-05 4.59E-03 upregulated
229534 Pbxip1 0.7084 6.2448 9.27E-05 4.59E-03 downregulated
71998 Slc25a35 0.9988 2.8898 9.37E-05 4.63E-03 downregulated
215798 Gpr126 1.0513 2.3657 9.39E-05 4.63E-03 downregulated
70769 Nolc1 -0.8714 7.3794 9.42E-05 4.64E-03 upregulated
12723 Clcn1 -1.5325 0.2336 9.48E-05 4.65E-03 upregulated
67887 Tmem66 0.5560 6.9394 9.50E-05 4.65E-03 downregulated
66249 Pno1 -0.8763 4.7608 9.50E-05 4.65E-03 upregulated
14205 Figf 1.7915 -0.2098 9.63E-05 4.69E-03 downregulated
13010 Cst3 0.6450 7.8822 9.64E-05 4.69E-03 downregulated
15165 Hcn1 1.5205 -0.1457 9.81E-05 4.76E-03 downregulated
59093 Pcbp3 -0.7135 4.5844 9.81E-05 4.76E-03 upregulated
319660 Agmo 1.2451 0.9363 9.89E-05 4.79E-03 downregulated
15370 Nr4a1 0.6336 6.0958 9.93E-05 4.79E-03 downregulated
328365 Zmiz1 -0.5276 7.7688 9.94E-05 4.79E-03 upregulated
98956 Nat10 -0.8885 6.2208 9.95E-05 4.79E-03 upregulated
227157 Mpp4 -2.5483 1.2596 9.99E-05 4.79E-03 upregulated
12722 Clca1 1.3850 1.1204 1.00E-04 4.79E-03 downregulated
237890 Slfn14-ps -4.3635 -3.4121 1.01E-04 4.81E-03 upregulated
101320 Dyrk4 1.4547 -0.2438 1.01E-04 4.81E-03 downregulated
72368 2310045N01Rik 0.7123 5.0023 1.02E-04 4.86E-03 downregulated
217410 Trib2 -0.5766 7.1988 1.02E-04 4.86E-03 upregulated
69072 Ebna1bp2 -0.7244 6.5884 1.03E-04 4.86E-03 upregulated
14184 Fgfr3 1.1652 1.6780 1.03E-04 4.87E-03 downregulated
17086 Ncr1 2.2364 -1.1791 1.04E-04 4.90E-03 downregulated
20528 Slc2a4 0.9537 3.6247 1.04E-04 4.90E-03 downregulated
266815 Mill1 1.4662 1.3190 1.06E-04 4.99E-03 downregulated
106757 Tmem146 -2.4163 0.7402 1.07E-04 5.00E-03 upregulated
131
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
328162 Trmt61a -1.0622 3.3577 1.07E-04 5.01E-03 upregulated
67177 Cdt1 -0.6777 6.3662 1.07E-04 5.01E-03 upregulated
11685 Alox12e 0.9011 4.8354 1.08E-04 5.03E-03 downregulated
57315 Wdr46 -0.6466 5.6521 1.08E-04 5.05E-03 upregulated
76051 Ganc 0.6766 5.1757 1.10E-04 5.10E-03 downregulated
14412 Slc6a13 -1.2923 1.1433 1.10E-04 5.11E-03 upregulated
16543 Mdfic 0.9400 3.7080 1.12E-04 5.18E-03 downregulated
67239 Rpf2 -0.7203 5.4750 1.15E-04 5.29E-03 upregulated
101612 Grwd1 -1.0771 4.7469 1.15E-04 5.30E-03 upregulated
78303 Hist3h2ba -1.3252 1.0740 1.16E-04 5.32E-03 upregulated
19227 Pthlh 1.7900 -0.0623 1.16E-04 5.32E-03 downregulated
108156 Mthfd1 -0.8215 7.0091 1.16E-04 5.32E-03 upregulated
83796 Smarcd2 0.4955 8.6879 1.16E-04 5.33E-03 downregulated
59028 Rcl1 -0.9334 4.5564 1.18E-04 5.37E-03 upregulated
29867 Cabp1 -2.9194 -0.5014 1.19E-04 5.42E-03 upregulated
57441 Gmnn -0.6332 6.2382 1.20E-04 5.43E-03 upregulated
14252 Flot2 0.5245 6.1303 1.20E-04 5.43E-03 downregulated
230558 C8a 2.9812 -2.8791 1.20E-04 5.43E-03 downregulated
50917 Galns -0.8210 4.7110 1.21E-04 5.44E-03 upregulated
22240 Dpysl3 0.9956 3.3312 1.21E-04 5.44E-03 downregulated
70835 Prss22 1.2583 0.3486 1.22E-04 5.49E-03 downregulated
13107 Cyp2f2 1.4728 2.8932 1.23E-04 5.53E-03 downregulated
26909 Exo1 -0.8939 5.5965 1.24E-04 5.53E-03 upregulated
66902 Mtap -0.7394 5.3013 1.24E-04 5.53E-03 upregulated
57741 Noc2l -0.7273 7.0442 1.24E-04 5.53E-03 upregulated
19385 Ranbp1 -0.7125 7.4743 1.24E-04 5.55E-03 upregulated
12649 Chek1 -0.8155 5.9514 1.27E-04 5.64E-03 upregulated
22634 Plagl1 1.1127 2.8822 1.28E-04 5.69E-03 downregulated
223838 Adamts20 -3.0499 2.6482 1.29E-04 5.72E-03 upregulated
218973 Wdhd1 -0.6506 6.5022 1.30E-04 5.73E-03 upregulated
78830 Slc25a12 0.5453 6.1114 1.32E-04 5.81E-03 downregulated
100503670 Rpl5 -0.7086 4.5746 1.32E-04 5.81E-03 upregulated
100705 Acacb 0.8684 4.1350 1.32E-04 5.82E-03 downregulated
12544 Cdc45 -0.6716 6.6898 1.33E-04 5.85E-03 upregulated
12290 Cacna1e -0.9287 6.7259 1.34E-04 5.86E-03 upregulated
57764 Ntn4 1.2774 1.7821 1.36E-04 5.93E-03 downregulated
