1

Supplementary Material Supplementary Materials and Methods

Plasmid construction

The HA tagged full length LDB1 cDNA (375 aa) was cloned into the pYM-IRES-Neo vector and

various mutations and deletions of LDB1 were created (by site directed mutagenesis) in this

background. The shRNA target sequence located in the LID coding sequence of Ldb1 cDNA was

altered without changing the amino acid coding sequence of LDB1 protein. The shRNA target

sequence is underlined and mutated nucleotides are bolded and underlined:

Wild type: cgacgaggacagctttaacaa

Mutant: tgatgaagattcattcaataa

The dimerization domain of LDB1 (amino acids 1-200) was deleted and the K365R (AAA to

CGA) mutation at 3’ end of Ldb1 cDNA was introduced to stabilize the protein thus created.

Deletions in the LDB1 DD included sequences encoding amino acids 31 to 43 (Δ1), 58-62 (Δ2),

86 to 97 (Δ3) or 173 to 192 (Δ4/5). DD-only proteins were created by deletion of LDB1 C-

terminal sequences encoding amino acids 201-375. Further details of cloning are available upon

request. For the LMO2 fusion (2xLMO2), LMO2 cDNAs were tethered in the sense orientation

through a flexible linker (22-aa polypeptide [GT(GGGS)4GGGT]) (Wang et al., 2008).

Co-immunoprecipitation

Protein complexes were precipitated with anti-HA agarose (Sigma, A2095) overnight at 4oC.

Agarose was washed 3 times with IP100 buffer (Brand et al., 2004). Bound proteins were eluted

with HA peptides (Sigma) and analyzed by western blot. For LDB1/FOG1 experiments, the

protein complexes were precipitated overnight at 4oC by antibodies against LDB1 or FOG1 and

Dynabeads® Protein G (Life Technologies, 10004D), then washed once with IP500 and two

times with IP100 and eluted as described (Brand et al., 2004).

2

RNA-seq library construction, sequencing and computational analysis

RNA-seq libraries were constructed for induced MEL WT cells, LDB1 KD cells and LDB1 KD

cells expressing either LDB1 FL or LDB1Δ4/5 using TruSeq RNA Sample Prep Kit V2

(Illumina) according to the manufacturers protocol. Three biological replicates of each cell type

were sequenced on a HiSeq 2000. Illumina TruSeq adapters from the 51-bp single-end reads

were clipped using cutadapt v.1.1 with default parameters (Martin, 2011). Clipped reads were

then mapped to the UCSC mm9 assembly with TopHat v1.4.1 (Trapnell et al., 2009) using

default parameters with the additional parameter--butterfly-search. The number of uniquely

mapped reads for each library ranged from 13 to 55M, with a median of 24M. Reads mapping to

repetitive regions defined by the UCSC RepeatMasker track were removed from further analysis.

Reads were counted in exons of the Ensembl release 67 GTF for mm9, using HTSeq 0.5.3p9.

Differential expression of genes for all pairwise comparisons was assessed with DESeq v1.10.1

(Anders and Huber, 2010).

We defined genes repressed by LDB1 KD as those that, in the DESeq analysis, had an adjusted

P value < 0.05 and a log2 fold change < -1 in gene expression between LDB1 KD cells and WT.

To define rescued genes, we first used the variance-stabilized transformation (VST) in the

DESeq package to obtain comparable scaled counts for all experiments. VST-scaled counts for

replicates were averaged to obtain per-treatment values. Then, rescued genes were defined as

those LDB1 KD repressed genes with equal or greater expression in LDB1 KD cells expressing

LDB1 FL than WT, or, if genes have lower expression in LDB1 KD cells expressing LDB1 FL

than WT, whose expression difference between WT and LDB1 FL expressing cells was less than

half the expression difference between WT and LDB1 KD cells. More precisely, a gene was

defined as rescued if it was an LDB1 KD repressed gene and satisfied the condition:

((WT - LDB1 FL) < 0) OR ((WT - LDB1 FL) < (0.5 * (WT - LDB1 KD)) AND ((WT - LDB1

FL) > 0)), where "WT", "LDB1 FL", and "LDB1 KD" are the averaged, VST- scaled counts for

each treatment.

