Corepressor-dependent silencing of fetal ... - Semantic Scholar

Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A Jian Xua, Daniel E. Bauera, Marc A. Kerenyia, Thuy D. Voa, Serena Houa, Yu-Jung Hsua, Huilan Yaob, Jennifer J. Trowbridgea, Gail Mandelb, and Stuart H. Orkina,c,1 a

Division of Hematology/Oncology, Boston Children’s Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115; bVollum Institute, Howard Hughes Medical Institute, Oregon Health and Science University, Portland, OR 97239; and cHoward Hughes Medical Institute, Boston, MA 02115

Contributed by Stuart H. Orkin, March 4, 2013 (sent for review February 11, 2013)

Reactivation of fetal hemoglobin (HbF) in adults ameliorates the severity of the common β-globin disorders. The transcription factor BCL11A is a critical modulator of hemoglobin switching and HbF silencing, yet the molecular mechanism through which BCL11A coordinates the developmental switch is incompletely understood. Particularly, the identities of BCL11A cooperating protein complexes and their roles in HbF expression and erythroid development remain largely unknown. Here we determine the interacting partner proteins of BCL11A in erythroid cells by a proteomic screen. BCL11A is found within multiprotein complexes consisting of erythroid transcription factors, transcriptional corepressors, and chromatinmodifying enzymes. We show that the lysine-specific demethylase 1 and repressor element-1 silencing transcription factor corepressor 1 (LSD1/CoREST) histone demethylase complex interacts with BCL11A and is required for full developmental silencing of mouse embryonic β-like globin genes and human γ-globin genes in adult erythroid cells in vivo. In addition, LSD1 is essential for normal erythroid development. Furthermore, the DNA methyltransferase 1 (DNMT1) is identified as a BCL11A-associated protein in the proteomic screen. DNMT1 is required to maintain HbF silencing in primary human adult erythroid cells. DNMT1 haploinsufficiency combined with BCL11A deficiency further enhances γ-globin expression in adult animals. Our findings provide important insights into the mechanistic roles of BCL11A in HbF silencing and clues for therapeutic targeting of BCL11A in β-hemoglobinopathies. gene regulation

| globin switching | hematopoiesis

etal hemoglobin (HbF, α2γ2) is a major genetic modifier of the phenotypic heterogeneity in patients with the major β-globin disorders sickle cell disease (SCD) and β-thalassemia (1). The synthesis of hemoglobin undergoes switching during ontogeny such that HbF is the predominant hemoglobin produced during fetal life and is gradually silenced and replaced by adult hemoglobin (HbA, α2β2) around birth. Because increased γ-globin expression in adults can substitute for the mutant or absent β-globin in SCD and β-thalassemia, respectively, the fetal-to-adult globin switch is critical to the pathogenesis of these conditions. As a result, intense efforts have been aimed to elucidate the molecular mechanisms of HbF silencing and to develop target-based therapeutics to enhance HbF production (2). Recent genomewide association studies (GWAS) led to the identification of a new HbF-associated locus on chromosome 2, located within the gene BCL11A (3). Subsequent studies demonstrated that BCL11A, a zinc-finger transcription factor, is a bona fide repressor of HbF expression (4–7). BCL11A protein is developmentally regulated and is required to maintain HbF silencing in human adult erythroid cells (4, 5). KO of BCL11A in mice carrying a human β-globin cluster transgene leads to profound delay in globin switching and impaired HbF silencing in adult erythroid cells (5, 8). Previously silenced γ-globin genes can also be reactivated by loss of BCL11A in adult animals (8). Most importantly, inactivation of BCL11A alone is sufficient to ameliorate the hematologic and pathologic defects associated with SCD through high-level HbF induction in humanized mouse models (8). These genetic studies provide persuasive evidence that BCL11A functions

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as a central modulator of HbF expression and globin switching in vivo. Further molecular studies revealed that BCL11A interacts with several erythroid regulators including GATA1, FOG1, and SOX6, and with the nucleosome remodeling and deacetylase complex (NuRD) (4, 6). BCL11A acts within the β-globin cluster by associating with several discrete regions, including sequences specifically deleted in patients with hereditary persistence of fetal hemoglobin (HPFH). However, BCL11A does not bind detectably to the γ-globin promoter in erythroid chromatin, suggesting that its mode of action is more complex than simply blocking transcription at the proximal promoter. This observation is consistent with a role of BCL11A in promoting long-range chromosomal interactions within the β-globin locus (6, 7, 9). Therefore, in an effort to elucidate further the precise molecular mechanisms by which BCL11A coordinates the switch, it is relevant to identify systematically BCL11A-interacting partner proteins, and use functional and genetic approaches to assess their individual roles in HbF regulation and erythroid cell maturation. In addition to providing important insights into the molecular mechanisms through which BCL11A controls hemoglobin switching, our data suggest opportunities, as well as challenges, for therapeutic induction of HbF in patients with the major hematologic disorders. Results BCL11A-Interacting Partner Proteins in Erythroid Cells. We characterized BCL11A-containing multiprotein complexes by a proteomic affinity screen using a metabolic biotin tagging approach (10). Murine erythroleukemia (MEL) cell lines that stably express the bacterial biotin ligase BirA and the recombinant BCL11A (XL isoform) containing a FLAG epitope tag together with a BirA recognition motif fused to its amino terminus were generated (FB-BCL11A; Fig. 1A). Clones were selected that express the recombinant BCL11A at levels similar to endogenous BCL11A (Fig. S1). Single streptavidin affinity purification or tandem anti-FLAG immunoaffinity and streptavidin affinity purification was performed on nuclear extracts from MEL cells stably expressing BirA alone or BirA together with FB-BCL11A. Copurified proteins were fractionated in SDS/PAGE, followed by liquid chromatography and tandem MS peptide sequencing (Fig. 1A). We identified numerous peptides of BCL11A and nearly all previously established interacting proteins, including FOG1, the entire NuRD complex, and the nuclear matrix protein MATRIN-3 (4). Besides previously recognized partners, we also identified a panel of previously undiscovered interacting complexes, including the hematopoietic regulators RUNX1 and IKZF1 (or IKAROS), several transcriptional corepressor complexes, and the SWI/SNF chromatin remodeling complex (Fig. 1B). The BCL11A-interacting corepressors include

Author contributions: J.X. and S.H.O. designed research; J.X., D.E.B., M.A.K., T.D.V., S.H., Y.-J.H., and J.J.T. performed research; H.Y. and G.M. contributed new reagents/analytic tools; J.X. and S.H.O. analyzed data; and J.X. and S.H.O. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. 1

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1303976110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1303976110

b

15, 8, 5

3, 6, 13, 5

Single streptavidin affinity purification Tandem α-FLAG and streptavidin affinity purification

Fig. 1. Identification of BCL11A-interacting proteins in erythroid cells by a proteomic affinity screen. (A) Schematic diagram of experimental design. (B) List of identified proteins is shown with the number of peptides obtained in each trial. Official gene symbols of the identified proteins are shown in parentheses. (C) Confirmation of interactions between BCL11A and identified proteins by coimmunoprecipitation in primary human erythroid cells.

the histone demethylase complex lysine-specific demethylase 1 (LSD1)/CoREST, the nuclear receptor corepressors NCoR/SMRT, the SIN3 deacetylase complex, the BCL6 corepressor BCOR, the DNA methyltransferase 1 (DNMT1), and histone demethylases (Fig. 1B). To identify BCL11A-interacting proteins in human erythroid cells, we performed a similar proteomic screen in human erythroleukemia (K562) cells. Remarkably, the identified partner proteins are largely similar to those found in MEL cells (Fig. 1B), suggesting that BCL11A acts within functionally conserved protein complexes. By coimmunoprecipitation (co-IP) analysis, we confirmed that both the epitope-tagged and the endogenous BCL11A associate with the identified partner proteins in erythroid cell lines and primary human adult erythroid cells, respectively (Fig. S1; Fig. 1C). Functional RNAi Screen in Primary Human Erythroid Cells. To assess the functional roles of BCL11A-interacting proteins in HbF regulation, we performed a lentiviral RNAi screen in primary adult human CD34+ hematopoietic stem/progenitor cells (HSPCs) undergoing ex vivo erythroid maturation (Fig. 2A). Primary CD34+ HSPCs were transduced with lentiviruses containing shRNAs Xu et al.

SCF, IL-3, IL-6, Flt-3 ligand

Epo, SCF, IL-3, Dexamethasone, β-estradiol

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Fig. 2. Functional RNAi screen of BCL11A-interacting proteins in primary human erythroid cells. (A) Schematic diagram of experimental design. (B) Expression of human γ-globin mRNA was measured by qRT-PCR. Data are plotted as percentage of γ-globin over total β-like human globin gene levels (two to five shRNAs per gene). Genes are ranked based on the level of γ-globin expression (highest to lowest). (C) Genes are ranked based on the level of e-globin expression (highest to lowest). (D) Expression of human total β-like globin mRNA normalized to GAPDH mRNA level. Results are the means ± SD of at least three independent experiments.