76954 St5 0.7563 5.3370 1.36E-04 5.93E-03 downregulated
328780 Prss34 -3.2521 -0.1346 1.37E-04 5.95E-03 upregulated
12652 Chga 2.1081 6.2775 1.37E-04 5.96E-03 downregulated
20390 Sftpd 1.4388 1.3926 1.37E-04 5.96E-03 downregulated
16009 Igfbp3 1.0188 3.0079 1.38E-04 5.96E-03 downregulated
12393 Runx2 1.1767 2.9634 1.38E-04 5.97E-03 downregulated
69719 Cad -1.0821 7.4429 1.38E-04 5.97E-03 upregulated
94040 Clmn 1.1038 3.0786 1.40E-04 6.01E-03 downregulated
215456 Gpat2 1.6953 0.4071 1.42E-04 6.11E-03 downregulated
23959 Nt5e 1.1456 2.3551 1.43E-04 6.11E-03 downregulated
66988 Lap3 -0.7455 5.8079 1.43E-04 6.11E-03 upregulated
226180 Ina -3.1791 0.4130 1.43E-04 6.11E-03 upregulated
12567 Cdk4 -0.5309 8.1499 1.43E-04 6.11E-03 upregulated
14450 Gart -0.7278 7.4665 1.44E-04 6.11E-03 upregulated
70024 Mcm10 -0.8079 6.2619 1.44E-04 6.11E-03 upregulated
20893 Bhlhe40 0.8720 3.7768 1.44E-04 6.11E-03 downregulated
19122 Prnp 0.7027 5.6803 1.44E-04 6.12E-03 downregulated
70747 Tspan2 1.0283 4.9712 1.45E-04 6.12E-03 downregulated
239027 Arhgap22 1.1815 1.9892 1.46E-04 6.15E-03 downregulated
132
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
215384 Fcgbp 0.7113 7.4546 1.47E-04 6.20E-03 downregulated
71897 Lypd6b -1.1353 3.6831 1.48E-04 6.20E-03 upregulated
217109 Utp18 -0.6176 6.0320 1.49E-04 6.23E-03 upregulated
15483 Hsd11b1 1.0083 4.5531 1.49E-04 6.23E-03 downregulated
229228 Nudt6 1.1192 1.7732 1.50E-04 6.26E-03 downregulated
69195 Tmem121 -1.3745 3.4350 1.51E-04 6.30E-03 upregulated
58206 Zbtb32 1.4437 -0.1802 1.52E-04 6.32E-03 downregulated
93694 Clec2d 0.8297 5.4885 1.54E-04 6.41E-03 downregulated
68458 Ppp1r14a 1.5143 -0.7566 1.56E-04 6.50E-03 downregulated
23792 Adam23 1.0467 3.8536 1.57E-04 6.50E-03 downregulated
72432 Spink5 1.1063 4.9818 1.58E-04 6.55E-03 downregulated
16619 Klk1b27 1.6339 -0.6017 1.59E-04 6.57E-03 downregulated
233081 Ffar1 4.0603 -3.6661 1.60E-04 6.59E-03 downregulated
66042 Sostdc1 2.1290 -0.9735 1.61E-04 6.61E-03 downregulated
14051 Eya4 1.0563 3.5989 1.61E-04 6.62E-03 downregulated
14961 H2-Ab1 0.7303 8.7878 1.65E-04 6.78E-03 downregulated
100201 Tmem64 0.6154 5.1928 1.66E-04 6.78E-03 downregulated
109168 Atl3 0.5227 7.1463 1.66E-04 6.78E-03 downregulated
27056 Irf5 0.8363 4.4498 1.68E-04 6.82E-03 downregulated
21938 Tnfrsf1b 0.7295 5.1399 1.68E-04 6.82E-03 downregulated
83557 Lin28a -2.2900 -1.7513 1.68E-04 6.82E-03 upregulated
56338 Txnip 0.7357 9.4901 1.68E-04 6.82E-03 downregulated
23834 Cdc6 -0.8204 6.3402 1.69E-04 6.85E-03 upregulated
72391 Cdkn3 1.0988 3.1219 1.70E-04 6.88E-03 downregulated
68028 Rpl22l1 -0.7363 5.5671 1.71E-04 6.88E-03 upregulated
28030 Gfm1 -0.6212 5.9770 1.71E-04 6.89E-03 upregulated
16891 Lipg -1.3701 0.7587 1.72E-04 6.89E-03 upregulated
71670 Acy3 -1.9177 1.3924 1.72E-04 6.89E-03 upregulated
60594 Capn12 -0.6741 4.6453 1.72E-04 6.89E-03 upregulated
15378 Hnf4a -4.0489 0.6614 1.72E-04 6.89E-03 upregulated
21942 Tnfrsf9 1.1374 2.7738 1.73E-04 6.91E-03 downregulated
14013 Mecom 1.1482 1.2212 1.73E-04 6.91E-03 downregulated
216643 Gabrp 1.3517 2.4855 1.75E-04 6.96E-03 downregulated
213742 Xist 1.4001 0.2586 1.75E-04 6.96E-03 downregulated
27410 Abca3 0.5171 6.9802 1.76E-04 6.96E-03 downregulated
20717 Serpina3m 1.4556 0.9086 1.79E-04 7.06E-03 downregulated
13476 Reep5 0.5225 6.5945 1.79E-04 7.06E-03 downregulated
101202 Hepacam2 1.3817 1.2865 1.80E-04 7.08E-03 downregulated
66942 Ddx18 -0.7535 5.4025 1.81E-04 7.14E-03 upregulated
110816 Pwp2 -0.9053 5.7008 1.82E-04 7.14E-03 upregulated
14343 Fut1 1.3381 1.1920 1.82E-04 7.14E-03 downregulated
15983 Ifrd2 -0.9110 5.5255 1.83E-04 7.18E-03 upregulated
100503605 Beta-s -1.1636 4.6900 1.84E-04 7.20E-03 upregulated
93843 Pnck -0.9522 2.4234 1.85E-04 7.22E-03 upregulated
69716 Trip13 -0.7199 4.9530 1.85E-04 7.22E-03 upregulated
