Supporting InformationXu et al. 10.1073/pnas.1303976110SI Materials and MethodsStreptavidin Immunoprecipitation and Western Blot. Streptavidinimmunoprecipitation (SA-IP) experiments in MEL or K562 stablecell lines were performed as described previously (1). Briefly,nuclear extracts were prepared from MEL-BirA (MBirA), MEL-FLAG-Bio-BCL11A (MBB1.4), K562-BirA (KBirA), and K562-FLAG-Bio-BCL11A (KBB2.4) stable cell lines, immunoprecipitatedwith streptavidin agarose beads (Invitrogen), and processed forWestern blot analysis. The following antibodies were used forcoimmunoprecipitation (co-IP) andWestern blot analysis: BCL11A(ab18688; Abcam), GATA1 (ab28839; Abcam), FOG1 (sc-9362;Santa Cruz Biotechnology), lysine-specific demethylase 1 (LSD1;ab17721; Abcam), CoREST (07-455; Millipore), NCOR1 (ab24552;Abcam), SMRT (04-1551; Millipore), KAISO (ab12723; Abcam),SIN3A (sc-994; Santa Cruz Biotechnology), TRIM28 (ab22553;Abcam), Mi-2β (provided by Stephen Smale, University ofCalifornia, Las Angeles, CA), MTA2 (sc-9447; Santa Cruz Bio-technology), HDAC1 (06-720; Millipore), HDAC2 (sc-7899; SantaCruz Biotechnology), MBD3 (sc-9402; Santa Cruz Biotechnology),SNF5, BRG1, BAF155 (provided by Charles Roberts, Dana-FarberCancer Institute, Boston), EZH2 (612666; BC Biosciences), SUZ12(ab12073; Abcam), SP1 (sc-14027; Santa Cruz Biotechnology),DNA methyltransferase 1 (DNMT1; 39204; Active Motif), andGAPDH (sc-26778; Santa Cruz Biotechnology).

Co-IP and Western Blot.Co-IP experiments of endogenous BCL11Aprotein in primary human erythroid cells were performed asdescribed (2). Briefly, 1–5 mg of nuclear extract proteins was

incubated with 5–25 μg of BCL11A or mouse IgG antibody over-night at 4 °C. The protein complexes were collected by protein G/A-agarose beads (Invitrogen), followed by four washes withBC139K buffer. The beads were boiled for 5 min in sample buffer(Bio-Rad), and the eluted material was used for Western blotanalysis.

Flow Cytometry.Cells were analyzed by flow cytometry as describedpreviously (3). Live cells were identified and gated by exclusion of7-amino-actinomycin D (7-AAD; BD Pharmingen). The cells wereanalyzed for expression of cell surface antigens with antibodiesspecific for CD34, CD71, and CD235a conjugated to phycoerythrin(PE), fluorescein isothiocyanate (FITC), or allophycocyanin (APC;BD Pharmingen). Data were analyzed using FlowJo software.

Cytospin. Cytocentrifuge preparations from cells at various stagesof differentiation were stained with May-Grunwald-Giemsa asdescribed previously (4).

Chemicals. Two LSD1 inhibitors, pargyline (Cayman Chemical)and tranylcypromine (TCP; Sigma-Aldrich), and three DNAmethylation inhibitors, 5-aza-2′-deoxycytidine (5-azaD; Sigma-Aldrich), Zebularine (Tocris Bioscience), and RG108 (TocrisBioscience), were used to treat primary human CD34+ hema-topoietic stem/progenitor cell–derived erythroid progenitors.Cells were incubated with inhibitors for 72 h before harvest foranalysis.

1. Xu J, et al. (2012) Combinatorial assembly of developmental stage-specific enhancerscontrols gene expression programs during human erythropoiesis. Dev Cell 23(4):796–811.

2. Xu J, et al. (2010) Transcriptional silencing of gamma-globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev 24(8):783–798.

3. Xu J, et al. (2011) Correction of sickle cell disease in adult mice by interference withfetal hemoglobin silencing. Science 334(6058):993–996.

4. Sankaran VG, et al. (2009) Developmental and species-divergent globin switching aredriven by BCL11A. Nature 460(7259):1093–1097.