PNAS | April 16, 2013 | vol. 110 | no. 16 | 6519

MEDICAL SCIENCES

a

A

BCoR

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9, 1, 3, 1 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 0, 2, 9, 0 0, 2, 3, 0

BRG1 BCoR TRIM28

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14, 22, 3 1, 1, 0 0, 1, 0 0, 1, 0 1, 1, 0 16, 16, 0 3, 6, 0

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2, 3, 3, 1 8, 1, 12, 2 7, 1, 11, 0 5, 2, 3, 0 10, 3, 3, 0 7, 3, 5, 0 16, 5, 10, 2 15, 3, 10, 0 12, 2, 12, 0 5, 1, 15, 0 2, 1, 5, 0

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ZBTB33 NCoR2

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17, 31, 6 11, 12, 5

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KDM3A SUZ12 KDM5D SIN3A EZH2 HDAC1 KDM3B HDAC2 KDM5A CHD4 SOX6 DNMT1

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CoREST (RCOR1) NCoR/SMRT complex NCoR (NCOR1) SMRT (NCOR2) TBLR1 TBL1 CORO2A KAISO (ZBTB33) SIN3 complex SIN3A SIN3B Other corepressors BCOR TRIM28 SWI/SNF complex SNF5 (SMARCB1) BRG1 (SMARCA4) BRM (SMARCA2) BAF57 (SMARCE1) BAF60A (SMARCD1) BAF60B (SMARCD2) BAF155 (SMARCC1) BAF170 (SMARCC2) SNF2H (SMARCA5) BAF180 (PB1) ASH2L Other nuclear factors DNMT1 JMJD1A (KDM3A) JMJD1B (KDM3B) JARID1A (KDM5A) CARM1 YLPM1 MSH2 Nuclear matrix MATRIN-3 (MATR3)

8, 8, 1 0, 0, 0 0, 0, 0 0, 0, 1 0, 0, 0 7, 7, 5 5, 4, 2 3, 3, 1 2, 1, 1 0, 0, 0 0, 0, 0 2, 1, 0

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targeting BCL11A or its interacting proteins during an expansion phase, followed by erythroid differentiation for 5–7 d. Knockdown of each gene was confirmed by qRT-PCR, and shRNAs that resulted in >75% decrease of target mRNA expression were selected for subsequent analyses (Table S1; Figs. S2 and S3). KLF1 (or EKLF) is a critical erythroid regulator required for adult β-globin transcription (11). Recently it was shown that KLF1 occupies the BCL11A promoter and activates its expression (12, 13). Consistent with previous analysis, knockdown of BCL11A or KLF1 results in marked increase in γ-globin expression and serves as positive controls for the RNAi screen. We first ranked the genes according to the level of human γ-globin expression (Fig. 2B). Notably, the top of the list consists of subunits of several transcriptional corepressor complexes, including the NuRD complex (CHD4, HDAC1, HDAC2, and MBD2), DNMT1, SIN3A, NCOR1, and the polycomb repressive complex 2 (PRC2; EZH2, EED, and SUZ12). shRNA-mediated depletion of BCL11A-interacting proteins also results in derepression of the human embryonic e-gene. We next ranked the genes according to the level of e-globin expression (Fig. 2C). The highest ranked genes include subunits of the PRC2 complex (EED, EZH1, EZH2, and SUZ12), several histone demethylases (KDM7A, KDM3A, and KDM5D) and SIN3A. Importantly, depletion of KLF1, but not BCL11A, significantly increases e-globin expression (Fig. 2C), indicating a distinct role of KLF1 in regulating human embryonic globin expression. These results demonstrate that BCL11A-interacting proteins are differentially required for silencing of human embryonic or fetal globin expression, suggesting that they may form distinct subcomplexes depending on the chromatin context.

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BCL11A Transcription factors FOG1 (ZFPM1) RUNX1 IKAROS (IKZF1) NuRD complex Mi-2β (CHD4) Mi-2α (CHD3) MTA1 MTA2 MTA3 HDAC1 HDAC2 RBBP4 RBBP7 MBD3 P66α (GATAD2A) P66β (GATAD2B) LSD1/CoREST complex LSD1 (KDM1A)

# of Peptides MEL


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demethylase LSD1, which is identified as a BCL11A-interacting protein (Fig. 1), we next examined the chromatin occupancy of LSD1 and its cofactor CoREST. Loss of BCL11A also impairs chromatin occupancy of the LSD1/CoREST complex within the β-globin locus, particularly at the e-promoter and the γδ-intergenic region (Fig. 3 E and F), suggesting that BCL11A is required for efficient recruitment or stable association of the LSD1/CoREST complex within the β-globin cluster. Similarly, the binding of the NuRD complex (Mi-2β and HDAC1) and EZH2 is also decreased on BCL11A knockdown (Fig. 3 G–I). Notably, RNA Pol II occupancy at the Aγ promoter is enhanced on knockdown, consistent with transcriptional reactivation of the γ-globin genes in the BCL11A-depleted adult erythroid cells (Figs. 2B and 3J). These results indicate that loss of BCL11A leads to impaired chromatin occupancy of a subset of its interacting corepressors in human adult erythroid cells.

Besides the reactivation of HbF expression, depletion of several BCL11A-interacting proteins impairs erythroid cell maturation ex vivo, as revealed by reduced expression of erythroid cell surface markers CD71 and CD235a (Fig. S3). Perturbation of total globin synthesis is a secondary consequence of defective red cell differentiation. Notably, depletion of BCL11A-interacting proteins results in variable effects on the synthesis of total human β-like globin mRNAs (Fig. 2D). Although knockdown of many genes has little effect, knockdown of several genes leads to a greater than twofold increase (NCOR1 and KDM5D) or decrease (KLF1, KDM5B, SOX6, RBBP7, LSD1/KDM1A, ZBTB33, and KDM4A) in expression of total β-like globin mRNAs (Fig. 2D; Fig. S3), suggesting that their gene products influence processes required for normal erythroid development. Chromatin Occupancy of BCL11A-Interacting Proteins. The identification of BCL11A-interacting proteins and subsequent functional RNAi screen led to prioritization of candidate HbF regulators for detailed mechanistic studies. If BCL11A and its interacting partners regulate HbF expression in a functional protein complex, ablation of one critical component of the complex may lead to destabilization of the larger complex and/or its chromatin occupancy. We next determined whether the chromatin occupancy of BCL11A-interacting proteins is dependent on the presence of BCL11A. ChIP was performed in primary human adult erythroid cells transduced with control or BCL11A lentiviral shRNA. In control cells, ChIP analysis revealed that BCL11A occupies several discrete regions within the human β-globin cluster, including the DNase I hypersensitive site 3 (HS3) within the locus control region (LCR), the e-globin promoter, and the γδ-intergenic region, consistent with previous analyses (4, 6). Knockdown of BCL11A abolishes its occupancy at the above regions, consistent with the near absence of BCL11A protein in the knockdown cells (Fig. 3B). Interestingly, loss of BCL11A leads to a marked increase of H3K4me2, a histone mark associated with active transcription, at the Aγ-globin promoter and the γδ-intergenic region, with a concomitant decrease of the H3K9me1 mark (Fig. 3 C and D). Because both H3K4me2 and H3K9me1 are substrates for the histone

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We next determined whether the LSD1/CoREST complex is required for the expression of β-like globin genes in vivo. Mouse strains carrying floxed alleles of Lsd1 or Rocr1 (encoding CoREST) were generated by gene targeting. We first examined the expression of mouse embryonic β-like globin genes (ey- and βh1-globin) in fetal livers of Lsd1- or Rcor1-deficient embryos. Erythroid-specific deletion of LSD1 by EpoR-Cre results in a 6.2- and 26.7-fold increase in expression of the ey- and βh1-globin mRNAs in embryonic day 12.5 (E12.5) fetal liver definitive erythroid cells, respectively (Fig. 4A; P < 0.05). Similarly, loss of CoREST results in a 13.0- and 15.4-fold increase in ey- and βh1-globin mRNAs in E13.5 fetal livers (Fig. 4B; P < 0.05). Because germ-line or erythroid-specific deletion of LSD1 is embryonic lethal, we studied LSD1 conditional KO (cKO) adult mice using the hematopoieticselective and IFN inducible Mx1-Cre allele (14). Acute deletion of LSD1 in adult bone marrow also results in a modest increase (5.8- and 10.6-fold) in expression of ey- and βh1-globin genes (Fig. 4D; P < 0.05). These results provide genetic evidence that the LSD1/CoREST complex is required to maintain full silencing of mouse embryonic β-like globin genes in definitive erythroid cells

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Fig. 3. Chromatin occupancy of BCL11A-interacting proteins in primary human erythroid cells. (A) In vivo chromatin occupancy in primary human erythroid cells transduced with lentiviral shRNAs against GFP (Control) or BCL11A (shBCL11A). Primers were designed to amplify discrete regions across the β-globin locus, including (a) HS3 within the LCR, (b) e-globin promoter, (c) Aγ-globin promoter, (d) +3-kb region downstream of Aγ-globin gene, (e) −1-kb region upstream of δ-globin gene, (f) δ-globin promoter, (g) β-globin promoter, and (h) 3′HS1 site. (B–K ) ChIPqPCR analysis of BCL11A, H3K4me2, H3K9me1, LSD1, CoREST, Mi-2β, HDAC1, EZH2, RNA Pol II, and rabbit IgG in primary human erythroid cells in the presence or absence of BCL11A knockdown, respectively. Results are means ± SD; *P < 0.05.

Xu et al.