16979 Lrrn1 1.7930 0.6933 1.86E-04 7.22E-03 downregulated
223920 Soat2 -1.5616 1.6587 1.87E-04 7.26E-03 upregulated
20862 Stfa2 -3.0432 -0.8253 1.88E-04 7.30E-03 upregulated
78286 Nav2 0.8549 8.0143 1.91E-04 7.38E-03 downregulated
12466 Cct6a -0.7548 7.8111 1.91E-04 7.38E-03 upregulated
16160 Il12b 1.3431 2.6547 1.93E-04 7.44E-03 downregulated
67187 Zmynd19 -1.2804 5.2223 1.93E-04 7.44E-03 upregulated
16011 Igfbp5 1.4015 7.6683 1.95E-04 7.50E-03 downregulated
18763 Pkd1 0.5301 7.2084 1.96E-04 7.52E-03 downregulated
52850 Sgsm1 1.3301 0.4304 1.96E-04 7.53E-03 downregulated
133
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
319934 Sbf2 -1.0066 5.8374 1.97E-04 7.55E-03 upregulated
104759 Pld4 0.8468 5.0520 1.99E-04 7.57E-03 downregulated
223649 Nrbp2 1.0366 4.6105 1.99E-04 7.57E-03 downregulated
19226 Pth 2.1517 5.8233 2.00E-04 7.62E-03 downregulated
11433 Acp5 0.5578 6.3684 2.01E-04 7.64E-03 downregulated
230779 Serinc2 1.2146 1.6111 2.03E-04 7.70E-03 downregulated
56802 Nespas -1.3931 0.5367 2.03E-04 7.71E-03 upregulated
620779 Gm12695 6.0075 -4.1258 2.06E-04 7.78E-03 downregulated
13175 Dclk1 1.0863 3.0732 2.06E-04 7.79E-03 downregulated
13433 Dnmt1 -0.5544 9.7892 2.07E-04 7.79E-03 upregulated
106952 Arap3 -0.9534 4.3081 2.08E-04 7.83E-03 upregulated
328829 9830107B12Rik -2.9481 -2.3788 2.09E-04 7.84E-03 upregulated
215446 Entpd3 1.1871 0.5753 2.09E-04 7.84E-03 downregulated
18025 Nfe2l3 0.9288 3.7747 2.09E-04 7.84E-03 downregulated
78245 Acbd7 -1.9445 -0.9879 2.10E-04 7.85E-03 upregulated
232187 Smyd5 -0.9382 5.0321 2.14E-04 8.00E-03 upregulated
66409 Rsl1d1 -0.6712 7.3186 2.16E-04 8.05E-03 upregulated
20715 Serpina3g 0.9769 4.0710 2.17E-04 8.06E-03 downregulated
231532 Arhgap24 1.1326 3.3692 2.17E-04 8.08E-03 downregulated
100504195 Micalcl -1.7681 -0.4638 2.19E-04 8.11E-03 upregulated
72572 Spats2 -0.6415 4.8163 2.21E-04 8.17E-03 upregulated
68436 Rpl34 -2.4863 4.0367 2.21E-04 8.18E-03 upregulated
55948 Sfn 0.8444 3.4701 2.22E-04 8.20E-03 downregulated
66659 Acp6 0.5916 5.2404 2.22E-04 8.20E-03 downregulated
227715 Exosc2 -0.6760 5.6582 2.24E-04 8.25E-03 upregulated
228151 4833423E24Rik 1.2014 2.3183 2.26E-04 8.29E-03 downregulated
76306 1110021L09Rik 1.3988 0.6806 2.26E-04 8.29E-03 downregulated
271786 Galnt13 4.0603 -3.6598 2.26E-04 8.29E-03 downregulated
382018 Unc13a -1.2835 2.0854 2.29E-04 8.37E-03 upregulated
109198 6030407O03Rik 1.6613 -1.2329 2.32E-04 8.47E-03 downregulated
14000 Drosha -0.5713 7.0711 2.33E-04 8.48E-03 upregulated
80905 Polh 0.6204 4.9708 2.33E-04 8.48E-03 downregulated
94212 Pag1 -0.4727 8.2853 2.33E-04 8.48E-03 upregulated
12737 Cldn1 0.9598 4.2715 2.38E-04 8.63E-03 downregulated
109620 Dsp 0.6038 7.5824 2.38E-04 8.63E-03 downregulated
71241 Dmrtc2 -3.7995 -1.6012 2.43E-04 8.78E-03 upregulated
73674 Wdr75 -0.7023 5.8140 2.44E-04 8.78E-03 upregulated
67731 Fbxo32 0.8164 5.5777 2.44E-04 8.78E-03 downregulated
75828 Hormad2 2.5040 -2.1669 2.44E-04 8.78E-03 downregulated
21934 Tnfrsf11a 0.9851 3.0683 2.46E-04 8.78E-03 downregulated
72147 Zbtb46 1.1663 1.6122 2.46E-04 8.78E-03 downregulated
52679 E2f7 0.6712 4.7989 2.46E-04 8.78E-03 downregulated
433016 Gm5483 -3.1054 -1.2641 2.46E-04 8.78E-03 upregulated
242286 Sdr16c6 1.7492 0.8590 2.46E-04 8.78E-03 downregulated
18140 Uhrf1 -0.6650 8.6369 2.46E-04 8.79E-03 upregulated
13557 E2f3 -0.8386 5.6603 2.51E-04 8.93E-03 upregulated
215303 Camk1g -1.1755 2.6199 2.52E-04 8.94E-03 upregulated
18035 Nfkbia 0.6184 7.5722 2.52E-04 8.94E-03 downregulated
142980 Tlr3 0.9898 2.7719 2.56E-04 9.06E-03 downregulated
107889 Gcm2 2.7262 -1.8166 2.58E-04 9.14E-03 downregulated
319263 Pcmtd1 0.6704 6.2543 2.59E-04 9.14E-03 downregulated
330010 Ttll10 1.2330 0.8773 2.59E-04 9.14E-03 downregulated
50518 a -1.3203 1.6227 2.60E-04 9.16E-03 upregulated