Xu et al. www.pnas.org/cgi/content/short/1303976110 1 of 11

MB

irAM

BB

1.4

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irAM

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WBα-BCL11A

α-GATA1

α-FOG1

α-LSD1

α-CoREST

α-SMRT

α-KAISO

α-SIN3A

α-TRIM28

α-Mi-2β

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KB

irAK

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WB

α-BCL11A

α-GATA1

α-LSD1

α-CoREST

α-SMRT

α-KAISO

α-SIN3A

α-TRIM28

α-Mi-2β

α-MTA2

α-HDAC1

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α-MBD3

α-SNF5

α-BRG1

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LSD

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oRE

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Rep

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α-SP1 α-SP1

BA

Fig. S1. Validation of interactions between BCL11A and identified partner proteins in erythroid cells. (A) SA-IP experiments were performed in MEL-BirA(MBirA) and MEL-FLAG-Bio-BCL11A (MBB1.4) stable cells. BCL11A-interacting protein complexes were purified by streptavidin immunoprecipitation followedby Western blot analysis; 2% of input nuclear extracts were analyzed as loading controls. (B) SA-IP experiments were performed in K562-BirA (MBirA) andK562-FLAG-Bio-BCL11A (KBB2.4) stable cells, followed by Western blot analysis.

Xu et al. www.pnas.org/cgi/content/short/1303976110 2 of 11

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SO

X6

β-gl

obin

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ativ

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GA

PD

H)

Fig. S2. Functional RNAi screen of BCL11A-interacting partner proteins. Expression of human β-like globin mRNAs was measured by qRT-PCR on shRNA-mediated knockdown of each gene. Data are plotted as relative mRNA level normalized to GAPDH mRNA level for human (A) fetal γ-globin, (B) embryonice-globin, and (C) adult β-globin genes, respectively. Results are the means ± SD of at least three independent experiments. The same data are plotted aspercentage of each globin gene over total β-like human globin gene levels and shown in Fig. 2.

Xu et al. www.pnas.org/cgi/content/short/1303976110 3 of 11

A

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E F

Fig. S3. Representative genes in functional RNAi screen in primary human adult eryrhoid cells. (A) Lentiviral shRNA-mediated knockdown of CHD4 (encodingMi2β) reactivates human γ-globin expression. Expression of Mi2βwas monitored by Western blot analysis (Upper). Expression of human γ-globin mRNA (as % oftotal β-like globin mRNAs) and total β-like globin mRNA (relative mRNA value normalized to GAPDH mRNA) was measured by qRT-PCR (Lower). Results are themeans ± SD of at least three independent experiments. (B) Erythroid maturation was assessed by flow cytometry analysis of cell surface marker (CD71 andCD235a) expression in primary human CD34+ HSPC-derived erythroid progenitor cells at day 5 of differentiation. (C) Lentiviral shRNA-mediated knockdown ofNCOR1 reactivates human γ-globin expression. (D) Erythroid maturation was assessed by flow cytometry analysis of cell surface marker expression onknockdown of NCOR1 expression. (E) Lentiviral shRNA-mediated knockdown of SIN3A reactivates human γ-globin expression. (F) Erythroid maturation wasassessed by flow cytometry analysis of cell surface marker expression on knockdown of SIN3A expression.

Xu et al. www.pnas.org/cgi/content/short/1303976110 4 of 11

A

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∗

Bcl11a::Lsd1 cKO (by EpoR-Cre)

Bcl11a::Lsd1 cKO (by Mx1-Cre)

Fig. S4. Differential peripheral blood analysis of LSD1 and BCL11A compound KO mice. (A) Differential peripheral blood (PB) counts in control (EpoR-Cre−),Bcl11a KO, and Bcl11a::Lsd1 compound KO (by EpoR-Cre) β-YAC mice. Results are shown as means ± SEM (n ≥ 5 per genotype; *P < 0.05). (B) Differential PBcounts in control (Mx1-Cre−), Bcl11a KO, and Bcl11a::Lsd1 compound KO (by Mx1-Cre) β-YAC mice (n ≥ 5 per genotype; *P < 0.05, **P < 0.01).