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Fig. 4. The LSD1/CoREST complex is required for silencing of mouse embryonic β-like globin genes. (A) Expression of mouse embryonic (ey and βh1) globin genes was monitored by qRT-PCR in E12.5 fetal livers of control (Lsd1+/+) and LSD1 KO (Lsd1−/−; by EpoR-Cre) embryos. All data are shown as percentage (%) of total mouse β-like globin expression. The fold changes of globin mRNAs are indicated. Results are means ± SD (n ≥ 5 per genotype; P < 0.05). (B) Expression of ey- and βh1-globin genes in E13.5 fetal livers of control and CoREST KO (Rcor1−/−) embryos (n ≥ 3 per genotype; P < 0.05). (C) Expression of ey- and βh1-globin genes in E12.5 fetal livers of control and BCL11A KO (Bcl11a−/−; by EpoR-Cre) embryos (n ≥ 3 per genotype; P < 0.01). (D) Expression of ey- and βh1globin genes in PB of control and LSD1;Mx1-Cre cKO mice (3 wk post-pIpC, 9–15 wk old; n ≥ 5 per genotype; P < 0.05). (E) Expression of ey- and βh1-globin genes in PB of control and BCL11A;Mx1-Cre cKO mice (3 wk post-pIpC, 9–15 wk old; n ≥ 3 per genotype; P < 0.01).

in vivo. However, deletion of BCL11A in mouse results in much greater increases (31.2- to 232.9-fold) of embryonic globin genes in definitive fetal liver and bone marrow erythroid cells (Fig. 4 C and E) (8), indicating that the extent of induction by LSD1 deletion is quite modest compared with that of BCL11A deletion (Fig. 4; compare A and B with C or D with E). LSD1 Cooperates with BCL11A in Silencing γ-Globin Expression. Because the repression of the endogenous ey- and βh1-globin genes is modestly and severely impaired in definitive fetal liver and bone marrow erythrocytes of Lsd1-deficient and Bcl11a-deficient mice, respectively (Fig. 4) (5), and LSD1 physically interacts with BCL11A (Fig. 1), we next examined whether LSD1 and BCL11A function collaboratively in silencing human γ-globin genes in vivo. We generated BCL11A and LSD1 compound KO mice carrying the human β-locus transgene [β-yeast artificial chromosome (β-YAC)]. Because erythroid-specific loss of LSD1 by EpoR-Cre results in embryonic lethality, we obtained compound BCL11A homozygous LSD1 heterozygous (Bcl11a−/−::Lsd1+/−) cKO adult mice. Erythroid-specific loss of BCL11A alone leads to 383-fold increase in γ-globin mRNA in the peripheral blood (from 0.022% in Bcl11a+/+::Lsd1+/+ control mice to 8.40% in Bcl11a−/−::Lsd1+/+ mice). The expression of γ-globin is further induced to 13.53% in Bcl11a−/−::Lsd1+/− compound KO adult mice (Fig. 5A; P = 0.0006). To determine the effect of complete loss of BCL11A and LSD1 on γ-globin silencing, we generated compound homozygous knockout adult mice using the inducible Mx1-Cre allele. Loss of LSD1 alone leads to a slight increase of γ-globin mRNA in the peripheral blood (from 0.025% in Bcl11a+/+::Lsd1+/+ mice to 0.04% in Bcl11a+/+::Lsd1−/− mice; Fig. 5B). Loss of BCL11A alone reactivates γ-globin mRNA to 12.88%. Combined loss of BCL11A and LSD1 further induces γ-globin mRNA to 18.19% in Bcl11a−/−:: Lsd1−/− compound KO adult mice (Fig. 5B; P = 0.03). These data demonstrate that depletion of LSD1 by itself has a modest effect on transgenic γ-globin expression in vivo. However, loss of LSD1 further enhances the effect of BCL11A deficiency in HbF derepression, suggesting that BCL11A serves as a major barrier to induction by LSD1 deficiency. Xu et al.

We initially observed that erythroid-specific loss of LSD1 resulted in embryonic lethality at E13.5 with severe anemia. We next examined hematologic parameters in LSD1 and BCL11A compound KO adult mice (by EpoR-Cre). Consistent with previous analysis (8), erythroid- or hematopoietic-specific loss of BCL11A has little effect on total red blood cell (RBC) number and hemoglobin (Hgb) level (Fig. S4). In contrast, the Bcl11a−/−::Lsd1+/− compound KO mice are mildly anemic (Fig. S4A), indicating that LSD1 is required for normal erythroid development. Hematopoieticselective loss of LSD1 also leads to profound decrease in total number of white blood cells (WBCs; Fig. S4B), consistent with a role for LSD1 in terminal differentiation of other hematopoietic lineages (15). To examine more directly the role of LSD1 in HbF silencing and erythropoiesis, we depleted its expression in primary human adult erythroid progenitors. Transduction of two independent lentiviral shRNAs against LSD1 leads to efficient knockdown of LSD1 protein in CD34+ HSPC-derived erythroid progenitors (Fig. 5C). Although knockdown of BCL11A expression leads to a substantial increase of human γ-globin mRNA (from 9.5% to 50.3% at day 7 of erythroid differentiation), depletion of LSD1 expression results in modest increases in γ-globin expression (sh1: 21.3%; sh5: 32.3%; Fig. 5D). Of note, depletion of LSD1 also results in ∼65% decrease in total β-globin mRNAs in day 7 erythroid progenitors; therefore, much of the observed increase in relative γ-globin expression is due to reduced β-globin expression rather than γ-globin induction per se (Fig. 5D, Lower). This finding is in contrast to the changes in relative globin expression on BCL11A knockdown, which are predominantly characterized by induction of γ-globin itself. Consistent with decreased total globin production, LSD1depleted cells retain expression of CD34 antigen and fail to express maturing erythroid cell-specific markers CD71 and CD235a (Fig. S5A). In fact, the majority of LSD1-depleted cells exhibit proerythroblast and basophilic erythroblast morphology, whereas the more mature polychromatophilic and orthochromatic erythroblasts are nearly absent at day 9 of differentiation (Fig. S5B). Collectively, these data indicate that LSD1 is required for both full HbF silencing and erythroid cell maturation. Several small molecule inhibitors have been used to target LSD1 by inhibiting its histone demethylase activity (16). We next tested two LSD1 inhibitors, pargyline and trans-2-phenylcyclopropylamine [tranylcypromine (TCP)], in reactivation of HbF expression in primary human adult erythroid cells. Pargyline treatment by itself has little effect on HbF expression. Combining pargyline treatment and BCL11A knockdown results in modest increases in HbF expression compared with BCL11A knockdown alone (Fig. S6A). In contrast, TCP treatment induces HbF expression in both control and BCL11A knockdown cells (Fig. S6A). However, TCP treatment results in a profound decrease in production of total β-like globin mRNAs (Fig. S6B). Additionally, TCP-treated cells fail to express CD71 and CD235a (Fig. S6D), indicating that the induction of HbF expression mediated by chemical inhibitors of LSD1 is also associated with impaired erythroid maturation. DNMT1 Is Required for Maintenance of HbF Silencing. The proteomic screen identified the DNA methyltransferase DNMT1 as another protein with intrinsic enzymatic activity in association with BCL11A (Fig. 1). DNA methylation plays an important role in globin gene expression, and DNA demethylating agents have been shown to induce HbF expression in various model systems and patients (8, 17, 18). However, the role of the methyltransferase DNMT1 in γ-globin silencing in an intact animal has not been previously assessed by formal genetic experiments. Thus, we determined whether DNMT1 is directly involved in HbF silencing in vitro and in vivo. Upon shRNA-mediated knockdown of DNMT1 expression in primary human adult erythroid cells, HbF expression is significantly induced, whereas the amount of total β-like globin mRNAs is modestly reduced (Figs. 2B and 5E). Similarly, inhibition of DNMT1 activity by chemical inhibitors leads to enhanced HbF expression in primary erythroid cells (Fig. S7). PNAS | April 16, 2013 | vol. 110 | no. 16 | 6521

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6.2

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Fig. 5. Compound KO of Bcl11a, Lsd1, or Dnmt1 enhances γ-globin expression. (A) Expression of γ-globin mRNA was measured by qRT-PCR in peripheral blood (PB) of Bcl11a::Lsd1 cKO animals (by EpoR-Cre; 8–12 wk old). Results are the means ± SD. P values were determined by a two-tailed t test. (B) Expression of γ-globin in PB of Bcl11a::Lsd1 cKO animals (by Mx1-Cre; 8–12 wk old). (C) Lentiviral shRNA-mediated knockdown of BCL11A and LSD1 proteins in primary human erythroid cells. (D) Knockdown of BCL11A or LSD1 results in increased γ-globin mRNAs (Upper). Knockdown of LSD1 reduces total β-like globin mRNAs (Lower). Data are shown as means ± SD; *P < 0.05, **P < 0.01. (E) shRNA-mediated knockdown of DNMT1 in primary human erythroid cells (Upper) results in increase of γ-globin mRNAs and modest decrease in total β-like globin mRNAs (Lower). Data are shown as means ± SD; *P < 0.05. (F) Expression of γ-globin in PB of Dnmt1::Bcl11a cKO animals (by EpoR-Cre; 8–12 wk old).

To assess the role of DNMT1 in HbF silencing in vivo, we generated DNMT1 and BCL11A compound erythroid-specific cKO β-YAC mice (by EpoR-Cre). Although homozygous loss of DNMT1 results in embryonic lethality, compound BCL11A homozygous DNMT1 heterozygous (Bcl11a−/−::Dnmt1+/−) KO mice are viable despite a mild anemic phenotype (Fig. S8). Of note, DNMT1 haploinsufficiency leads to a 2.1-fold increase of γ-globin mRNA in the absence of BCL11A (Fig. 5F; from 11.16% in Bcl11a−/−::Dnmt1+/+ mice to 23.58% in Bcl11a−/−::Dnmt1+/− mice, P = 1.9 × 10−6). Collectively, these data provide important genetic evidence that DNMT1 haploinsufficiency in combination with BCL11A deficiency can further enhance HbF expression. Discussion We characterized interacting partner proteins of BCL11A in primary human erythroid cells that may participate in HbF regulation. We infer that BCL11A acts within multiprotein complexes consisting of transcriptional corepressors and chromatin-modifying subunits. Knockdown of several BCL11A-interacting corepressor proteins induces HbF expression in primary human erythroid cells, whereby the effect on total hemoglobin production and erythroid maturation is variable. The chromatin occupancy of several BCL11A-interacting complexes, including the LSD1/CoREST and NuRD complexes, within the β-globin cluster is dependent on the presence of BCL11A in human erythroid cells. These results suggest that BCL11A coordinates the hemoglobin switch and HbF silencing by assembling transcriptional corepressor complexes within the β-globin cluster. The identification of BCL11A-interacting proteins provides clues to target BCL11A and/or its cofactors for therapeutic HbF induction in β-hemoglobinopathies. To define favorable molecular targets for HbF induction, it is imperative to evaluate several criteria, including quantitative effects on HbF expression, limited effects on expression of nonglobin genes and on erythroid maturation, minimal impact outside the erythroid lineage, and feasibility of therapeutic intervention (2). Several favorable features, such as potency in HbF silencing, dose dependence, and minimal influence on erythropoiesis, recommend BCL11A as a target. However, its roles in other cell lineages, including B lymphocytes and the central nervous system, may present challenges (19, 20). As transcription factors have traditionally been viewed as undruggable due to the lack of catalytic domains, alternative strategies involving interference with protein–protein interactions or targeting partner proteins with enzymatic activities may be considered. Our results show that BCL11A interacts with several chromatinmodifying enzymes, such as the LSD1/CoREST demethylase complex, DNMT1, and the NuRD complex. Small molecule inhibitors for these enzymes are in various phases of clinical development, 6522 | www.pnas.org/cgi/doi/10.1073/pnas.1303976110