Next, of the genes that were rescued by LDB1 FL, we defined a gene to be 4/5-dependent if it

was repressed in LDB1 KD cells expressing LDB1Δ4/5 compared to LDB1 KD cells expressing

3

LDB1 FL (that is, log2 fold change < -1 and adjusted P value < 0.05). Otherwise, it was defined

as 4/5-independent. Since the transcribed sequence for LDB1 differs between LDB1 delta 4/5

and LDB1 FL, we removed LDB1 from consideration in the downstream RNA-seq rescue and

4/5-dependence analysis. Finally, genes were defined to be LDB1 bound if there was an LDB1

peak in induced MEL cells within 1kb of the start position of the gene, inside gene body or

within 1 kb of the end position of the gene ((Soler et al., 2010); BED file downloaded from the

PSU genome browser, http://main.genome-browser.bx.psu.edu). The OMIM database was

downloaded from a URL provided by omim.org (September 2013). A combination of Ensembl's

BioMart (mapping human HGNC to orthologous mouse Ensembl IDs) and the "mousecorr" field

in the OMIM database were used to identify human homologs of mouse genes. An OMIM entry

was considered orthologous in mouse if one of these methods successfully mapped the HGNC

human gene symbol to a mouse Ensembl gene.

Primers

ChIP primers Alas2 + 2kb Fw AGGGCAGGACTTTGCCTCTAATCTAlas2 + 2kb Rev AGATGTCCCAGTTCCTGCAGGTTTGypA Fw GTCCTCGCAGTTATGCAGAC GypA Rev GGCCTCTATCCGTTGACACA α-globin HS26 Fw TGACCATAGTCAACAGCAGGT α-globin HS26 Rev GCTCGTTCAAGCATCTCCAT A730036l17Rik Fw TCAAAACTCTGCCTCCTCCC A730036l17Rik Rev GATAGGTGAAAAGGCGCCAG Kctd14 Fw CTTAGAGTTCCTCAGGGGCG Kctd14 Rev GGGTACACAACGCTGTCTTG Ubash3a Fw TTTTCTTCACAGCCTCAGCC Ubash3a Rev CCATGTCTTCCTGCTTGCTG CD24a Fw AACAAAGGAAACTTGGGCCG CD24a Rev CTCTGGCACAGCTAGGGTTT Treml1 Fw TGTCAGCCTCGATGGATAGC Treml1 Rev GCTGGGCAGTAGTAGGTTCT Ypel3 Fw CAGCCCTTTCTCTCCTTCCA Ypel3 Rev TCCCAGCCGTTGTCTTTGAT

qRT-PCR primers α-globin Fw GCTGAAGCCCTGGAAAGGAT α-globin Rev GGCTTACATCAAAGTGAGGAAAGT Alas2 Fw CCATCTTAAGGCAACCAAGGC

4

Alas2 Rev ACAGCATGAAAGGACAATGGC GypA Fw ATTCATGTCTCAACTTATCACA GypA Rev CCAATGTGTGGTGAGACAGGCT A730036l17Rik Fw GCATTCAGGACTTGCTCTGG A730036l17Rik Rev GTCCTCACACTTGGCTTGTG Kctd14 Fw ATGCCACAGATCTTCGGTGA Kctd14 Rev AGGACCTCCAGGTTTTCTCTG Ubash3a Fw GCCAGTAAAGACGCTGACAC Ubash3a Rev ACCTCTTCCTGGAAAGCTCG CD24a Fw AGTAACGCTACCACCAGAGG CD24a Rev GTTTCCTGGCCTGAGTCTCT Treml1 Fw GATCCACCATCAAGCGAACC Treml1 Rev TCCTGGGAAGACAACTGTGG Ypel3 Fw CATGTGGCTTCAGCCTGG Ypel3 Rev GTACAGGAGCTGGGTCCTTC Neo Fw TCGACGTTGTCACTGAAGCG Neo Rev GGATACTTTCTCGGCAGGAGC Ldb1’UTR Fw GGGGTACTCATGTGGATGCCTGT Ldb1 3’UTR Rev ACCCAGAACCTGGGGTAAGAACG Hprt Fw TGACACTGGTAAAACAATGCAAACT Hprt Rev AACAAAGTCTGGCCTGTATCCAA

All other primers have been described (Song et al., 2007; Handoko et al., 2011).