Xu et al. www.pnas.org/cgi/content/short/1303976110 5 of 11

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shGFP shBCL11A LSD1 sh1 LSD1 sh5A

shGFP shBCL11A LSD1 sh1 LSD1 sh5

Day

5D

ay 9

B

Fig. S5. Depletion of LSD1 expression in primary human CD34+ HSPCs leads to impaired erythroid differentiation. (A) Erythroid maturation was assessed byflow cytometry analysis of cell surface marker (CD34, CD71, and CD235a) expression in primary human CD34+ HSPC-derived erythroid progenitor cells at day 5of differentiation. Cells were transduced with lentiviruses containing shRNAs again GFP (shGFP, negative control), BCL11A (shBCL11A), and LSD1 (sh1 and sh5),respectively. (B) Representative cytospin images are shown for cells at days 5 and 9 of differentiation. Under normal culture conditions, the majority of thedifferentiating erythroid progenitors acquired proerythroblast morphology at day 5 of differentiation. At day 9 of differentiation, the majority of cells werepolychromatophilic and orthochromatic. Depletion of BCL11A expression by lentiviral shRNA had no effect on erythroid maturation. Depletion of LSD1 re-sulted in marked decrease in total cell number and polychromatophilic/orthochromatic erythroid progenitors.

Xu et al. www.pnas.org/cgi/content/short/1303976110 6 of 11

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∗∗ ∗∗ ∗∗

∗ ∗∗∗

∗

Control shBCL11A

∗∗

∗∗∗∗ ∗∗ ∗∗ ∗∗

A

Fig. S6. Inhibition of LSD1 activity leads to increased γ-globin expression and impaired erythroid differentiation in primary human erythroid cells. (A) Ex-pression of human γ-globin mRNA was measured by qRT-PCR in primary human erythroid progenitor cells treated with two LSD1 inhibitors: pargyline and TCP.Data are shown as means ± SD; *P < 0.05. (B) Inhibition of LSD1 results in decrease in total mRNA level of β-like globin genes (Lower) in primary humanerythroid progenitor cells. Data are shown as means ± SD; *P < 0.05, **P < 0.01. (C) Erythroid maturation was assessed by flow cytometry analysis of cell surfacemarker (CD71 and CD235a) expression in primary human erythroid progenitor cells at day 5 of differentiation in the presence or absence of pargyline. (D)Erythroid maturation was assessed by flow cytometry analysis of cell surface marker (CD71 and CD235a) expression in primary human erythroid progenitor cellsat day 5 of differentiation in the presence or absence of TCP.

Xu et al. www.pnas.org/cgi/content/short/1303976110 7 of 11

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∗∗ ∗

Fig. S7. Inhibition of DNMT1 activity leads to increased γ-globin expression in primary human erythroid cells. (A) Expression of human γ-globin mRNA wasmeasured by qRT-PCR in primary human erythroid progenitor cells treated with three DNAmethylation inhibitors in the presence or absence of BCL11A shRNA.(B) Expression of total human β-like globin mRNA was measured by qRT-PCR in primary human erythroid progenitor cells treated with DNA methylationinhibitors in the presence or absence of BCL11A shRNA. Data are shown as means ± SD; *P < 0.05.

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Bcl11a::Dnmt1 cKO (by EpoR-Cre)

Fig. S8. Differential peripheral blood analysis of DNMT1 and BCL11A compound KO mice. Differential PB counts in control (EpoR-Cre−), Bcl11a KO, andBcl11a::Dnmt1 compound KO (by EpoR-Cre) β-YAC mice (n ≥ 5 per genotype; *P < 0.05).

Xu et al. www.pnas.org/cgi/content/short/1303976110 8 of 11

Table S1. List of lentiviral shRNA sequences used in this study

Gene name shRNA ID TRC number shRNA sequences

GFP shGFP SHC005 CCGGTACAACAGCCACAACGTCTATCTCGAGATAGACGTTGTGGCTGTTGTATTTTTBCL11A sh49 TRCN0000033449 CCGGCGCACAGAACACTCATGGATTCTCGAGAATCCATGAGTGTTCTGTGCGTTTTTG

sh51 TRCN0000033451 CCGGCCAGAGGATGACGATTGTTTACTCGAGTAAACAATCGTCATCCTCTGGTTTTTGsh53 TRCN0000033453 CCGGGCATAGACGATGGCACTGTTACTCGAGTAACAGTGCCATCGTCTATGCTTTTTG

KLF1 sh1 TRCN0000230814 CCGGTGCACATGAAGCGCCACCTTTCTCGAGAAAGGTGGCGCTTCATGTGCATTTTTGsh4 TRCN0000230812 CCGGCCCTCCTTCCTGAGTTGTTTGCTCGAGCAAACAACTCAGGAAGGAGGGTTTTTGsh5 TRCN0000230813 CCGGCAGAGGATCCAGGTGTGATAGCTCGAGCTATCACACCTGGATCCTCTGTTTTTG