including those in current medical practice. In principle, targeting enzymatic partner proteins of BCL11A constitutes a strategy for indirectly targeting BCL11A function. Our in-depth characterization of the roles of two BCL11Ainteracting enzymatic partners, LSD1 and DNMT1, in HbF regulation provides additional insights. KO of LSD1 in mice leads to derepression of mouse embryonic β-like globin genes and transgenic human γ-globin genes in definitive erythroid cells. Interference with LSD1 by shRNA or small molecule inhibitors reactivates HbF expression in primary human adult erythroid cells. Consistent with a role of LSD1 in HbF silencing, while this paper was under review, it was reported that RNAi or chemical inhibition of LSD1 enhances γ-globin expression in human erythroid cells and quite modestly β-locus transgenic mice (21). Although these findings illustrate favorable features of LSD1 as a potential target, we also demonstrate that LSD1 serves critical roles more broadly in erythroid cell maturation and overall globin expression. LSD1 homozygous KO mice are embryonic lethal with severe anemia. Deletion of LSD1 in adult bone marrow results in impaired function of hematopoietic stem cells, neutropenia, and markedly reduced number of leukocytes (15), indicating that LSD1 is necessary for specification and terminal differentiation of several hematopoietic lineages. Similarly, interference with LSD1 by shRNAs or inhibitors impairs erythroid maturation from human CD34+ HSPCs ex vivo (Figs. S5 and S6). Accordingly, its potential as a therapeutic target for HbF induction is compromised by its multifaceted roles in hematopoiesis. Our study also shows that DNMT1 haploinsufficiency augments HbF expression elicited by BCL11A deficiency. However, complete loss of DNMT1 is incompatible with normal hematopoietic development (22). Therefore, the therapeutic window appears to be relatively narrow whereby DNMT1 activity may be inhibited without perturbing normal erythroid functions. The nucleosome remodeling and histone deacetylase (NuRD) complex is also identified as a BCL11A-interacting corepressor complex. The NuRD complex consists of several enzymatic subunits, including the ATPase subunit Mi-2β and the histone deacetylases (HDAC1 and HDAC2). Depletion of Mi-2β results in a profound increase in γ-globin expression in primary human erythroid cells (the top-ranked gene CHD4; Fig. 2B; Fig. S3). Consistent with a role in HbF silencing, knockdown or KO of Mi-2β reactivates γ-globin genes in the β-YAC transgenic mice (23, 24). However, the degree of γ-globin induction is substantially less than that seen with BCL11A deficiency (8). Additionally, depletion of HDAC1 and HDAC2 reactivates γ-globin expression in primary human erythroid cells (Fig. 2B). HDAC1 and HDAC2 have also been identified as HbF inducers by a chemical genetic screen (25). These results collectively provide strong support for important Xu et al.

Materials and Methods

analyzed on a HEMAVET HV950 hematology system (Drew Scientific). All experiments were performed with the approval of the Children’s Hospital Boston Animal Ethics Committee. Cell Culture. Primary adult human CD34+ HSPC-derived erythroid progenitors were generated ex vivo as described (6). The MEL-BirA (MBirA), MEL-FLAGBio-BCL11A (MBB1.4), K562-BirA (KBirA), and K562-FLAG-Bio-BCL11A (KBB2.4) stable cell lines were generated as described (10). Protein Affinity Purification and Proteomic Analysis. BCL11A-interacting multiprotein complexes were purified and characterized as described (29). Copurified proteins were separated by SDS/PAGE, followed by whole lane LC-MS/MS using an LTQ linear ion-trap mass spectrometer. A subtractive approach including parallel pull-down in parental MEL-BirA (MBirA) or K562-BirA (KBirA) cells was used. Lentiviral RNAi. Lentiviral shRNA clones in the pLKO.1-puro vector were obtained from Sigma-Aldrich (two to five shRNAs per gene; Table S1). Primary human CD34+ HSPCs were transduced by spin infection at day 3 of expansion. Cells were washed three times with PBS and seeded in fresh media 24 h after infection. Selection (1 μg/mL puromycin) was initiated at 48 h after infection, followed by erythroid differentiation for 5–7 d. ChIP. ChIP was performed as described previously (29) using the following antibodies: H3K4me2 (07-030; Millipore), H3K9me1 (ab9045; Abcam), BCL11A (ab19487 and ab18688; Abcam), LSD1 (ab17721; Abcam), CoREST (07-455; Millipore), Mi-2β (provided by Stephen Smale, University of California, Los Angeles, CA), HDAC1 (06-720; Millipore), EZH2 (07-689; Millipore), RNA Pol II (sc-899; Santa Cruz Biotechnology), and normal rabbit IgG (sc-2027; Santa Cruz Biotechnology). RNA Isolation and qRT-PCR. RNA was extracted using the QIAamp RNA Blood Mini Kit or RNeasy Plus Mini Kit (Qiagen). RNA was reverse-transcribed and analyzed with the iQ SYBR Green Supermix using the iCycler Real-Time PCR Detection System (Bio-Rad). Primer sequences are listed in Table S2.

Experimental Animals. The β-globin locus transgenic (β-YAC) mouse strain was created as described (26). Mice containing a Bcl11a floxed allele were created as described (8). Mice containing an Lsd1 floxed allele (M.A.K. and S.H.O.) or a Rocr1 (encoding CoREST) floxed allele (H.Y. and G.M.) were generated through gene targeting approaches. Dnmt1fl/fl mice were created as previously described (22, 27). To obtain the Bcl11a cKO mice, the Bcl11afl/fl mice were crossed with the EpoR-Cre knock-in mice (28) or the Mx1-Cre transgenic mice (14). These mice were crossed with Lsd1fl/fl or Dnmt1fl/fl mice to create compound cKO mice. PolyI:ployC (pIpC) was prepared in PBS and administrated via i.p. injection daily at a dose of 25 μg/kg for 7 d. Peripheral blood was isolated via the retroorbital plexus and

ACKNOWLEDGMENTS. We thank K. Peterson for providing β-YAC mice. We thank D. R. Higgs, D. A. Williams, L. I. Zon, A. B. Cantor, V. G. Sankaran, H. Huang, and A. Woo for helpful advice and discussions. This work was supported by funding from the National Institutes of Health (to S.H.O. and G.M.), including a Center of Excellence in Molecular Hematology Award from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (to S.H.O.). S.H.O. and G.M. are Investigators of the Howard Hughes Medical Institute (HHMI). J.X. is an HHMI-Helen Hay Whitney Foundation fellow and is supported by NIDDK Career Development Award K01DK093543.

1. Stamatoyannopoulos G (2005) Control of globin gene expression during development and erythroid differentiation. Exp Hematol 33(3):259–271. 2. Bauer DE, Kamran SC, Orkin SH (2012) Reawakening fetal hemoglobin: Prospects for new therapies for the β-globin disorders. Blood 120(15):2945–2953. 3. Thein SL, Menzel S, Lathrop M, Garner C (2009) Control of fetal hemoglobin: New insights emerging from genomics and clinical implications. Hum Mol Genet 18(R2):R216–R223. 4. Sankaran VG, et al. (2008) Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 322(5909):1839–1842. 5. Sankaran VG, et al. (2009) Developmental and species-divergent globin switching are driven by BCL11A. Nature 460(7259):1093–1097. 6. Xu J, et al. (2010) Transcriptional silencing of gamma-globin by BCL11A involves longrange interactions and cooperation with SOX6. Genes Dev 24(8):783–798. 7. Jawaid K, Wahlberg K, Thein SL, Best S (2010) Binding patterns of BCL11A in the globin and GATA1 loci and characterization of the BCL11A fetal hemoglobin locus. Blood Cells Mol Dis 45(2):140–146. 8. Xu J, et al. (2011) Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing. Science 334(6058):993–996. 9. Sankaran VG, et al. (2011) A functional element necessary for fetal hemoglobin silencing. N Engl J Med 365(9):807–814. 10. Kim J, Cantor AB, Orkin SH, Wang J (2009) Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells. Nat Protoc 4(4):506–517. 11. Miller IJ, Bieker JJ (1993) A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Krüppel family of nuclear proteins. Mol Cell Biol 13(5):2776–2786. 12. Borg J, et al. (2010) Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet 42(9):801–805. 13. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM (2010) KLF1 regulates BCL11A expression and gamma- to beta-globin gene switching. Nat Genet 42(9):742–744. 14. Kühn R, Schwenk F, Aguet M, Rajewsky K (1995) Inducible gene targeting in mice. Science 269(5229):1427–1429. 15. Sprüssel A, et al. (2012) Lysine-specific demethylase 1 restricts hematopoietic progenitor proliferation and is essential for terminal differentiation. Leukemia 26(9):2039–2051.