17002 Ltf -1.2818 5.7648 2.60E-04 9.16E-03 upregulated
80797 Clca2 1.2311 3.1995 2.61E-04 9.18E-03 downregulated
134
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
66214 1190002H23Rik -0.4907 7.0187 2.62E-04 9.19E-03 upregulated
30054 Rnf17 -3.8454 1.3744 2.63E-04 9.21E-03 upregulated
100038577 Gm10790 3.2817 -3.0001 2.66E-04 9.29E-03 downregulated
11520 Plin2 0.5226 5.9847 2.66E-04 9.30E-03 downregulated
17873 Gadd45b 0.9936 3.5277 2.71E-04 9.45E-03 downregulated
110829 Lims1 0.4909 7.6155 2.71E-04 9.45E-03 downregulated
230233 Ikbkap -0.5133 6.3878 2.72E-04 9.45E-03 upregulated
11522 Adh1 1.0606 2.6291 2.74E-04 9.52E-03 downregulated
69692 Hddc2 -0.7778 4.1578 2.75E-04 9.52E-03 upregulated
12833 Col6a1 0.7382 6.0365 2.76E-04 9.54E-03 downregulated
628100 Fbxo39 5.9159 -4.2173 2.76E-04 9.54E-03 downregulated
234779 Plcg2 0.7475 4.7618 2.79E-04 9.62E-03 downregulated
50723 Icosl 0.9863 3.8829 2.83E-04 9.73E-03 downregulated
229697 Cym -3.7639 1.7843 2.84E-04 9.73E-03 upregulated
18788 Serpinb2 1.0490 3.0958 2.84E-04 9.73E-03 downregulated
68964 1500010J02Rik -0.5122 6.1808 2.85E-04 9.73E-03 upregulated
387247 Mirlet7d 2.1123 -2.3418 2.85E-04 9.73E-03 downregulated
19826 Rnps1 -0.8077 4.5224 2.85E-04 9.73E-03 upregulated
474160 BC033916 2.1747 -2.2907 2.86E-04 9.73E-03 downregulated
22348 Slc32a1 -3.3344 -3.5310 2.86E-04 9.73E-03 upregulated
75805 Nln -0.9936 4.3665 2.86E-04 9.73E-03 upregulated
12483 Cd22 0.9107 3.0934 2.86E-04 9.73E-03 downregulated
67949 Mki67ip -0.6432 6.1077 2.87E-04 9.74E-03 upregulated
21389 Tbx6 1.0680 2.9227 2.88E-04 9.75E-03 downregulated
18619 Penk 1.3509 2.3784 2.89E-04 9.79E-03 downregulated
16880 Lifr 0.6994 7.1190 2.90E-04 9.81E-03 downregulated
52874 D19Bwg1357e -0.6577 6.4852 2.92E-04 9.83E-03 upregulated
14998 H2-DMa 0.7039 5.3501 2.92E-04 9.84E-03 downregulated
226695 Ifi205 1.1072 2.5282 2.94E-04 9.90E-03 downregulated
53896 Slc7a10 1.6021 -0.5940 2.95E-04 9.92E-03 downregulated
18201 Nsmaf 0.4705 7.6625 2.96E-04 9.92E-03 downregulated
217169 Tns4 1.1003 4.0940 2.98E-04 9.99E-03 downregulated
66102 Cxcl16 0.6719 5.0472 3.00E-04 1.00E-02 downregulated
252868 Odf4 -6.2819 -3.8599 3.01E-04 1.00E-02 upregulated
75751 Ipo4 -0.7262 6.0402 3.06E-04 1.02E-02 upregulated
380660 Acss3 1.6261 -0.4543 3.07E-04 1.02E-02 downregulated
21955 Tnnt1 -1.4769 3.5522 3.11E-04 1.03E-02 upregulated
11539 Adora1 1.3705 -0.5001 3.11E-04 1.03E-02 downregulated
225288 Fhod3 1.1091 1.7492 3.12E-04 1.04E-02 downregulated
15951 Ifi204 0.7448 4.8892 3.13E-04 1.04E-02 downregulated
217995 Heatr1 -0.6754 7.4163 3.14E-04 1.04E-02 upregulated
66141 Ifitm3 0.6818 5.7797 3.16E-04 1.04E-02 downregulated
15469 Prmt1 -0.6322 7.4528 3.16E-04 1.04E-02 upregulated
223227 Sox21 -1.6497 -0.4963 3.17E-04 1.04E-02 upregulated
74127 Krt80 1.1440 2.1506 3.18E-04 1.04E-02 downregulated
22163 Tnfrsf4 1.0994 3.6571 3.20E-04 1.05E-02 downregulated
207607 Ccdc40 -1.1867 1.9568 3.21E-04 1.05E-02 upregulated
11815 Apod 0.9566 3.1869 3.23E-04 1.06E-02 downregulated
244579 Tox3 1.1915 1.3330 3.29E-04 1.08E-02 downregulated
14797 Aes 0.4516 8.9436 3.30E-04 1.08E-02 downregulated
433940 BC057022 -0.7898 3.5960 3.30E-04 1.08E-02 upregulated
17089 Lyar -0.8403 6.1458 3.32E-04 1.08E-02 upregulated
230917 Tmem201 -0.5794 5.8032 3.32E-04 1.08E-02 upregulated
76795 Tbc1d9b 0.4990 6.8061 3.35E-04 1.09E-02 downregulated
83673 Snhg1 -0.7949 5.4641 3.35E-04 1.09E-02 upregulated
135
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
434128 Pnmal2 1.0469 3.6246 3.35E-04 1.09E-02 downregulated
18762 Prkcz -0.9523 3.0987 3.37E-04 1.09E-02 upregulated
20973 Syngr2 0.5248 5.8546 3.39E-04 1.09E-02 downregulated
22061 Trp63 0.8499 5.8500 3.40E-04 1.10E-02 downregulated
12035 Bcat1 -1.4395 4.8420 3.41E-04 1.10E-02 upregulated