CHD4 sh4 TRCN0000021362 CCGGGCTGACACAGTTATTATCTATCTCGAGATAGATAATAACTGTGTCAGCTTTTTsh5 TRCN0000021363 CCGGGCGGGAGTTCAGTACCAATAACTCGAGTTATTGGTACTGAACTCCCGCTTTTT

DNMT1 sh2 TRCN0000021891 CCGGGCCCAATGAGACTGACATCAACTCGAGTTGATGTCAGTCTCATTGGGCTTTTTsh3 TRCN0000021893 CCGGCGACTACATCAAAGGCAGCAACTCGAGTTGCTGCCTTTGATGTAGTCGTTTTTsh5 TRCN0000021892 CCGGCGAGAAGAATATCGAACTCTTCTCGAGAAGAGTTCGATATTCTTCTCGTTTTT

SIN3A sh1 TRCN0000021774 CCGGCGTGAACATCTAGCACAGAAACTCGAGTTTCTGTGCTAGATGTTCACGTTTTTsh2 TRCN0000021775 CCGGCCCTGAGTTGTTTAATTGGTTCTCGAGAACCAATTAAACAACTCAGGGTTTTTsh3 TRCN0000021776 CCGGGCTACGTCTCAAAGAACCTATCTCGAGATAGGTTCTTTGAGACGTAGCTTTTT

HDAC1 sh2 TRCN0000004814 CCGGCGTTCTTAACTTTGAACCATACTCGAGTATGGTTCAAAGTTAAGAACGTTTTTsh3 TRCN0000004816 CCGGGCCGGTCATGTCCAAAGTAATCTCGAGATTACTTTGGACATGACCGGCTTTTTsh5 TRCN0000004818 CCGGGCTGCTCAACTATGGTCTCTACTCGAGTAGAGACCATAGTTGAGCAGCTTTTT

NCOR1 sh3 TRCN0000060655 CCGGCGCAGTATTGTCCAAATTATTCTCGAGAATAATTTGGACAATACTGCGTTTTTGsh4 TRCN0000060656 CCGGGCCATCAAACACAATGTCAAACTCGAGTTTGACATTGTGTTTGATGGCTTTTTGsh5 TRCN0000060657 CCGGGCTCTCAAAGTTCAGACTCTTCTCGAGAAGAGTCTGAACTTTGAGAGCTTTTTG

EZH2 sh1 TRCN0000286227 CCGGTATTGCCTTCTCACCAGCTGCCTCGAGGCAGCTGGTGAGAAGGCAATATTTTTGsh2 TRCN0000040074 CCGGGCTAGGTTAATTGGGACCAAACTCGAGTTTGGTCCCAATTAACCTAGCTTTTTGsh4 TRCN0000286290 CCGGCGGAAATCTTAAACCAAGAATCTCGAGATTCTTGGTTTAAGATTTCCGTTTTTG

HDAC2 sh1 TRCN0000004822 CCGGGCAGACTCATTATCTGGTGATCTCGAGATCACCAGATAATGAGTCTGCTTTTTsh2 TRCN0000004823 CCGGGCAAATACTATGCTGTCAATTCTCGAGAATTGACAGCATAGTATTTGCTTTTTsh3 TRCN0000004819 CCGGCAGTCTCACCAATTTCAGAAACTCGAGTTTCTGAAATTGGTGAGACTGTTTTTsh4 TRCN0000004820 CCGGCCAGCGTTTGATGGACTCTTTCTCGAGAAAGAGTCCATCAAACGCTGGTTTTT

MBD2 sh1 TRCN0000013319 CCGGGCCTAGTAAATTACAGAAGAACTCGAGTTCTTCTGTAATTTACTAGGCTTTTTsh2 TRCN0000013320 CCGGGTAGCAATGATGAGACCCTTTCTCGAGAAAGGGTCTCATCATTGCTACTTTTTsh3 TRCN0000013321 CCGGGTACGCAAGAAATTGGAAGAACTCGAGTTCTTCCAATTTCTTGCGTACTTTTTsh5 TRCN0000013318 CCGGGCTTAATGAAAGGGTTTGTAACTCGAGTTACAAACCCTTTCATTAAGCTTTTT

