Journal Club 5/14/15Dysregulation of Gene Expression as a Cause of Cockayne Syndrome Neurological Disease.
Wang Y, et al. PNAS. 2014.
Agenda
Background Cockayne Syndrome
Clinical manifestations Known pathophysiology
Rationale for the study
Experimental findings Gene expression in fibroblasts iPS reprogramming Neuroblast differentiation Cerebellar gene expression
Discussion of results Importance Further Directions
Cockayne Syndrome (and me)
dysmorphic
Microcephalic Very small
Hypomyelination
deafRetinal disease
Abnormal liver enzymes
Developmental delay
Ataxia
Joint contracture
sSpine differences
What causes Cockayne Syndrome?
Cockayne Syndrome
Autosomal Recessive Loss of function
Mutations in ERCC6 (CSB) = 80%
Mutations in ERCC8 (CSA) = 20%
What does this do?
UV big, bulky DNA damage
CSA & B help perform nucleotide excision repair
Restore ability of transcription
UV
Does this make sense? Problem 1: CS ≠ XP
XP is in fact a broader defect than CS, but the symptoms don’t overlap
Relatedly, Problem 2: Lack of skin cancer Some aren’t sun sensitive at
all
Problem 3: Neurologic disease How does this fit with UV
exposure? Non-UV damage requiring
NER < UV damage by 3 orders of magnitude
Problem 4: Time course Pre/neonatal onset of
symptoms
Problem 5: sensitivity to other DNA damage, oxidative stress
UV
So, what else? Secondary mitochondrial
disease Free radical generation Mitochondrial DNA repair
Transcription regulation effects Evidence:
Known to interact with RNAPI, RNAPII
Brooks, PJ. DNA Repair. 2014
Hypothesis: Transcription regulation is a major feature of mutations in CSBGoals1. Demonstrate change in regulation2. Determine affected tissues3. Determine relationship between dysregulation and disease
Genes are dysregulated as a result of CSB mutationHypothesis 1.
Cell lines
Fibroblast line from patient with genetically proven Cockayne syndrome (CS1AN) Immortalized with Sv40 Rescued cell line
BAC rescue (BAC-CSB) Conditional tetra/doxy promotor rescue (CSB-TetON)
Experiment 1: Comparison of expression
1,200 “dysregulated genes”
Statistically significantly different between control v. Bac and control v. tetON
>1.5 fold difference
What is in common?
Noted mostly neuronal genes
Confirmed by RT-PCR
Why does this happen
Genome wide CHiP-Seq for CSB RNAPII
Looked at genes that are downregulated in CSB Loss of CSB binding Loss of RNAPII binding
Summary, experiment 1
What have we shown? There is selective dysregulation of multiple genes Many of the downregulated genes are neuronal Downregulation happens because mutant CSB does not
bind to the gene target, and RNAPII subsequently doesn’t bind
What are the problems (immortalized with Sv40) Looking at neuronal genes in fibroblasts
Next step: try to reprogram to NPC
Genes that are dysregulated in fibroblasts are meaningful for neuronal functionHypothesis 2
Experiment 2: reprogramming FCL
• shRNA knockdown of PTBP1 or• Overexpression of miR-9/124• And introduction of three neuronal transcription regulators• Key event: transition from PTB to nPTB
Unable to transduce mutant cells
32 GenesSelected for neuronal functionConsistently upregulated in WT and not in CSB
Summary, experiment 2
Unable to transduce cells with loss of function of CSB into neurons
Fibroblast to neuronal transduction is not normal physiology Patients with CS obviously have neurons
What meaning does this have for CSB-mutant neurons?
Experiment 3: neuroblast differentiation
SH-SY5Y CSB knockdown
RA
Pahlman, et al. Cell diff. 1984
Attempted differentiation
Experiment 4: neuronal maintenance
Knocked down CSB in differentiated SH-
SY5Y
• Loss of long neurites
• Cell death
Summary of experiments 2 & 3
Knockdown of CSB affects Neuroblast differentiation into neurons Neuronal maintenance of established neurons
Remaining questions Why does this happen? Is this laboratory model applicable to patients with CS?
Defects in neuronal differentiation and maintenance are due to genetic dysregulationHypothesis 3
Transcriptome analysis of neuronal differentiation
Is there a difference in CSB k.d.?
Overall, no
Selected by K-means clustering specific differences in expression
Genes identified 100 genes Different at every
time point P<0.01
Did not do multiple comparison adjustment
17 in neuronal ontology
Summary experiment 4
Some evidence that neuronal problems are due to transcriptional dysregulation These is pretty weak Their stats are even weaker
Now what? Mice with CSB K.O. have no neuronal phenotype So, turn to human brain
Gene dysregulation will be demonstrable in human brains from CS patientsHypothesis 4
Experiment 5: Tissue
Human cerebellum Patients with molecularly
confirmed CS Does not specify gene Does not specify
mutation type “matched” controls
Extracted RNA
Hierarchical, nonsupervised clustering
Selected genes >2-fold dysregulated
What are the functions of the dysregulated genes?
Exocytosis machinery
Synaptotagmins Voltage dependent
calcium channels Maybe explains
Brain calcifications Calcium-dependent
myelination
Preservation of cerebellar signature
Summary 1
In models of CSB mutation there is genetic dysregulation Resulting from abnormal CSB binding Abnormal RNAPII recruitment
Genetic dysregulation specifically targets Neuronal genes Important for neuronal secretion, synaptic density and
neuronal differentiation
Evidence supports dysregulation as a major cause of neuronal dysfunction in Cockayne Syndrome
Generalizing the resultsSome side experiments
How similar are the models to each other?
How similar is CSA?
Does reduce overlap, but does not change enriched ontology
But what about mice?Mice are interesting because they don’t have neuronal dysfunction
Genes differentially regulated between mice and humans with CSB mutations may be interesting targets for understanding the neuronal disease
Conclusions There is compelling evidence that CSB is important for
transcriptional regulation
Intriguing identification of models of neuronal dysfunction in CS
Large dataset with some overlap is best used for hypothesis generation
Further questions Mechanism of CSB targeting to genes CSA transcriptional analysis Effects in additional tissue types Understanding of specific genetic dysregulation important
in disease