Antibodies

Antibodies used for western blots were against HA (Roche, 11867423001), Tubulin (Sigma,

T5168), LDB1 (Santa Cruz Biotechnology, sc-11198), TAL1 (Santa Cruz Biotechnology, sc-

12984), FOG1 (Santa Cruz Biotechnology, sc-9361), ETO2 (Santa Cruz Biotechnology, sc-

9739), GATA1 (Santa Cruz Biotechnology, sc-265), LMO2 (R&D, AF2726) and appropriate

HRP-fused secondary antibodies from Santa Cruz Biotechnology or Goat TrueBlot®: Anti-Goat

IgG HRP (Rockland Immunochemicals, 18-8814-33).

Antibodies from Santa Cruz Biotechnology were used in ChIP for GATA1 (sc-1233), BRG1 (sc-

10768), Pol II (sc-899), FOG1 (Santa Cruz Biotechnology,sc-9361), MTA2 (sc-9474), HDAC1

(sc-7872), CBP (sc-369) and LAMB1 (sc-6217). Other antibodies were against AcH3 (Millipore,

06-599), H3 (Abcam, ab1791), and HA (Millipore, 05-904).

For immuno-staining antibody against LAMB1 (Santa Cruz Biotechnology, sc-6216) and

secondary anti-Goat DyLight 594 (Jackson ImmunoResearch, 805-515-180) were been used.

5

Supplementary References

Anders S, Huber W. 2010. Differential expression analysis for sequence count data. Genome. Biol. 11:

R106.

Brand M, Ranish JA, Kummer NT, Hamilton J, Igarashi K, Francastel C, Chi TH, Crabtree GR, Aebersold

R, Groudine M. 2004. Dynamic changes in transcription factor complexes during erythroid

differentiation revealed by quantitative proteomics. Nat. Struct. Mol Biol. 11: 73-80.

Handoko L, Xu H, Li G, Ngan CY, Chew E, Schnapp M, Lee CW, Ye C, Ping JL, Mulawadi F et al. 2011.

CTCF-mediated functional chromatin interactome in pluripotent cells. Nat Genet 43: 630-638.

Martin M. 2011. Cutadapt removes adaptor sequences from high-throughput sequencing reads.

EMBnet. journal 17: 10-12.

Soler E, Andrieu-Soler C, de Boer E., Bryne JC, Thongjuea S, Stadhouders R, Palstra RJ, Stevens M,

Kockx C, van IW et al. 2010. The genome-wide dynamics of the binding of Ldb1 complexes during

erythroid differentiation. Genes Dev 24: 277-289.

Song S-H, Hou C, Dean A. 2007. A positive role for NLI/Ldb1 in long-range -globin locus control

region function. Mol. Cell 28: 810-822.

Trapnell C, Pachter L, Salzberg SL. 2009. TopHat: discovering splice junctions with RNA-Seq.

Bioinformatics. 25: 1105-1111.

Wang J, Levasseur DN, Orkin SH. 2008. Requirement of Nanog dimerization for stem cell self-renewal

and pluripotency. Proc. Natl. Acad. Sci U. S. A. 105: 6326-6331.

Supplementary Figure Legends

Figure S1. LDB1 FL fully rescues β-globin expression in induced LDB1 KD MEL cells. (A)

Diagram of LDB1 FL cDNA expressed in induced LDB1 KD MEL cells. Colored rectangles:

purple, HA tag; green, DD domain; orange, nuclear localization signal (NLS); yellow, LID. (B)

Western blot of protein extracts from three induced LDB1 FL-expressing LDB1 KD MEL cell

lines with LDB1 or HA antibodies. α-tubulin served as loading control. (C) β-globin and

6

endogenous LDB1 gene expression in three induced LDB1 FL-expressing LDB1 KD MEL cell

lines. Induced cells containing an empty vector (Empty) served as a control. Expression in

induced WT MEL cells was set to 1. Error bars indicate SEM, N=3 biological replicates.