EED sh1 TRCN0000021204 CCGGGCAAACTTTATGTTTGGGATTCTCGAGAATCCCAAACATAAAGTTTGCTTTTTsh2 TRCN0000021205 CCGGCCAGAGACATACATAGGAATTCTCGAGAATTCCTATGTATGTCTCTGGTTTTTsh3 TRCN0000021206 CCGGGCAGCATTCTTATAGCTGTTTCTCGAGAAACAGCTATAAGAATGCTGCTTTTTsh4 TRCN0000021207 CCGGCCTATAACAATGCAGTGTATACTCGAGTATACACTGCATTGTTATAGGTTTTTsh5 TRCN0000021208 CCGGCCAGTGAATCTAATGTGACTACTCGAGTAGTCACATTAGATTCACTGGTTTTT

KDM3A sh1 TRCN0000021149 CCGGCCCAAGATGTATAATGCTTATCTCGAGATAAGCATTATACATCTTGGGTTTTTsh2 TRCN0000021150 CCGGCCCTAATAACTGTTCAGGAAACTCGAGTTTCCTGAACAGTTATTAGGGTTTTTsh3 TRCN0000021151 CCGGGCTGGTATTTAGACCGATCATCTCGAGATGATCGGTCTAAATACCAGCTTTTTsh4 TRCN0000021152 CCGGGCTTTGATTGTGAAGCATTTACTCGAGTAAATGCTTCACAATCAAAGCTTTTTsh5 TRCN0000021153 CCGGCCATACGTTTAACAGCACAATCTCGAGATTGTGCTGTTAAACGTATGGTTTTT

SUZ12 sh2 TRCN0000038725 CCGGGCTTACGTTTACTGGTTTCTTCTCGAGAAGAAACCAGTAAACGTAAGCTTTTTGsh3 TRCN0000038726 CCGGCCAAACCTCTTGCCACTAGAACTCGAGTTCTAGTGGCAAGAGGTTTGGTTTTTGsh4 TRCN0000038727 CCGGCGGAATCTCATAGCACCAATACTCGAGTATTGGTGCTATGAGATTCCGTTTTTGsh5 TRCN0000038728 CCGGGCTGACAATCAAATGAATCATCTCGAGATGATTCATTTGATTGTCAGCTTTTTG

KDM5B sh1 TRCN0000014759 CCGGGCTCCCTTACTTTAGATGATACTCGAGTATCATCTAAAGTAAGGGAGCTTTTTsh2 TRCN0000014760 CCGGCCTCTCCAAGATGTGGATATACTCGAGTATATCCACATCTTGGAGAGGTTTTTsh3 TRCN0000014761 CCGGCCTGAGGAAGAGGAGTATCTTCTCGAGAAGATACTCCTCTTCCTCAGGTTTTTsh4 TRCN0000014762 CCGGCGAGATGGAATTAACAGTCTTCTCGAGAAGACTGTTAATTCCATCTCGTTTTTsh5 TRCN0000014758 CCGGCCCACCAATTTGGAAGGCATTCTCGAGAATGCCTTCCAAATTGGTGGGTTTTT

RBBP4 sh2 TRCN0000115869 CCGGCCCTTGTATCATCGCAACAAACTCGAGTTTGTTGCGATGATACAAGGGTTTTTGsh3 TRCN0000115870 CCGGGCCTTTCTTTCAATCCTTATACTCGAGTATAAGGATTGAAAGAAAGGCTTTTTGsh4 TRCN0000115868 CCGGCGGCAGTAGTAGAAGATGTTTCTCGAGAAACATCTTCTACTACTGCCGTTTTTGsh5 TRCN0000115871 CCGGGCAGACTGAATGTCTGGGATTCTCGAGAATCCCAGACATTCAGTCTGCTTTTTG

KDM5A sh1 TRCN0000014628 CCGGCCAGGTACTTAATGCCCTAAACTCGAGTTTAGGGCATTAAGTACCTGGTTTTTsh2 TRCN0000014629 CCGGCCAGACTTACAGGGACACTTACTCGAGTAAGTGTCCCTGTAAGTCTGGTTTTTsh3 TRCN0000014630 CCGGCGGACCGACATTGGTGTATATCTCGAGATATACACCAATGTCGGTCCGTTTTTsh4 TRCN0000014631 CCGGCCCATGCAGAAGAAATGTCTTCTCGAGAAGACATTTCTTCTGCATGGGTTTTTsh5 TRCN0000014632 CCGGCCTTGAAAGAAGCCTTACAAACTCGAGTTTGTAAGGCTTCTTTCAAGGTTTTT

SOX6 sh1 TRCN0000017990 CCGGCCAGTGAACTTCTTGGAGAAACTCGAGTTTCTCCAAGAAGTTCACTGGTTTTTsh2 TRCN0000017988 CCGGCCAACACTTGTCAGTACCATTCTCGAGAATGGTACTGACAAGTGTTGGTTTTTsh3 TRCN0000017989 CCGGGCCACACATTAAGCGACCAATCTCGAGATTGGTCGCTTAATGTGTGGCTTTTT

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Table S1. Cont.