208748 Prrg3 1.0588 3.0274 3.43E-04 1.10E-02 downregulated
52348 Vps37a 0.5500 6.9651 3.43E-04 1.10E-02 downregulated
192897 Itgb4 0.6487 5.6110 3.48E-04 1.11E-02 downregulated
13661 Ehf 0.9427 3.4772 3.49E-04 1.11E-02 downregulated
71817 Tmem50a 0.5342 7.1994 3.49E-04 1.11E-02 downregulated
12821 Col17a1 0.9935 2.9188 3.49E-04 1.11E-02 downregulated
12504 Cd4 0.8699 9.9388 3.50E-04 1.11E-02 downregulated
56149 Grasp 0.9192 2.9734 3.51E-04 1.12E-02 downregulated
53313 Atp2a3 0.5299 9.2167 3.52E-04 1.12E-02 downregulated
14958 H1f0 -1.2878 5.9549 3.54E-04 1.12E-02 upregulated
21681 Alyref -0.6297 6.9233 3.55E-04 1.12E-02 upregulated
56758 Mbnl1 -0.4430 9.6540 3.57E-04 1.13E-02 upregulated
53311 Mybph 1.6060 -1.0781 3.59E-04 1.13E-02 downregulated
22072 Prss2 -4.4621 4.4098 3.59E-04 1.13E-02 upregulated
73410 1700065D16Rik -1.2821 0.3396 3.61E-04 1.14E-02 upregulated
216781 Trim58 1.8168 -1.6683 3.62E-04 1.14E-02 downregulated
20536 Slc4a3 1.1920 1.4622 3.63E-04 1.14E-02 downregulated
235534 Acpl2 -1.0584 2.0481 3.63E-04 1.14E-02 upregulated
17386 Mmp13 1.3136 0.4418 3.65E-04 1.14E-02 downregulated
76787 Ppfia3 1.1889 0.2627 3.72E-04 1.16E-02 downregulated
15526 Hspa9 -0.6704 8.2981 3.73E-04 1.16E-02 upregulated
55987 Cpxm2 1.0398 2.8722 3.74E-04 1.17E-02 downregulated
26878 B3galt2 1.2596 0.5196 3.77E-04 1.18E-02 downregulated
26939 Polr3e -0.7242 5.7444 3.81E-04 1.19E-02 upregulated
70762 Dclk2 -0.5144 6.7953 3.82E-04 1.19E-02 upregulated
18160 Npr1 1.0115 2.9885 3.84E-04 1.19E-02 downregulated
223726 Mpped1 2.8489 -2.9991 3.85E-04 1.19E-02 downregulated
18124 Nr4a3 1.1065 3.1758 3.90E-04 1.21E-02 downregulated
13852 Stx2 0.6997 4.4943 3.91E-04 1.21E-02 downregulated
55985 Cxcl13 1.5163 0.6672 3.92E-04 1.21E-02 downregulated
19116 Prlr 1.4790 0.4791 3.94E-04 1.22E-02 downregulated
27374 Prmt5 -0.6436 7.0294 3.96E-04 1.22E-02 upregulated
11421 Ace 0.8854 3.6298 4.01E-04 1.23E-02 downregulated
54610 Tbc1d8 0.6600 5.5053 4.01E-04 1.23E-02 downregulated
54713 Fezf2 0.9761 3.6843 4.07E-04 1.25E-02 downregulated
225348 Wdr36 -0.5599 6.4851 4.09E-04 1.25E-02 upregulated
237403 Lingo3 -4.3635 -3.4209 4.11E-04 1.26E-02 upregulated
53333 Tomm40 -0.6464 6.1659 4.14E-04 1.26E-02 upregulated
12192 Zfp36l1 0.6523 7.0487 4.14E-04 1.26E-02 downregulated
14107 Fat1 0.6416 5.3519 4.19E-04 1.28E-02 downregulated
69617 Pitrm1 -0.5202 7.4037 4.20E-04 1.28E-02 upregulated
66412 Arrdc4 0.8584 4.7550 4.22E-04 1.28E-02 downregulated
100038464 Gm15441 0.7073 7.2839 4.22E-04 1.28E-02 downregulated
399603 Fam84b 0.5882 5.5414 4.29E-04 1.30E-02 downregulated
239134 Gucy1b2 1.7980 -1.8391 4.39E-04 1.33E-02 downregulated
103551 E130012A19Rik -1.5376 2.3961 4.42E-04 1.34E-02 upregulated
24015 Abce1 -0.5450 7.9383 4.51E-04 1.36E-02 upregulated
22427 Wrn 0.5084 6.3054 4.51E-04 1.36E-02 downregulated
23849 Klf6 0.6111 6.0660 4.53E-04 1.37E-02 downregulated
107522 Ece2 -0.9887 2.9990 4.55E-04 1.37E-02 upregulated
136
Gene ID Gene Symbol log FC log CPM P Value padj Direction in KO
108052 Slc14a1 1.0852 3.6304 4.55E-04 1.37E-02 downregulated
22329 Vcam1 0.4706 7.9493 4.56E-04 1.37E-02 downregulated
243864 Mill2 1.3138 -0.1412 4.57E-04 1.37E-02 downregulated
69809 1810046K07Rik 1.6062 -0.7230 4.57E-04 1.37E-02 downregulated
19041 Ppl 0.9613 3.6364 4.57E-04 1.37E-02 downregulated
66422 Dctpp1 -0.7869 5.2804 4.59E-04 1.37E-02 upregulated
12870 Cp 0.7858 4.2210 4.60E-04 1.37E-02 downregulated
74735 Trim14 0.8401 3.7980 4.63E-04 1.38E-02 downregulated
56296 Dmrtb1 1.2784 0.0128 4.64E-04 1.38E-02 downregulated
12261 C1qbp -0.8536 6.7242 4.64E-04 1.38E-02 upregulated
170761 Pdzd3 -1.3419 0.7229 4.64E-04 1.38E-02 upregulated
100198 H6pd 0.7739 6.8996 4.66E-04 1.38E-02 downregulated
56843 Trpm5 1.1309 2.3860 4.67E-04 1.38E-02 downregulated
270096 Mon1b 0.6591 5.6537 4.67E-04 1.38E-02 downregulated