Figure S2. K365R mutation stabilizes LDB1ΔDD. (A) Western blotting of protein extracts

from LDB1 KD MEL cells expressing LDB1 FL and LDB1ΔDD with antibodies against HA. α-

tubulin served as loading control. (B) Expression level of transgenic RNA measured by real-time

PCR with primers to neo-resistant gene. Expression level in LDB1 FL was set to 1. Error bars

indicate SEM, N=3. (C) Western blotting of protein extracts from LDB1 KD MEL cells

expressing LDB1ΔDD incubated at 30o C overnight or treated with 26S proteasome inhibitor

MG132 with antibodies against HA. α-tubulin served as loading control. (D) Western blotting of

protein extracts from LDB1 KD MEL cells expressing LDB1 FL, LDB1 FL K365R, LDB1ΔDD

and LDB1ΔDD K365R with antibodies against HA. α-tubulin served as loading control.

Figure S3. Heterologous dimerizing or physical tethering of LMO2 fails to rescue activation

of β-globin gene expression. (A) Expression level of the β-globin gene in induced MEL LDB1

KD cell lines expressing LDB1 FL, LMO-Lex, 2xLMO2 or GATA1-DD. (B) Locus-wide

crosslinking frequencies analyzed by 3C-qPCR using the LCR HS2 as the anchor fragment (red

vertical bar). Relative crosslinking frequencies observed in induced LDB1 KD MEL cell lines

expressing LDB1 FL, LMO-DD, LMO-Lex, 2xLMO2 or GATA1-DD or containing an empty

expression vector are shown. The X-axis shows the genomic coordinates of the interacting

fragments and globin gene locations in the locus. Yellow triangles mark BglII restriction sites.

Error bars in both panels indicate SEM, N=3 biological replicates.

Figure S4. Conserved and structural elements in DD domain of LDB1. Alignment of amino

acid sequences of LDB1 homologs from M. musculus, D.melanogaster and C.elegans. Yellow

color marks coincidence in all sequences, blue – in two, green - conserved substitutions.

Cylinders mark potential helical regions.

Figure S5. Inducible deletion of endogenous LDB1 in E14.5 fetal liver cells. (A)

Experimental strategy of endogenous Ldb1 deletion and overexpression of transgenic versions of

7

LDB1. (B) Ldb1 deletion in E14.5 Ldbfl/fl fetal liver cells with and without Mx1CreDNA was

analyzed by PCR of genomic DNA for the presence of the Mx1Cre, Ldb1 deleted allele (Ldb1Δ)

and floxed allele (Ldb1fl) before and after IFN-β treatment. (C) Expression level of Ldb1 and β-

globin genes in Mx1Cre Ldb1fl/fl and control Ldb1fl/fl E14.5 fetal liver cells after 3 days of IFN-β

treatment. Expression level in control cells was set to 1. Error bars indicate SEM, N=3 biological

replicates.

Figure S6. DD4/5 region is not required for LDB1 dimerization. (A) Diagram of cDNAs of

DD and deleted versions without the LDB1 C-terminal LID that were expressed in wild type

MEL cells. Designations are the same as Figure 2A. (B) Western blots of protein extracts from

representative wild type MEL cells expressing the indicated proteins. α-tubulin served as loading

control. (C) Immunoprecipitation (IP) was performed with an antibody to the HA tag using

nuclear extracts from induced WT MEL cells expressing the DD (left panel). IP material was

analyzed by western blot with antibodies to LDB1, LMO2 and TAL1. The DD interacted with

endogenous LDB1 and through this interaction with LDB1 complex members LMO2 and TAL1.

Antibodies to the HA tag and ETO2 served as positive and negative controls. IP and western

blotting for cells expressing DDΔ1 or DDΔ4/5 were performed in the same way (right panels).

(D) β-globin expression in induced MEL cells expressing DD, DDΔ1 or DDΔ4/5 compared to

induced WT MEL cells (set to 1). Error bars indicate SEM, N=3 biological replicates.