Gene name shRNA ID TRC number shRNA sequences

EZH1 sh1 TRCN0000002439 CCGGGCTACTCGGAAAGGAAACAAACTCGAGTTTGTTTCCTTTCCGAGTAGCTTTTTsh2 TRCN0000002440 CCGGGCTCTTCTTTGATTACAGGTACTCGAGTACCTGTAATCAAAGAAGAGCTTTTTsh3 TRCN0000002441 CCGGCCGCCGTGGTTTGTATTCATTCTCGAGAATGAATACAAACCACGGCGGTTTTTsh4 TRCN0000002442 CCGGCAACAGAACTTTATGGTAGAACTCGAGTTCTACCATAAAGTTCTGTTGTTTTTsh5 TRCN0000010708 CCGGGCTTCCTCTTCAACCTCAATACTCGAGTATTGAGGTTGAAGAGGAAGCTTTTT

IKZF1 sh2 TRCN0000107871 CCGGGCGGAGGATTTACGAATGCTTCTCGAGAAGCATTCGTAAATCCTCCGCTTTTTGsh3 TRCN0000107872 CCGGCCGTTGGTAAACCTCACAAATCTCGAGATTTGTGAGGTTTACCAACGGTTTTTGsh4 TRCN0000107873 CCGGGCCGAAGCTATAAACAGCGAACTCGAGTTCGCTGTTTATAGCTTCGGCTTTTTGsh5 TRCN0000107874 CCGGCGCCAAACGTAAGAGCTCTATCTCGAGATAGAGCTCTTACGTTTGGCGTTTTTG

KDM5D sh1 TRCN0000022114 CCGGCGATCACATTACGAACGCATTCTCGAGAATGCGTTCGTAATGTGATCGTTTTTsh2 TRCN0000022115 CCGGGCCACATTGGAAGCCATAATTCTCGAGAATTATGGCTTCCAATGTGGCTTTTTsh3 TRCN0000022116 CCGGCCAGTGCTAGATCAGTCTGTTCTCGAGAACAGACTGATCTAGCACTGGTTTTTsh4 TRCN0000022117 CCGGCGCGTCCAAAGGCTAAATGAACTCGAGTTCATTTAGCCTTTGGACGCGTTTTTsh5 TRCN0000022118 CCGGCAGCCCTTTCTTGAAAGGAAACTCGAGTTTCCTTTCAAGAAAGGGCTGTTTTT

KDM4B sh1 TRCN0000018014 CCGGGCCCATCATCCTGAAGAAGTACTCGAGTACTTCTTCAGGATGATGGGCTTTTTsh2 TRCN0000018013 CCGGCCGGCCACATTACCCTCCAAACTCGAGTTTGGAGGGTAATGTGGCCGGTTTTTsh3 TRCN0000018015 CCGGGCGGCATAAGATGACCCTCATCTCGAGATGAGGGTCATCTTATGCCGCTTTTTsh4 TRCN0000018016 CCGGGTGGAAGCTGAAATGCGTGTACTCGAGTACACGCATTTCAGCTTCCACTTTTT

KDM7A sh1 TRCN0000253856 CCGGTTAGACCTGGACACCTTATTACTCGAGTAATAAGGTGTCCAGGTCTAATTTTTGsh2 TRCN0000253855 CCGGTATGGGATCAACAGGTATTTACTCGAGTAAATACCTGTTGATCCCATATTTTTGsh3 TRCN0000253854 CCGGTGGATTTGATGTCCCTATTATCTCGAGATAATAGGGACATCAAATCCATTTTTGsh4 TRCN0000253852 CCGGGCAGTTGTATCGCTATGATAACTCGAGTTATCATAGCGATACAACTGCTTTTTGsh5 TRCN0000253853 CCGGAGGCTCCCTTCACCTACATTTCTCGAGAAATGTAGGTGAAGGGAGCCTTTTTTG