16. Lee MG, Wynder C, Schmidt DM, McCafferty DG, Shiekhattar R (2006) Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol 13(6):563–567. 17. DeSimone J, Heller P, Hall L, Zwiers D (1982) 5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons. Proc Natl Acad Sci USA 79(14):4428–4431. 18. Ley TJ, et al. (1982) 5-azacytidine selectively increases gamma-globin synthesis in a patient with beta+ thalassemia. N Engl J Med 307(24):1469–1475. 19. Liu P, et al. (2003) Bcl11a is essential for normal lymphoid development. Nat Immunol 4(6):525–532. 20. Leid M, et al. (2004) CTIP1 and CTIP2 are differentially expressed during mouse embryogenesis. Gene Expr Patterns 4(6):733–739. 21. Shi L, Cui S, Engel JD, Tanabe O (2013) Lysine-specific demethylase 1 is a therapeutic target for fetal hemoglobin induction. Nat Med 19(3):291–294. 22. Trowbridge JJ, Snow JW, Kim J, Orkin SH (2009) DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell 5(4):442–449. 23. Costa FC, Fedosyuk H, Chazelle AM, Neades RY, Peterson KR (2012) Mi2β is required for γ-globin gene silencing: Temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice. PLoS Genet 8(12):e1003155. 24. Amaya ML, et al. (2013) Mi2β-mediated silencing of the fetal γ-globin gene in adult erythroid cells. Blood, 10.1182/blood-2012-11-466227. 25. Bradner JE, et al. (2010) Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease. Proc Natl Acad Sci USA 107(28):12617–12622. 26. Peterson KR, et al. (1995) Use of yeast artificial chromosomes (YACs) in studies of mammalian development: Production of beta-globin locus YAC mice carrying human globin developmental mutants. Proc Natl Acad Sci USA 92(12):5655–5659. 27. Jackson-Grusby L, et al. (2001) Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat Genet 27(1):31–39. 28. Heinrich AC, Pelanda R, Klingmüller U (2004) A mouse model for visualization and conditional mutations in the erythroid lineage. Blood 104(3):659–666. 29. Xu J, et al. (2012) Combinatorial assembly of developmental stage-specific enhancers controls gene expression programs during human erythropoiesis. Dev Cell 23(4):796–811.

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roles of several enzymatic subunits within the NuRD complex in globin expression, yet the extent to which various effects can be ascribed directly to action in concert with BCL11A or independently are difficult to discern. Our studies used two model systems, including the primary human erythroid culture and the β-YAC transgenic mice, to evaluate HbF regulation and erythroid maturation. To interpret results meaningfully, it is important to acknowledge the potential limitations unique to each system. Although cultured primary human erythroid cells model several important aspects of human erythroid development such as an adult-stage pattern of globin profile, the background level of γ-globin mRNA is much higher than that present in adult erythroid cells of normal individuals. Moreover, CD34+ HSPCs appear relatively permissive for HbF induction by various agents, thus rendering them sensitive but not necessarily specific indicators to assay for the effects of manipulations inducing HbF (2). Changes in growth kinetics and differentiation status of CD34+ HSPCs affect HbF expression. In contrast to primary human cells, the baseline expression of transgenic γ-globin gene in adult β-YAC mice is far lower than anticipated from normal individuals, possibly due to differences in the transacting environment between mouse and human (5). Thus, interventions seeking to derepress γ-globin expression face a much greater quantitative hurdle. Although some manipulations, such as inhibition of LSD1 or DNMT1, may induce transgenic γ-globin expression several fold above this low level, the effects are nonetheless small compared with those elicited by BCL11A KO. Care needs to be exercised in extrapolating findings in the available model systems to nominate targets for therapeutic manipulation.

Supporting Information Xu et al. 10.1073/pnas.1303976110 SI Materials and Methods Streptavidin Immunoprecipitation and Western Blot. Streptavidin

immunoprecipitation (SA-IP) experiments in MEL or K562 stable cell lines were performed as described previously (1). Briefly, nuclear extracts were prepared from MEL-BirA (MBirA), MELFLAG-Bio-BCL11A (MBB1.4), K562-BirA (KBirA), and K562FLAG-Bio-BCL11A (KBB2.4) stable cell lines, immunoprecipitated with streptavidin agarose beads (Invitrogen), and processed for Western blot analysis. The following antibodies were used for coimmunoprecipitation (co-IP) and Western 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 of California, Las Angeles, CA), MTA2 (sc-9447; Santa Cruz Biotechnology), HDAC1 (06-720; Millipore), HDAC2 (sc-7899; Santa Cruz Biotechnology), MBD3 (sc-9402; Santa Cruz Biotechnology), SNF5, BRG1, BAF155 (provided by Charles Roberts, Dana-Farber Cancer Institute, Boston), EZH2 (612666; BC Biosciences), SUZ12 (ab12073; Abcam), SP1 (sc-14027; Santa Cruz Biotechnology), DNA methyltransferase 1 (DNMT1; 39204; Active Motif), and GAPDH (sc-26778; Santa Cruz Biotechnology).

incubated with 5–25 μg of BCL11A or mouse IgG antibody overnight at 4 °C. The protein complexes were collected by protein G/ A-agarose beads (Invitrogen), followed by four washes with BC139K buffer. The beads were boiled for 5 min in sample buffer (Bio-Rad), and the eluted material was used for Western blot analysis. Flow Cytometry. Cells were analyzed by flow cytometry as described

previously (3). Live cells were identified and gated by exclusion of 7-amino-actinomycin D (7-AAD; BD Pharmingen). The cells were analyzed for expression of cell surface antigens with antibodies specific 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 stages of differentiation were stained with May-Grunwald-Giemsa as described previously (4).

Co-IP and Western Blot. Co-IP experiments of endogenous BCL11A protein in primary human erythroid cells were performed as described (2). Briefly, 1–5 mg of nuclear extract proteins was

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

1. Xu J, et al. (2012) Combinatorial assembly of developmental stage-specific enhancers controls 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 longrange 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 with fetal hemoglobin silencing. Science 334(6058):993–996. 4. Sankaran VG, et al. (2009) Developmental and species-divergent globin switching are driven by BCL11A. Nature 460(7259):1093–1097.

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α-GATA1

α-GATA1

α-LSD1 α-CoREST α-NCOR1 α-SMRT α-KAISO α-SIN3A α-TRIM28 α-Mi-2β

Other LSD1/ Co-Rep. NCoR/SMRT CoREST

Other LSD1/ Co-Rep. NCoR/SMRT CoREST

α-FOG1

α-NCOR1 α-SMRT α-KAISO α-SIN3A α-TRIM28

NuRD

α-MTA2 α-HDAC1 α-HDAC2 α-MBD3

α-SNF5

α-SNF5

α-BRG1 α-BAF155 α-EZH2 α-SUZ12 α-SP1

SWI/SNF

α-MBD3

PRC2

NuRD PRC2

SWI/SNF

α-HDAC2

α-CoREST

α-Mi-2β

α-MTA2 α-HDAC1

α-LSD1

α-BRG1 α-BAF155 α-EZH2 α-SUZ12 α-SP1

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 followed by Western blot analysis; 2% of input nuclear extracts were analyzed as loading controls. (B) SA-IP experiments were performed in K562-BirA (MBirA) and K562-FLAG-Bio-BCL11A (KBB2.4) stable cells, followed by Western blot analysis.


2 of 11

A

20

BRG1 BCoR TRIM28

KDM3B KDM4A

ZBTB33 NCoR2

0.16

ε-globin mRNA (relative to GAPDH)

B


0

SIN3A HDAC1 NCoR1

10


γ-globin mRNA (relative to GAPDH)

30

0.12 0.08

SIN3A HDAC1 NCoR1


ZBTB33 NCoR2

KDM3B KDM4A

BRG1 BCoR TRIM28

SIN3A HDAC1 NCoR1


ZBTB33 NCoR2

KDM3B KDM4A

BRG1 BCoR TRIM28

β-globin mRNA (relative to GAPDH)

C


0


0.04

50 40 30 20 10 0

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


3 of 11

B sh5

CHD4 sh1 sh2 sh3 sh4

shGFP

shBCL11A

A

shGFP

shBCL11A

CHD4 sh4

80

4

60

7.97

100

80

3

40

Mi-2β

CHD4 sh5

60

3.71

40

60

4.24

2

Counts

20

GAPDH

9.21

40

20 1

20

0

0 10

0

10

1

10

2

10

3

10

4

0

0

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

3

10

4

10

4

CD34 100

120

40 20 0 50

40

120

90

80

90

40

20

60

30

0 10

10

10

2

10

3

10

4

20

0

10

0

10

1

10

2

10

3

10

4

0 10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

CD71

30

80

80

20

30

0

97.1

shBCL11A

Counts

sh5

sh4

CHD4

20

20

0

0 10 0

10 1

10 2

96.6

40

10 3

10 4

20

10

20

0

10 0

10 1

10 2

10 3

10 4

84.9

40

0 10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

CD235a

D

NCOR1

shGFP

NCoR1 sh4

NCoR1 sh3

NCoR1 sh5

60 4

sh5

150

sh1 sh2 sh3 sh4

shGFP

shBCL11A

shGFP

40

60

83.1

60

60

10

C

40

10

30

0 1

90.7 60

98.9

60

0

87.4

30

97.5

Counts


γ-globin mRNA (%)

100 80 60

30 3

40 100

20

7.97

4.04

2

2.08

4.12

20

NCOR1 Counts

50

GAPDH

0

10

0 10 0

10 1

10 2

10 3

10 4

1

0 10 0

10 1

10 2

10 3

0 10 0

10 4

10 1

10 2

10 3

10 4

10 0

10 1

10 2

10 3

10 4

CD34 250 40

120 200

98.3

90

60

20

100

2

Counts

30

40

10

50

0

0 10

0

10

1

10

2

10

3

10

4

0 10

0

10

1

10

2

10

3

10

4

0 10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

CD71

30

80

200

60

150

4 30

20 10

80.2

98.5

sh5

100

20

50

0

0

2

10

Counts

sh4

sh3

shBCL11A

40

10 0

10 1

10 2

10 3

10 4

1

0

0 10 0

10 1

10 2 FL2-H: CD235

10 3

10 4

10 0

10 1

10 2

10 3

10 4

10

0

10

1

10

2

10

3

10

4

CD235

F

shGFP

SIN3A sh1

SIN3A sh2

SIN3A sh3

200

60

150

150

150

sh5

SIN3A sh1 sh2 sh3 sh4

shGFP

NCOR1

72

3

20

97.1

shGFP

E

72

4

97.5

40 20 0 50

0

85.8

30

150

shBCL11A


γ-globin mRNA (%)