18846 Plxna3 -0.9568 4.2283 4.69E-04 1.38E-02 upregulated
268396 Sh3pxd2b 0.6900 4.7521 4.70E-04 1.38E-02 downregulated
100038854 BC117090 -2.6309 -1.8051 4.70E-04 1.38E-02 upregulated
72049 Tnfrsf13c 1.4280 0.2550 4.74E-04 1.39E-02 downregulated
13088 Cyp2b10 6.5242 -3.6024 4.76E-04 1.40E-02 downregulated
329839 Gm829 3.0386 -3.2121 4.78E-04 1.40E-02 downregulated
21949 Tnfsf8 1.2237 2.1800 4.82E-04 1.41E-02 downregulated
11980 Atp8a1 0.5168 6.9920 4.87E-04 1.42E-02 downregulated
22419 Wnt5b -1.1089 3.5768 4.88E-04 1.42E-02 upregulated
12700 Cish 0.8719 3.8198 4.92E-04 1.43E-02 downregulated
12986 Csf3r -1.1417 3.0741 4.93E-04 1.44E-02 upregulated
66957 Serpinb11 1.2034 2.1794 4.95E-04 1.44E-02 downregulated
78753 Lipm 1.1840 1.6181 4.96E-04 1.44E-02 downregulated
171285 Havcr2 0.9408 3.2013 4.97E-04 1.44E-02 downregulated
75188 1700009J07Rik 2.1869 -1.7969 4.97E-04 1.44E-02 downregulated
74318 Hopx 1.4060 1.4033 4.97E-04 1.44E-02 downregulated
71390 5430425J12Rik -2.0144 -1.6983 4.99E-04 1.44E-02 upregulated
13522 Adam28 1.3827 1.6287 5.00E-04 1.44E-02 downregulated
56361 Pus1 -0.8586 4.8350 5.01E-04 1.44E-02 upregulated
14025 Bcl11a 1.0475 2.6977 5.03E-04 1.45E-02 downregulated
104458 Rars -0.5383 6.7416 5.03E-04 1.45E-02 upregulated
100504464 E230016K23Rik 1.5335 -1.0503 5.05E-04 1.45E-02 downregulated
54635 Pdgfc 0.9292 2.8011 5.08E-04 1.46E-02 downregulated
107586 Ovol2 1.7841 -1.4809 5.08E-04 1.46E-02 downregulated
269016 Sh3rf2 1.1806 0.7854 5.20E-04 1.49E-02 downregulated
109624 Cald1 0.7834 4.7538 5.23E-04 1.49E-02 downregulated
71864 1700026J04Rik -1.6520 -1.0742 5.25E-04 1.50E-02 upregulated
12043 Bcl2 0.8686 5.2397 5.26E-04 1.50E-02 downregulated
137
APPENDIX D
GENE ONTOLOGY ANALYSIS 808 DIFFERENTIALLY-REGULATED TRANSCRIPTS IN
MBNL1-/-
THYMUS BENJAMINI FDR≤0.1
Term Count %
Fold
Enrichment
Benjamini
FDR p-Value Biological Process
GO:0006955~immune response 56 7.0 2.851 9.18E-09 3.53E-12
GO:0002376~immune system process 77 9.7 2.334 6.49E-09 5.00E-12
GO:0006952~defense response 46 5.8 2.462 3.47E-05 4.01E-08
GO:0050896~response to stimulus 152 19.1 1.496 6.54E-05 1.01E-07
GO:0048002~antigen processing and
presentation of peptide antigen 11 1.4 7.536 5.39E-04 1.04E-06
GO:0019884~antigen processing and
presentation of exogenous antigen 10 1.3 8.564 5.20E-04 1.20E-06
GO:0009605~response to external stimulus 54 6.8 2.023 4.86E-04 1.31E-06
GO:0002478~antigen processing and
presentation of exogenous peptide antigen 9 1.1 9.383 7.87E-04 2.42E-06
GO:0019882~antigen processing and
presentation 16 2.0 4.410 7.74E-04 2.68E-06
GO:0002504~antigen processing and
presentation of peptide or polysaccharide
antigen via MHC class II 8 1.0 10.096 1.77E-03 6.81E-06
GO:0032502~developmental process 158 19.8 1.381 1.75E-03 7.40E-06
GO:0048583~regulation of response to
stimulus 33 4.1 2.398 1.71E-03 7.89E-06
GO:0002682~regulation of immune system
process 32 4.0 2.383 2.53E-03 1.27E-05
GO:0050778~positive regulation of immune
response 19 2.4 3.350 2.53E-03 1.36E-05
GO:0022613~ribonucleoprotein complex
biogenesis 19 2.4 3.325 2.61E-03 1.51E-05
GO:0050776~regulation of immune response 23 2.9 2.887 2.46E-03 1.52E-05
GO:0002684~positive regulation of immune
system process 24 3.0 2.794 2.49E-03 1.63E-05
GO:0006950~response to stress 80 10.0 1.620 2.41E-03 1.67E-05
GO:0048731~system development 121 15.2 1.431 3.77E-03 2.76E-05
GO:0019886~antigen processing and
presentation of exogenous peptide antigen
via MHC class II 7 0.9 10.490 3.69E-03 2.84E-05
GO:0002495~antigen processing and
presentation of peptide antigen via MHC
class II 7 0.9 10.490 3.69E-03 2.84E-05
GO:0048584~positive regulation of response
to stimulus 22 2.8 2.836 3.94E-03 3.19E-05
GO:0048856~anatomical structure
development 127 15.9 1.403 4.94E-03 4.19E-05