RBBP7 sh1 TRCN0000038885 CCGGCCTCCAGAACTCCTGTTTATTCTCGAGAATAAACAGGAGTTCTGGAGGTTTTTGsh2 TRCN0000038886 CCGGCGTGTCATCAATGAAGAATATCTCGAGATATTCTTCATTGATGACACGTTTTTGsh3 TRCN0000038887 CCGGGCACAGTTTGATGCTTCCCATCTCGAGATGGGAAGCATCAAACTGTGCTTTTTGsh5 TRCN0000038884 CCGGCGTTTCTATATGACCTGGTTACTCGAGTAACCAGGTCATATAGAAACGTTTTTG

KDM4C sh1 TRCN0000022054 CCGGGCCCAAGTCTTGGTATGCTATCTCGAGATAGCATACCAAGACTTGGGCTTTTTsh2 TRCN0000022055 CCGGCCTTGCATACATGGAGTCTAACTCGAGTTAGACTCCATGTATGCAAGGTTTTTsh3 TRCN0000022056 CCGGGCCTCTGACATGCGATTTGAACTCGAGTTCAAATCGCATGTCAGAGGCTTTTTsh4 TRCN0000022057 CCGGGCACCTATCTATGGTGCAGATCTCGAGATCTGCACCATAGATAGGTGCTTTTT

BAF155 sh1 TRCN0000015628 CCGGGCAGGATATTAGCTCCTTATACTCGAGTATAAGGAGCTAATATCCTGCTTTTTsh2 TRCN0000015629 CCGGCCCACCACATTTACCCATATTCTCGAGAATATGGGTAAATGTGGTGGGTTTTTsh3 TRCN0000015630 CCGGGCTATGATACTTGGGTCCATACTCGAGTATGGACCCAAGTATCATAGCTTTTTsh5 TRCN0000015632 CCGGCCTAGCTGTTTATCGACGGAACTCGAGTTCCGTCGATAAACAGCTAGGTTTTT

LSD1 (KDM1A) sh1 TRCN0000046068 CCGGGCCTAGACATTAAACTGAATACTCGAGTATTCAGTTTAATGTCTAGGCTTTTTGsh5 TRCN0000046072 CCGGCCACGAGTCAAACCTTTATTTCTCGAGAAATAAAGGTTTGACTCGTGGTTTTTG

ZBTB33 sh1 TRCN0000017838 CCGGCCCTTCCATGTTAGCACTTTACTCGAGTAAAGTGCTAACATGGAAGGGTTTTTsh2 TRCN0000017840 CCGGCGGTGAAGATACTTATGATATCTCGAGATATCATAAGTATCTTCACCGTTTTT

NCOR2 sh2 TRCN0000060704 CCGGCCTCTATTACTACCTGACTAACTCGAGTTAGTCAGGTAGTAATAGAGGTTTTTGsh5 TRCN0000060707 CCGGGCAGTGTAAGAACTTCTACTTCTCGAGAAGTAGAAGTTCTTACACTGCTTTTTG

KDM3B sh1 TRCN0000017093 CCGGCCCTAGTTCATCGCAACCTTTCTCGAGAAAGGTTGCGATGAACTAGGGTTTTTsh2 TRCN0000017095 CCGGGCGATCTTTGTAGAATTTGATCTCGAGATCAAATTCTACAAAGATCGCTTTTTsh3 TRCN0000017096 CCGGGCTGTTAATGTGATGGTGTATCTCGAGATACACCATCACATTAACAGCTTTTTsh4 TRCN0000017097 CCGGCCTTGTAGATAAACTGGGTTTCTCGAGAAACCCAGTTTATCTACAAGGTTTTT

KDM4A sh1 TRCN0000013493 CCGGGCTGCAGTATTGAGATGCTAACTCGAGTTAGCATCTCAATACTGCAGCTTTTTsh3 TRCN0000013495 CCGGGCACCGAGTTTGTCTTGAAATCTCGAGATTTCAAGACAAACTCGGTGCTTTTTsh4 TRCN0000013496 CCGGCCGAAACTTCAGTAGATACATCTCGAGATGTATCTACTGAAGTTTCGGTTTTTsh5 TRCN0000013497 CCGGGCCTTGGATCTTTCTGTGAATCTCGAGATTCACAGAAAGATCCAAGGCTTTTT