100 80 60

6

40 100

7.97

100

100

0.21

0.54

0.32

20

SIN3A

Counts

50

GAPDH

0

0

0 10 0

10 1

10 2

10 3

10 4

50

50

10 0

10 1

10 2

10 3

10 4

0 10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

CD34 120

250

100

120

90.5

90

97.5

200

80

90

89.1

98.7

60

150

40

100

60

60

40 20 0 60

30

Counts

30

50

20

0

0 10

0

10

1

10

2

10

3

10

4

0 10

0

10

1

10

2

10

3

10

4

0 10 0

10 1

10 2

10 3

10 4

40

80

10 1

10 2

10 3

10 4

10 3

10 4

120

60

20

sh3

98.1

90

40 60

20

20

20

Counts

sh2

sh1

77.5

40

97.1

40

SIN3A

60

75

60

shBCL11A

0

10 0

CD71

shGFP


γ-globin mRNA (%)

100 80 60

0

0 10 0

10 1

10 2

10 3

10 4

30

0 10 0

10 1

10 2

10 3

10 4

0 10 0

10 1

10 2

10 3

10 4

10 0

10 1

10 2

CD235

Fig. S3. Representative genes in functional RNAi screen in primary human adult eryrhoid cells. (A) Lentiviral shRNA-mediated knockdown of CHD4 (encoding Mi2β) reactivates human γ-globin expression. Expression of Mi2β was monitored by Western blot analysis (Upper). Expression of human γ-globin mRNA (as % of total 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 the means ± SD of at least three independent experiments. (B) Erythroid maturation was assessed by flow cytometry analysis of cell surface marker (CD71 and CD235a) expression in primary human CD34+ HSPC-derived erythroid progenitor cells at day 5 of differentiation. (C) Lentiviral shRNA-mediated knockdown of NCOR1 reactivates human γ-globin expression. (D) Erythroid maturation was assessed by flow cytometry analysis of cell surface marker expression on knockdown of NCOR1 expression. (E) Lentiviral shRNA-mediated knockdown of SIN3A reactivates human γ-globin expression. (F) Erythroid maturation was assessed 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

Bcl11a::Lsd1 cKO (by EpoR-Cre)

3

8 4 0

-/+/-

B

6

12

6

0 Bcl11a +/+ -/Lsd1 +/+ +/+

12

∗

WBCs (x10 /mL)

Hgb (g/dL)

9

16

∗

9

RBCs (x10 /mL)

12

+/+ -/+/+ +/+

-/+/-

9 6 3 0 +/+ -/+/+ +/+

-/+/-

Bcl11a::Lsd1 cKO (by Mx1-Cre) 16

6 3

0 Bcl11a +/+ -/Lsd1 +/+ +/+

-/-/-

∗

8 4 0 +/+ -/- -/+/+ +/+ -/-

6

12

12

WBCs (x10 /mL)

∗

Hgb (g/dL)

9

9

RBCs (x10 /mL)

12

9

∗

6

∗∗

3 0 +/+ -/+/+ +/+

-/-/-

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 PB counts 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).


5 of 11

A

shGFP

shBCL11A

LSD1 sh1

LSD1 sh5

40

60 80

60 30

60 40

40

7.97

40

20

3.71

30.8

30.5

20

Counts

0

0

0 10

0

10

1

10

2

10

3

10

4

20

10

20

10

0

10

1

10

2

10

3

10

0 10 0

4

10 1

10 2

10 3

10 4

10 0

10 1

10 2

10 3

10 4

CD34 60

30

120

120

90

90

40

20

97.5

98.9

60

31.9

35.2

60 20

10

Counts

30

30

0

0 10

0

10

1

10

2

10

3

10

4

0

0 10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

0

10

1

10

2

10

3

10

4

10

4

CD71 80

80

80 30

60

60

60 20

97.1

40

40

20

0

0

Counts

20

10 0

10 1

10 2

10 3

96.6

10 4

26.9

17

40

10 20

0 10 0

10 1

10 2

10 3

10 4

0 10 0

10 1

10 2

10 3

10 4

10

0

10

1

10

2

10

3

CD235a

shGFP

shBCL11A

LSD1 sh1

LSD1 sh5

Day 9

Day 5

B

Fig. S5. Depletion of LSD1 expression in primary human CD34+ HSPCs leads to impaired erythroid differentiation. (A) Erythroid maturation was assessed by flow cytometry analysis of cell surface marker (CD34, CD71, and CD235a) expression in primary human CD34+ HSPC-derived erythroid progenitor cells at day 5 of 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 the differentiating erythroid progenitors acquired proerythroblast morphology at day 5 of differentiation. At day 9 of differentiation, the majority of cells were polychromatophilic and orthochromatic. Depletion of BCL11A expression by lentiviral shRNA had no effect on erythroid maturation. Depletion of LSD1 resulted in marked decrease in total cell number and polychromatophilic/orthochromatic erythroid progenitors.


6 of 11

A

Control

γ-globin mRNA (%)

60

shBCL11A

∗

∗

∗

∗

50 40

∗∗

∗∗

30

∗∗

∗

20 10 0

Control

0.3

1

3

5

10

Pargyline (mM) 100


B

25

TCP (μM)

Control

shBCL11A

80 60 40

∗

∗

20

∗∗

0

Control

0.3

1

3

5

Pargyline (mM)

∗∗ ∗∗

∗∗ ∗∗

10

25

TCP (μM)

C

0

10

1

10

2

10

3

10

4

10

Counts

Counts

0 mM Pargyline 0.3 mM Pargyline 1 mM Pargyline 3 mM Pargyline

CD71

0

10

1

10

2

10

3

10

4

10

CD235a

D

0

10

CD71

1

10

2

10

3

10

4

10

Counts

Counts

0 μM TCP 5 μM TCP 10 μM TCP 25 μM TCP

0

10

1

10

2

10

3

10

4

10

CD235a

Fig. S6. Inhibition of LSD1 activity leads to increased γ-globin expression and impaired erythroid differentiation in primary human erythroid cells. (A) Expression 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 human erythroid 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 surface marker (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 cells at day 5 of differentiation in the presence or absence of TCP.


7 of 11

Control shBCL11A

80

100

∗

60 40 20 0

0

0.001 0.01

0.1

1

10

100

Control shBCL11A

80

γ-globin mRNA (%)

100

γ-globin mRNA (%)

γ-globin mRNA (%)

A ∗

60 40 20 0

0

1

5-azaD (μM)

10

100

1000

Control shBCL11A

80

∗

60 40 20 0

0

Zebularine (μM)

1

10

100

1000

RG108 (μM)

Control shBCL11A

0

0.001 0.01

0.1

1

10

5-azaD (μM)

120 100 80 60 40 20 0


120 100 80 60 40 20 0



B 120 100 80 60 40 20 0

Control shBCL11A

0

1

10

100

1000

Zebularine (μM)

Control shBCL11A

0

1

10

100

1000

RG108 (μM)

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

Bcl11a::Dnmt1 cKO (by EpoR-Cre)

3 -/+/-

WBCs (x10 /mL)

12

6

∗

6

0 Bcl11a +/+ -/Dnmt1 +/+ +/+

12

16 Hgb (g/dL)

9

9

RBCs (x10 /mL)

12

8 4 0

+/+ -/+/+ +/+

-/+/-

9 6 3 0 +/+ -/+/+ +/+

-/+/-

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


8 of 11

Table S1. List of lentiviral shRNA sequences used in this study Gene name GFP BCL11A

KLF1

CHD4 DNMT1

SIN3A

HDAC1

NCOR1

EZH2

HDAC2

MBD2

EED

KDM3A

SUZ12

KDM5B

RBBP4

KDM5A

SOX6

shRNA ID

TRC number

shRNA sequences

shGFP sh49 sh51 sh53 sh1 sh4 sh5 sh4 sh5 sh2 sh3 sh5 sh1 sh2 sh3 sh2 sh3 sh5 sh3 sh4 sh5 sh1 sh2 sh4 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3

SHC005 TRCN0000033449 TRCN0000033451 TRCN0000033453 TRCN0000230814 TRCN0000230812 TRCN0000230813 TRCN0000021362 TRCN0000021363 TRCN0000021891 TRCN0000021893 TRCN0000021892 TRCN0000021774 TRCN0000021775 TRCN0000021776 TRCN0000004814 TRCN0000004816 TRCN0000004818 TRCN0000060655 TRCN0000060656 TRCN0000060657 TRCN0000286227 TRCN0000040074 TRCN0000286290 TRCN0000004822 TRCN0000004823 TRCN0000004819 TRCN0000004820 TRCN0000013319 TRCN0000013320 TRCN0000013321 TRCN0000013318 TRCN0000021204 TRCN0000021205 TRCN0000021206 TRCN0000021207 TRCN0000021208 TRCN0000021149 TRCN0000021150 TRCN0000021151 TRCN0000021152 TRCN0000021153 TRCN0000038725 TRCN0000038726 TRCN0000038727 TRCN0000038728 TRCN0000014759 TRCN0000014760 TRCN0000014761 TRCN0000014762 TRCN0000014758 TRCN0000115869 TRCN0000115870 TRCN0000115868 TRCN0000115871 TRCN0000014628 TRCN0000014629 TRCN0000014630 TRCN0000014631 TRCN0000014632 TRCN0000017990 TRCN0000017988 TRCN0000017989