GO:0042254~ribosome biogenesis 16 2.0 3.425 6.89E-03 6.12E-05
GO:0006954~inflammatory response 24 3.0 2.558 7.14E-03 6.62E-05
GO:0002252~immune effector process 17 2.1 3.235 7.01E-03 6.77E-05
GO:0044085~cellular component biogenesis 47 5.9 1.824 9.38E-03 9.43E-05
GO:0048513~organ development 100 12.5 1.442 1.24E-02 1.30E-04
GO:0042127~regulation of cell proliferation 42 5.3 1.872 1.26E-02 1.37E-04
GO:0046649~lymphocyte activation 21 2.6 2.636 1.25E-02 1.40E-04
138
Term Count %
Fold
Enrichment
Benjamini
FDR p-Value Biological Process (continued)
GO:0007275~multicellular organismal
development 140 17.6 1.332 1.54E-02 1.80E-04
GO:0048518~positive regulation of
biological process 91 11.4 1.461 1.52E-02 1.83E-04
GO:0030154~cell differentiation 94 11.8 1.437 2.03E-02 2.52E-04
GO:0016072~rRNA metabolic process 12 1.5 3.836 2.01E-02 2.58E-04
GO:0048869~cellular developmental process 97 12.2 1.422 2.17E-02 2.87E-04
GO:0002443~leukocyte mediated immunity 13 1.6 3.502 2.26E-02 3.07E-04
GO:0045321~leukocyte activation 22 2.8 2.409 2.35E-02 3.29E-04
GO:0045597~positive regulation of cell
differentiation 19 2.4 2.603 2.57E-02 3.71E-04
GO:0001775~cell activation 23 2.9 2.242 4.21E-02 6.28E-04
GO:0009611~response to wounding 29 3.6 2.004 4.23E-02 6.48E-04
GO:0045072~regulation of interferon-
gamma biosynthetic process 5 0.6 10.899 4.92E-02 7.77E-04
GO:0045595~regulation of cell
differentiation 31 3.9 1.926 4.81E-02 7.78E-04
GO:0002521~leukocyte differentiation 16 2.0 2.702 5.11E-02 8.47E-04
GO:0002253~activation of immune response 12 1.5 3.346 5.03E-02 8.53E-04
GO:0051239~regulation of multicellular
organismal process 52 6.5 1.603 4.92E-02 8.54E-04
GO:0030098~lymphocyte differentiation 14 1.8 2.945 5.10E-02 9.06E-04
GO:0006364~rRNA processing 11 1.4 3.564 5.19E-02 9.43E-04
GO:0016064~immunoglobulin mediated
immune response 10 1.3 3.806 6.02E-02 1.12E-03
GO:0002449~lymphocyte mediated
immunity 11 1.4 3.471 6.11E-02 1.16E-03
GO:0016485~protein processing 12 1.5 3.197 6.41E-02 1.25E-03
GO:0002520~immune system development 25 3.1 2.032 6.91E-02 1.38E-03
GO:0051605~protein maturation by peptide
bond cleavage 10 1.3 3.689 6.93E-02 1.41E-03
GO:0019724~B cell mediated immunity 10 1.3 3.689 6.93E-02 1.41E-03
GO:0051094~positive regulation of
developmental process 20 2.5 2.241 7.52E-02 1.56E-03
GO:0002711~positive regulation of T cell
mediated immunity 5 0.6 9.222 7.44E-02 1.57E-03
GO:0045087~innate immune response 13 1.6 2.913 7.55E-02 1.63E-03
GO:0002526~acute inflammatory response 11 1.4 3.256 8.60E-02 1.90E-03
GO:0006935~chemotaxis 13 1.6 2.860 8.50E-02 1.91E-03
GO:0042330~taxis 13 1.6 2.860 8.50E-02 1.91E-03
GO:0051604~protein maturation 12 1.5 2.997 9.21E-02 2.12E-03
GO:0002250~adaptive immune response 11 1.4 3.140 1.06E-01 2.50E-03
GO:0002460~adaptive immune response
based on somatic recombination of
immune receptors built from
immunoglobulin superfamily domains 11 1.4 3.140 1.06E-01 2.50E-03
GO:0031328~positive regulation of cellular
biosynthetic process 38 4.8 1.651 1.23E-01 2.98E-03
GO:0006259~DNA metabolic process 31 3.9 1.766 1.24E-01 3.05E-03
GO:0007610~behavior 30 3.8 1.776 1.28E-01 3.21E-03
GO:0042110~T cell activation 13 1.6 2.687 1.27E-01 3.23E-03
GO:0006730~one-carbon metabolic process 13 1.6 2.664 1.33E-01 3.46E-03
139
Term Count %
Fold
Enrichment
Benjamini
FDR p-Value Biological Process (continued)
GO:0009891~positive regulation of
biosynthetic process 38 4.8 1.636 1.34E-01 3.53E-03
GO:0048519~negative regulation of
biological process 76 9.5 1.374 1.38E-01 3.71E-03
GO:0060348~bone development 13 1.6 2.642 1.36E-01 3.71E-03
Cellular Component
GO:0005730~nucleolus 42 5.3 3.240 1.96E-08 5.60E-11
GO:0042611~MHC protein complex 13 1.6 5.979 1.98E-04 1.13E-06
GO:0009986~cell surface 32 4.0 2.509 5.29E-04 4.54E-06