BRG1 sh1 TRCN0000015548 CCGGCCATATTTATACAGCAGAGAACTCGAGTTCTCTGCTGTATAAATATGGTTTTTsh2 TRCN0000015549 CCGGCCCGTGGACTTCAAGAAGATACTCGAGTATCTTCTTGAAGTCCACGGGTTTTTsh4 TRCN0000015551 CCGGCCGAGGTCTGATAGTGAAGAACTCGAGTTCTTCACTATCAGACCTCGGTTTTTsh5 TRCN0000015552 CCGGCGGCAGACACTGTGATCATTTCTCGAGAAATGATCACAGTGTCTGCCGTTTTT

BCOR sh1 TRCN0000033460 CCGGGCCAAATAAGTATTCACTGAACTCGAGTTCAGTGAATACTTATTTGGCTTTTTGsh2 TRCN0000033461 CCGGCCACGAAACTTATACTTTCAACTCGAGTTGAAAGTATAAGTTTCGTGGTTTTTGsh3 TRCN0000033462 CCGGGCTCTATATTTCTGTCTCCAACTCGAGTTGGAGACAGAAATATAGAGCTTTTTGsh5 TRCN0000033459 CCGGCCCGCATATTTCGCTGCAATTCTCGAGAATTGCAGCGAAATATGCGGGTTTTTG

TRIM28 sh2 TRCN0000017999 CCGGCCTGGCTCTGTTCTCTGTCCTCTCGAGAGGACAGAGAACAGAGCCAGGTTTTTsh3 TRCN0000018001 CCGGCTGAGACCAAACCTGTGCTTACTCGAGTAAGCACAGGTTTGGTCTCAGTTTTT

The gene name, TRC number (Sigma-Aldrich), and sequences of shRNAs are shown.

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Table S2. List of primers used in this study

Primer name Species Region/gene Application Primer sequence (5′→3′)Productsize (bp)

a-fwd Human HS3 ChIP ATAGACCATGAGTAGAGGGCAGAC 142a-rev TGATCCTGAAAACATAGGAGTCAAb-fwd Human HBE1 ChIP GCCAGAACTTCGGCAGTAAA 98b-rev GGCCTGAGAGCTTGCTAGTGc-fwd Human HBG1 ChIP TTACTGCGCTGAAACTGTGG 130c-rev CAGTGGTTTCTAAGGAAAAAGTGCd-fwd Human HBG1 +3kb ChIP AATGACCTAATGCCCAGCAC 80d-rev AGTGTTGGGGGAGAAGTGTGe-fwd Human HBD -1kb ChIP GCAACAGAAGCCCAGCTATT 104e-rev GTGGCATGGTTTGATTTGTGf-fwd Human HBD ChIP TGTAGAGGAGAACAGGGTTT 181f-rev CTGCCTTTTATGCTGGTCCTg-fwd Human HBB ChIP TGCTCCTGGGAGTAGATTGG 161g-rev TGGTATGGGGCCAAGAGATAh-fwd Human 3′HS1 ChIP TCTTCAGCCATCCCAAGACT 137h-rev TGGTCTTTTCTGGACACCACHBE1-RT2-fwd Human HBE1 RT-PCR GCAAGAAGGTGCTGACTTCC 142HBE1-RT2-rev ACCATCACGTTACCCAGGAGHBG-RT-fwd Human HBG1/HBG2 RT-PCR TGGATGATCTCAAGGGCAC 209HBG-RT-rev TCAGTGGTATCTGGAGGACAHBB-RT-fwd Human HBB RT-PCR CTGAGGAGAAGTCTGCCGTTA 146HBB-RT-rev AGCATCAGGAGTGGACAGAThGAPDH-RT-fwd Human GAPDH RT-PCR ACCCAGAAGACTGTGGATGG 125hGAPDH-RT-rev TTCAGCTCAGGGATGACCTTey-RT-fwd Mouse Hbb-y RT-PCR TGGCCTGTGGAGTAAGGTCAA 120ey-RT-rev GAAGCAGAGGACAAGTTCCCAbh1-RT-fwd Mouse Hbb-bh1 RT-PCR TGGACAACCTCAAGGAGACC 145bh1-RT-rev ACCTCTGGGGTGAATTCCTTbmaj/min-RT-

fwdMouse Hbb-b1/Hbb-

b2RT-PCR TTTAACGATGGCCTGAATCACTT 132

bmaj/min-RT-rev CAGCACAATCACGATCATATTGCmGapdh-RT-fwd Mouse Gapdh RT-PCR TGGTGAAGGTCGGTGTGAAC 123mGapdh-RT-rev CCATGTAGTTGAGGTCAATGAAGG

fwd, forward; rev, reverse.

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