CCGGTACAACAGCCACAACGTCTATCTCGAGATAGACGTTGTGGCTGTTGTATTTTT CCGGCGCACAGAACACTCATGGATTCTCGAGAATCCATGAGTGTTCTGTGCGTTTTTG CCGGCCAGAGGATGACGATTGTTTACTCGAGTAAACAATCGTCATCCTCTGGTTTTTG CCGGGCATAGACGATGGCACTGTTACTCGAGTAACAGTGCCATCGTCTATGCTTTTTG CCGGTGCACATGAAGCGCCACCTTTCTCGAGAAAGGTGGCGCTTCATGTGCATTTTTG CCGGCCCTCCTTCCTGAGTTGTTTGCTCGAGCAAACAACTCAGGAAGGAGGGTTTTTG CCGGCAGAGGATCCAGGTGTGATAGCTCGAGCTATCACACCTGGATCCTCTGTTTTTG CCGGGCTGACACAGTTATTATCTATCTCGAGATAGATAATAACTGTGTCAGCTTTTT CCGGGCGGGAGTTCAGTACCAATAACTCGAGTTATTGGTACTGAACTCCCGCTTTTT CCGGGCCCAATGAGACTGACATCAACTCGAGTTGATGTCAGTCTCATTGGGCTTTTT CCGGCGACTACATCAAAGGCAGCAACTCGAGTTGCTGCCTTTGATGTAGTCGTTTTT CCGGCGAGAAGAATATCGAACTCTTCTCGAGAAGAGTTCGATATTCTTCTCGTTTTT CCGGCGTGAACATCTAGCACAGAAACTCGAGTTTCTGTGCTAGATGTTCACGTTTTT CCGGCCCTGAGTTGTTTAATTGGTTCTCGAGAACCAATTAAACAACTCAGGGTTTTT CCGGGCTACGTCTCAAAGAACCTATCTCGAGATAGGTTCTTTGAGACGTAGCTTTTT CCGGCGTTCTTAACTTTGAACCATACTCGAGTATGGTTCAAAGTTAAGAACGTTTTT CCGGGCCGGTCATGTCCAAAGTAATCTCGAGATTACTTTGGACATGACCGGCTTTTT CCGGGCTGCTCAACTATGGTCTCTACTCGAGTAGAGACCATAGTTGAGCAGCTTTTT CCGGCGCAGTATTGTCCAAATTATTCTCGAGAATAATTTGGACAATACTGCGTTTTTG CCGGGCCATCAAACACAATGTCAAACTCGAGTTTGACATTGTGTTTGATGGCTTTTTG CCGGGCTCTCAAAGTTCAGACTCTTCTCGAGAAGAGTCTGAACTTTGAGAGCTTTTTG CCGGTATTGCCTTCTCACCAGCTGCCTCGAGGCAGCTGGTGAGAAGGCAATATTTTTG CCGGGCTAGGTTAATTGGGACCAAACTCGAGTTTGGTCCCAATTAACCTAGCTTTTTG CCGGCGGAAATCTTAAACCAAGAATCTCGAGATTCTTGGTTTAAGATTTCCGTTTTTG CCGGGCAGACTCATTATCTGGTGATCTCGAGATCACCAGATAATGAGTCTGCTTTTT CCGGGCAAATACTATGCTGTCAATTCTCGAGAATTGACAGCATAGTATTTGCTTTTT CCGGCAGTCTCACCAATTTCAGAAACTCGAGTTTCTGAAATTGGTGAGACTGTTTTT CCGGCCAGCGTTTGATGGACTCTTTCTCGAGAAAGAGTCCATCAAACGCTGGTTTTT CCGGGCCTAGTAAATTACAGAAGAACTCGAGTTCTTCTGTAATTTACTAGGCTTTTT CCGGGTAGCAATGATGAGACCCTTTCTCGAGAAAGGGTCTCATCATTGCTACTTTTT CCGGGTACGCAAGAAATTGGAAGAACTCGAGTTCTTCCAATTTCTTGCGTACTTTTT CCGGGCTTAATGAAAGGGTTTGTAACTCGAGTTACAAACCCTTTCATTAAGCTTTTT CCGGGCAAACTTTATGTTTGGGATTCTCGAGAATCCCAAACATAAAGTTTGCTTTTT CCGGCCAGAGACATACATAGGAATTCTCGAGAATTCCTATGTATGTCTCTGGTTTTT CCGGGCAGCATTCTTATAGCTGTTTCTCGAGAAACAGCTATAAGAATGCTGCTTTTT CCGGCCTATAACAATGCAGTGTATACTCGAGTATACACTGCATTGTTATAGGTTTTT CCGGCCAGTGAATCTAATGTGACTACTCGAGTAGTCACATTAGATTCACTGGTTTTT CCGGCCCAAGATGTATAATGCTTATCTCGAGATAAGCATTATACATCTTGGGTTTTT CCGGCCCTAATAACTGTTCAGGAAACTCGAGTTTCCTGAACAGTTATTAGGGTTTTT CCGGGCTGGTATTTAGACCGATCATCTCGAGATGATCGGTCTAAATACCAGCTTTTT CCGGGCTTTGATTGTGAAGCATTTACTCGAGTAAATGCTTCACAATCAAAGCTTTTT CCGGCCATACGTTTAACAGCACAATCTCGAGATTGTGCTGTTAAACGTATGGTTTTT CCGGGCTTACGTTTACTGGTTTCTTCTCGAGAAGAAACCAGTAAACGTAAGCTTTTTG CCGGCCAAACCTCTTGCCACTAGAACTCGAGTTCTAGTGGCAAGAGGTTTGGTTTTTG CCGGCGGAATCTCATAGCACCAATACTCGAGTATTGGTGCTATGAGATTCCGTTTTTG CCGGGCTGACAATCAAATGAATCATCTCGAGATGATTCATTTGATTGTCAGCTTTTTG CCGGGCTCCCTTACTTTAGATGATACTCGAGTATCATCTAAAGTAAGGGAGCTTTTT CCGGCCTCTCCAAGATGTGGATATACTCGAGTATATCCACATCTTGGAGAGGTTTTT CCGGCCTGAGGAAGAGGAGTATCTTCTCGAGAAGATACTCCTCTTCCTCAGGTTTTT CCGGCGAGATGGAATTAACAGTCTTCTCGAGAAGACTGTTAATTCCATCTCGTTTTT CCGGCCCACCAATTTGGAAGGCATTCTCGAGAATGCCTTCCAAATTGGTGGGTTTTT CCGGCCCTTGTATCATCGCAACAAACTCGAGTTTGTTGCGATGATACAAGGGTTTTTG CCGGGCCTTTCTTTCAATCCTTATACTCGAGTATAAGGATTGAAAGAAAGGCTTTTTG CCGGCGGCAGTAGTAGAAGATGTTTCTCGAGAAACATCTTCTACTACTGCCGTTTTTG CCGGGCAGACTGAATGTCTGGGATTCTCGAGAATCCCAGACATTCAGTCTGCTTTTTG CCGGCCAGGTACTTAATGCCCTAAACTCGAGTTTAGGGCATTAAGTACCTGGTTTTT CCGGCCAGACTTACAGGGACACTTACTCGAGTAAGTGTCCCTGTAAGTCTGGTTTTT CCGGCGGACCGACATTGGTGTATATCTCGAGATATACACCAATGTCGGTCCGTTTTT CCGGCCCATGCAGAAGAAATGTCTTCTCGAGAAGACATTTCTTCTGCATGGGTTTTT CCGGCCTTGAAAGAAGCCTTACAAACTCGAGTTTGTAAGGCTTCTTTCAAGGTTTTT CCGGCCAGTGAACTTCTTGGAGAAACTCGAGTTTCTCCAAGAAGTTCACTGGTTTTT CCGGCCAACACTTGTCAGTACCATTCTCGAGAATGGTACTGACAAGTGTTGGTTTTT CCGGGCCACACATTAAGCGACCAATCTCGAGATTGGTCGCTTAATGTGTGGCTTTTT


9 of 11

Table S1. Cont. Gene name EZH1

IKZF1

KDM5D

KDM4B

KDM7A

RBBP7

KDM4C

BAF155

LSD1 (KDM1A) ZBTB33 NCOR2 KDM3B

KDM4A

BRG1

BCOR

TRIM28

shRNA ID

TRC number

shRNA sequences

sh1 sh2 sh3 sh4 sh5 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh4 sh5 sh1 sh2 sh3 sh5 sh1 sh2 sh3 sh4 sh1 sh2 sh3 sh5 sh1 sh5 sh1 sh2 sh2 sh5 sh1 sh2 sh3 sh4 sh1 sh3 sh4 sh5 sh1 sh2 sh4 sh5 sh1 sh2 sh3 sh5 sh2 sh3

TRCN0000002439 TRCN0000002440 TRCN0000002441 TRCN0000002442 TRCN0000010708 TRCN0000107871 TRCN0000107872 TRCN0000107873 TRCN0000107874 TRCN0000022114 TRCN0000022115 TRCN0000022116 TRCN0000022117 TRCN0000022118 TRCN0000018014 TRCN0000018013 TRCN0000018015 TRCN0000018016 TRCN0000253856 TRCN0000253855 TRCN0000253854 TRCN0000253852 TRCN0000253853 TRCN0000038885 TRCN0000038886 TRCN0000038887 TRCN0000038884 TRCN0000022054 TRCN0000022055 TRCN0000022056 TRCN0000022057 TRCN0000015628 TRCN0000015629 TRCN0000015630 TRCN0000015632 TRCN0000046068 TRCN0000046072 TRCN0000017838 TRCN0000017840 TRCN0000060704 TRCN0000060707 TRCN0000017093 TRCN0000017095 TRCN0000017096 TRCN0000017097 TRCN0000013493 TRCN0000013495 TRCN0000013496 TRCN0000013497 TRCN0000015548 TRCN0000015549 TRCN0000015551 TRCN0000015552 TRCN0000033460 TRCN0000033461 TRCN0000033462 TRCN0000033459 TRCN0000017999 TRCN0000018001