GO:0009897~external side of plasma
membrane 25 3.1 2.902 4.83E-04 5.52E-06
GO:0005576~extracellular region 106 13.3 1.509 9.84E-04 1.41E-05
GO:0044459~plasma membrane part 102 12.8 1.494 1.84E-03 3.16E-05
GO:0005886~plasma membrane 163 20.5 1.341 1.68E-03 3.35E-05
GO:0044421~extracellular region part 56 7.0 1.730 3.47E-03 7.95E-05
GO:0031981~nuclear lumen 61 7.7 1.652 5.10E-03 1.31E-04
GO:0043232~intracellular non-membrane-
bounded organelle 112 14.1 1.396 6.74E-03 1.93E-04
GO:0043228~non-membrane-bounded
organelle 112 14.1 1.396 6.74E-03 1.93E-04
GO:0042613~MHC class II protein complex 5 0.6 11.957 1.63E-02 5.17E-04
GO:0031012~extracellular matrix 27 3.4 2.090 1.61E-02 5.58E-04
GO:0005578~proteinaceous extracellular
matrix 26 3.3 2.093 1.87E-02 7.00E-04
GO:0042612~MHC class I protein complex 8 1.0 4.555 3.96E-02 1.62E-03
GO:0005771~multivesicular body 4 0.5 10.628 1.11E-01 5.04E-03
GO:0005615~extracellular space 35 4.4 1.638 1.05E-01 5.05E-03
GO:0070013~intracellular organelle lumen 66 8.3 1.393 1.03E-01 5.28E-03
GO:0031974~membrane-enclosed lumen 68 8.5 1.385 1.01E-01 5.43E-03
GO:0043233~organelle lumen 66 8.3 1.389 1.02E-01 5.82E-03
GO:0044428~nuclear part 71 8.9 1.368 9.81E-02 5.88E-03
GO:0030057~desmosome 5 0.6 5.979 1.33E-01 8.55E-03
GO:0031226~intrinsic to plasma membrane 36 4.5 1.560 1.37E-01 9.20E-03
Molecular Function
GO:0005509~calcium ion binding 70 8.8 1.966 6.34E-05 8.24E-08
GO:0030246~carbohydrate binding 35 4.4 2.605 2.42E-04 6.29E-07
GO:0019955~cytokine binding 14 1.8 3.753 2.03E-02 8.00E-05
GO:0004857~enzyme inhibitor activity 25 3.1 2.427 1.99E-02 1.04E-04
GO:0030414~peptidase inhibitor activity 20 2.5 2.681 2.60E-02 1.71E-04
GO:0005488~binding 524 65.7 1.080 2.24E-02 1.76E-04
GO:0005529~sugar binding 20 2.5 2.607 2.67E-02 2.46E-04
GO:0005515~protein binding 291 36.5 1.173 2.90E-02 3.06E-04
GO:0001871~pattern binding 16 2.0 2.949 2.82E-02 3.35E-04
GO:0030247~polysaccharide binding 16 2.0 2.949 2.82E-02 3.35E-04
GO:0004866~endopeptidase inhibitor
activity 18 2.3 2.637 3.62E-02 4.79E-04
GO:0008236~serine-type peptidase activity 21 2.6 2.337 4.72E-02 6.91E-04
GO:0017171~serine hydrolase activity 21 2.6 2.326 4.60E-02 7.33E-04
GO:0004869~cysteine-type endopeptidase
inhibitor activity 8 1.0 4.839 6.37E-02 1.11E-03
GO:0004252~serine-type endopeptidase
activity 19 2.4 2.371 6.01E-02 1.13E-03
GO:0004175~endopeptidase activity 32 4.0 1.793 9.89E-02 2.03E-03
140
Term Count %
Fold
Enrichment
Benjamini
FDR p-Value Molecular Function (continued)
GO:0042605~peptide antigen binding 5 0.6 8.425 1.03E-01 2.27E-03
GO:0008201~heparin binding 11 1.4 3.126 1.11E-01 2.58E-03
GO:0005539~glycosaminoglycan binding 13 1.6 2.690 1.28E-01 3.20E-03
141
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BIOGRAPHICAL SKETCH
Marina M. Scotti dual majored in Chemistry with a Biochemistry focus and
Microbiology and Cell Science to obtain her Bachelor of Science degree from the University of
Florida in 2004. During her undergraduate education she worked as research volunteer in the
laboratory of Dr. Naohiro Terada studying the underlying molecular mechanisms responsible for
maintaining pluripotency. Subsequently, she worked as a Research Associate in Pre-clinical
research and Development for Applied Genetics Technologies Corporation (AGTC). She joined
the University of Florida’s College of Medicine graduate Interdisciplinary Program in
Biomedical Sciences in 2010. Her research interests focus on immune system dysregulation and
autoimmunity in the neuromuscular disease myotonic dystrophy. She completed her doctoral
research in the laboratory of Dr. Maurice S. Swanson in the Department of Molecular Genetics
and Microbiology within the Genetics Concentration and received her Doctorate of Philosophy
in Medical Sciences in May 2016.