Figure S7. Association of LAMB1 with the β-globin locus. ChIP was performed with a

LAMB1 antibody and chromatin from uninduced LDB1 KD MEL cells expressing LDB1 FL and

induced LDB1 KD MEL cells expressing LDB1 FL, LDB1Δ4/5 or LMO-DD. LAD served as

positive control and Trem151a served as a negative control (Handoko et al., 2011). Error bars

indicate SEM, N=3 biological replicates. Values are compared to the value for uninduced LDB1

KD cells expressing LDB1 FL and * indicates P<0.05 by Student’s t-test.

Figure S8. Failure to recruit BRG1 affects DNase I sensitivity. Nuclei from induced LDB1

KD MEL cells expressing LDB1 FL, LDB1Δ4/5, LMO-DD or containing an empty expression

vector were digested with increasing amounts of DNase I (from left to right: 15, 30, 45 U).

DNase I sensitivity was normalized by signal from coding region of actB gene. Values are

8

compared to the value for LDB1 FL and * indicates P<0.05 by Student’s t-test. (A) HS2 region

DNase I sensitivity. (B) β-globin promoter DNAseI sensitivity. Error bars in both panels indicate

SEM, N=3 biological replicates.

Figure S9. RNA-seq analysis of genes directly regulated by LDB1 in induced MEL cells.

(A) Strategy to identify genes that are directly regulated by LDB1 and sensitive to the presence

of the 4/5 region in the DD of LDB1. Bar plots show representative expression profiles of genes

in each class (see Supplementary Materials and Methods for details). The solid horizontal line in

the second panel indicates one-half the expression difference between WT and Ldb1 KD. (B)

Effect of knocking down Ldb1 on the 94 LDB1-occupied 4/5 dependent genes and 148 LDB1-

occupied 4/5 independent genes. Genes are ranked by log2 fold change in Ldb1 KD vs WT, and

erythroid genes are highlighted in red.

Figure S10. UCSC genome browser screenshots of selected LDB1-dependent genes. RNA-

seq signal (top) and induced MEL cells ChIP-seq signal and peaks (bottom) over representative

genes that are 4/5-independent (A), 4/5-dependent (B) and 4/5-dependent with LDB1 only peak

(C). Each RNA-seq track represents combined coverage for all replicates, divided by the number

of million reads in the merged library (tags per million, TPM). Induced MEL cells ChIP-seq data

(Soler et al., 2010), were downloaded from the PSU genome browser ( http://main.genome-

browser.bx.psu.edu). As described at http://main.genome-browser.bx.psu.edu/cgi-

bin/hgTrackUi?g=grosveldTfbs&db=mm9, ChIP-seq signal is in read depth, and peaks were

called with MACS. Bottom point equals zero.

Figure S11. OMIM database analysis. (A) Strategy to identify disease-associated human

homologs of mouse LDB1-activated and occupied genes. (B) Enrichment of disease-associated

genes among LDB1 4/5-dependent, LDB1 4/5-independent and all other human genes with

mouse homologues. The number of genes in each group was compared with the group of all

human genes using a Fisher’s exact test and the P values are shown. (C) Blood related diseases

among LDB1 4/5-dependent and LB1 4/5-independent disease-associated genes.

ALDB1 FL

Krivega_FigS1

αLDB1αHAαTub

HA-LDB1 FL

DD NLS LID

B

1

1.2

1.4β-globin Endogenous Ldb1C

αTubon

0

0.2

0.4

0.6

0.8

LDB1 FL

Exp

ress

io

LDB1 FL

1.5

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A B

Krivega_FigS2

αHA

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1

Relative mRNA leve

Transgenic mRNA level

C D

αHA

αTub

αHA

αTub

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Krivega_FigS3

A1.4

0 4

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0.8

1

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0

0.2

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‐glo

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slinkingfreq

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C.elegansD.melanogaster

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1521 genesrepressed by LDB1 KD

496 genesrescued by LDB1 FL

WT

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349 genes4/5-independent

148 genes occupied by LDB1

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147 genes4/5-dependent

93 genes occupied by LDB1

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4/5-dependentdisease related genes

4/5-independentdisease related genes

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blood related diseases

others