CCGGGCTACTCGGAAAGGAAACAAACTCGAGTTTGTTTCCTTTCCGAGTAGCTTTTT CCGGGCTCTTCTTTGATTACAGGTACTCGAGTACCTGTAATCAAAGAAGAGCTTTTT CCGGCCGCCGTGGTTTGTATTCATTCTCGAGAATGAATACAAACCACGGCGGTTTTT CCGGCAACAGAACTTTATGGTAGAACTCGAGTTCTACCATAAAGTTCTGTTGTTTTT CCGGGCTTCCTCTTCAACCTCAATACTCGAGTATTGAGGTTGAAGAGGAAGCTTTTT CCGGGCGGAGGATTTACGAATGCTTCTCGAGAAGCATTCGTAAATCCTCCGCTTTTTG CCGGCCGTTGGTAAACCTCACAAATCTCGAGATTTGTGAGGTTTACCAACGGTTTTTG CCGGGCCGAAGCTATAAACAGCGAACTCGAGTTCGCTGTTTATAGCTTCGGCTTTTTG CCGGCGCCAAACGTAAGAGCTCTATCTCGAGATAGAGCTCTTACGTTTGGCGTTTTTG CCGGCGATCACATTACGAACGCATTCTCGAGAATGCGTTCGTAATGTGATCGTTTTT CCGGGCCACATTGGAAGCCATAATTCTCGAGAATTATGGCTTCCAATGTGGCTTTTT CCGGCCAGTGCTAGATCAGTCTGTTCTCGAGAACAGACTGATCTAGCACTGGTTTTT CCGGCGCGTCCAAAGGCTAAATGAACTCGAGTTCATTTAGCCTTTGGACGCGTTTTT CCGGCAGCCCTTTCTTGAAAGGAAACTCGAGTTTCCTTTCAAGAAAGGGCTGTTTTT CCGGGCCCATCATCCTGAAGAAGTACTCGAGTACTTCTTCAGGATGATGGGCTTTTT CCGGCCGGCCACATTACCCTCCAAACTCGAGTTTGGAGGGTAATGTGGCCGGTTTTT CCGGGCGGCATAAGATGACCCTCATCTCGAGATGAGGGTCATCTTATGCCGCTTTTT CCGGGTGGAAGCTGAAATGCGTGTACTCGAGTACACGCATTTCAGCTTCCACTTTTT CCGGTTAGACCTGGACACCTTATTACTCGAGTAATAAGGTGTCCAGGTCTAATTTTTG CCGGTATGGGATCAACAGGTATTTACTCGAGTAAATACCTGTTGATCCCATATTTTTG CCGGTGGATTTGATGTCCCTATTATCTCGAGATAATAGGGACATCAAATCCATTTTTG CCGGGCAGTTGTATCGCTATGATAACTCGAGTTATCATAGCGATACAACTGCTTTTTG CCGGAGGCTCCCTTCACCTACATTTCTCGAGAAATGTAGGTGAAGGGAGCCTTTTTTG CCGGCCTCCAGAACTCCTGTTTATTCTCGAGAATAAACAGGAGTTCTGGAGGTTTTTG CCGGCGTGTCATCAATGAAGAATATCTCGAGATATTCTTCATTGATGACACGTTTTTG CCGGGCACAGTTTGATGCTTCCCATCTCGAGATGGGAAGCATCAAACTGTGCTTTTTG CCGGCGTTTCTATATGACCTGGTTACTCGAGTAACCAGGTCATATAGAAACGTTTTTG CCGGGCCCAAGTCTTGGTATGCTATCTCGAGATAGCATACCAAGACTTGGGCTTTTT CCGGCCTTGCATACATGGAGTCTAACTCGAGTTAGACTCCATGTATGCAAGGTTTTT CCGGGCCTCTGACATGCGATTTGAACTCGAGTTCAAATCGCATGTCAGAGGCTTTTT CCGGGCACCTATCTATGGTGCAGATCTCGAGATCTGCACCATAGATAGGTGCTTTTT CCGGGCAGGATATTAGCTCCTTATACTCGAGTATAAGGAGCTAATATCCTGCTTTTT CCGGCCCACCACATTTACCCATATTCTCGAGAATATGGGTAAATGTGGTGGGTTTTT CCGGGCTATGATACTTGGGTCCATACTCGAGTATGGACCCAAGTATCATAGCTTTTT CCGGCCTAGCTGTTTATCGACGGAACTCGAGTTCCGTCGATAAACAGCTAGGTTTTT CCGGGCCTAGACATTAAACTGAATACTCGAGTATTCAGTTTAATGTCTAGGCTTTTTG CCGGCCACGAGTCAAACCTTTATTTCTCGAGAAATAAAGGTTTGACTCGTGGTTTTTG CCGGCCCTTCCATGTTAGCACTTTACTCGAGTAAAGTGCTAACATGGAAGGGTTTTT CCGGCGGTGAAGATACTTATGATATCTCGAGATATCATAAGTATCTTCACCGTTTTT CCGGCCTCTATTACTACCTGACTAACTCGAGTTAGTCAGGTAGTAATAGAGGTTTTTG CCGGGCAGTGTAAGAACTTCTACTTCTCGAGAAGTAGAAGTTCTTACACTGCTTTTTG CCGGCCCTAGTTCATCGCAACCTTTCTCGAGAAAGGTTGCGATGAACTAGGGTTTTT CCGGGCGATCTTTGTAGAATTTGATCTCGAGATCAAATTCTACAAAGATCGCTTTTT CCGGGCTGTTAATGTGATGGTGTATCTCGAGATACACCATCACATTAACAGCTTTTT CCGGCCTTGTAGATAAACTGGGTTTCTCGAGAAACCCAGTTTATCTACAAGGTTTTT CCGGGCTGCAGTATTGAGATGCTAACTCGAGTTAGCATCTCAATACTGCAGCTTTTT CCGGGCACCGAGTTTGTCTTGAAATCTCGAGATTTCAAGACAAACTCGGTGCTTTTT CCGGCCGAAACTTCAGTAGATACATCTCGAGATGTATCTACTGAAGTTTCGGTTTTT CCGGGCCTTGGATCTTTCTGTGAATCTCGAGATTCACAGAAAGATCCAAGGCTTTTT CCGGCCATATTTATACAGCAGAGAACTCGAGTTCTCTGCTGTATAAATATGGTTTTT CCGGCCCGTGGACTTCAAGAAGATACTCGAGTATCTTCTTGAAGTCCACGGGTTTTT CCGGCCGAGGTCTGATAGTGAAGAACTCGAGTTCTTCACTATCAGACCTCGGTTTTT CCGGCGGCAGACACTGTGATCATTTCTCGAGAAATGATCACAGTGTCTGCCGTTTTT CCGGGCCAAATAAGTATTCACTGAACTCGAGTTCAGTGAATACTTATTTGGCTTTTTG CCGGCCACGAAACTTATACTTTCAACTCGAGTTGAAAGTATAAGTTTCGTGGTTTTTG CCGGGCTCTATATTTCTGTCTCCAACTCGAGTTGGAGACAGAAATATAGAGCTTTTTG CCGGCCCGCATATTTCGCTGCAATTCTCGAGAATTGCAGCGAAATATGCGGGTTTTTG CCGGCCTGGCTCTGTTCTCTGTCCTCTCGAGAGGACAGAGAACAGAGCCAGGTTTTT 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

a-fwd a-rev b-fwd b-rev c-fwd c-rev d-fwd d-rev e-fwd e-rev f-fwd f-rev g-fwd g-rev h-fwd h-rev HBE1-RT2-fwd HBE1-RT2-rev HBG-RT-fwd HBG-RT-rev HBB-RT-fwd HBB-RT-rev hGAPDH-RT-fwd hGAPDH-RT-rev ey-RT-fwd ey-RT-rev bh1-RT-fwd bh1-RT-rev bmaj/min-RTfwd bmaj/min-RT-rev mGapdh-RT-fwd mGapdh-RT-rev

Human

HS3

ChIP

Human

HBE1

ChIP

Human

HBG1

ChIP

Human

HBG1 +3kb

ChIP

Human

HBD -1kb

ChIP

Human

HBD

ChIP

Human

HBB

ChIP

Human

3′HS1

ChIP

Human

HBE1

RT-PCR

Human

HBG1/HBG2

RT-PCR

Human

HBB

RT-PCR

Human

GAPDH

RT-PCR

Mouse

Hbb-y

RT-PCR

Mouse

Hbb-bh1

RT-PCR

Mouse

Hbb-b1/Hbbb2

RT-PCR

Mouse

Gapdh

RT-PCR

Primer sequence (5′→3′) ATAGACCATGAGTAGAGGGCAGAC TGATCCTGAAAACATAGGAGTCAA GCCAGAACTTCGGCAGTAAA GGCCTGAGAGCTTGCTAGTG TTACTGCGCTGAAACTGTGG CAGTGGTTTCTAAGGAAAAAGTGC AATGACCTAATGCCCAGCAC AGTGTTGGGGGAGAAGTGTG GCAACAGAAGCCCAGCTATT GTGGCATGGTTTGATTTGTG TGTAGAGGAGAACAGGGTTT CTGCCTTTTATGCTGGTCCT TGCTCCTGGGAGTAGATTGG TGGTATGGGGCCAAGAGATA TCTTCAGCCATCCCAAGACT TGGTCTTTTCTGGACACCAC GCAAGAAGGTGCTGACTTCC ACCATCACGTTACCCAGGAG TGGATGATCTCAAGGGCAC TCAGTGGTATCTGGAGGACA CTGAGGAGAAGTCTGCCGTTA AGCATCAGGAGTGGACAGAT ACCCAGAAGACTGTGGATGG TTCAGCTCAGGGATGACCTT TGGCCTGTGGAGTAAGGTCAA GAAGCAGAGGACAAGTTCCCA TGGACAACCTCAAGGAGACC ACCTCTGGGGTGAATTCCTT TTTAACGATGGCCTGAATCACTT CAGCACAATCACGATCATATTGC TGGTGAAGGTCGGTGTGAAC CCATGTAGTTGAGGTCAATGAAGG

Product size (bp) 142 98 130 80 104 181 161 137 142 209 146 125 120 145 132

123

fwd, forward; rev, reverse.


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