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 aDivision 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 as a central modulator of HbF expression and globin switching severity of the common β-globin disorders. The transcription factor in vivo. Further molecular studies revealed that BCL11A inter- BCL11A is a critical modulator of hemoglobin switching and HbF acts with several erythroid regulators including GATA1, FOG1, silencing, yet the molecular mechanism through which BCL11A and SOX6, and with the nucleosome remodeling and deace- coordinates the developmental switch is incompletely understood. tylase complex (NuRD) (4, 6). BCL11A acts within the β-globin Particularly, the identities of BCL11A cooperating protein complexes cluster by associating with several discrete regions, including fi and their roles in HbF expression and erythroid development re- sequences speci cally deleted in patients with hereditary per- sistence of fetal hemoglobin (HPFH). However, BCL11A does main largely unknown. Here we determine the interacting partner γ proteins of BCL11A in erythroid cells by a proteomic screen. BCL11A not bind detectably to the -globin promoter in erythroid chro- is found within multiprotein complexes consisting of erythroid tran- matin, suggesting that its mode of action is more complex than simply blocking transcription at the proximal promoter. This scription factors, transcriptional corepressors, and chromatin- fi observation is consistent with a role of BCL11A in promoting modifying enzymes. We show that the lysine-speci c demethylase long-range chromosomal interactions within the β-globin locus 1 and repressor element-1 silencing transcription factor corepressor 1 (6, 7, 9). Therefore, in an effort to elucidate further the precise (LSD1/CoREST) histone demethylase complex interacts with BCL11A molecular mechanisms by which BCL11A coordinates the switch, and is required for full developmental silencing of mouse embryonic it is relevant to identify systematically BCL11A-interacting partner β-like globin genes and human γ-globin genes in adult erythroid proteins, and use functional and genetic approaches to assess their cells in vivo. In addition, LSD1 is essential for normal erythroid individual roles in HbF regulation and erythroid cell maturation. In development. Furthermore, the DNA methyltransferase 1 (DNMT1) addition to providing important insights into the molecular mech- is identified as a BCL11A-associated protein in the proteomic screen. anisms through which BCL11A controls hemoglobin switching, DNMT1 is required to maintain HbF silencing in primary human adult our data suggest opportunities, as well as challenges, for therapeutic erythroid cells. DNMT1 haploinsufficiency combined with BCL11A induction of HbF in patients with the major hematologic disorders. deficiency further enhances γ-globin expression in adult animals. Our findings provide important insights into the mechanistic roles Results of BCL11A in HbF silencing and clues for therapeutic targeting of BCL11A-Interacting Partner Proteins in Erythroid Cells. We charac- BCL11A in β-hemoglobinopathies. terized BCL11A-containing multiprotein complexes by a proteomic affinity screen using a metabolic biotin tagging approach (10). Mu- gene regulation | globin switching | hematopoiesis rine erythroleukemia (MEL) cell lines that stably express the bac- terial biotin ligase BirA and the recombinant BCL11A (XL isoform) containing a FLAG epitope tag together with a BirA recognition etal hemoglobin (HbF, α2γ2) is a major genetic modifier of the β motif fused to its amino terminus were generated (FB-BCL11A; Fig. Fphenotypic heterogeneity in patients with the major -globin A disorders sickle cell disease (SCD) and β-thalassemia (1). The 1 ). Clones were selected that express the recombinant BCL11A synthesis of hemoglobin undergoes switching during ontogeny at levels similar to endogenous BCL11A (Fig. S1). Single streptavidin affinity purification or tandem anti-FLAG such that HbF is the predominant hemoglobin produced during fi fi fi fetal life and is gradually silenced and replaced by adult hemo- immunoaf nity and streptavidin af nity puri cation was performed α β γ on nuclear extracts from MEL cells stably expressing BirA alone globin (HbA, 2 2) around birth. Because increased -globin ex- fi pression in adults can substitute for the mutant or absent β-globin or BirA together with FB-BCL11A. Copuri ed proteins were β fractionated in SDS/PAGE, followed by liquid chromatography in SCD and -thalassemia, respectively, the fetal-to-adult globin A fi switch is critical to the pathogenesis of these conditions. As a re- and tandem MS peptide sequencing (Fig. 1 ). We identi ed nu- sult, intense efforts have been aimed to elucidate the molecular merous peptides of BCL11A and nearly all previously established mechanisms of HbF silencing and to develop target-based ther- interacting proteins, including FOG1, the entire NuRD complex, and the nuclear matrix protein MATRIN-3 (4). Besides previously apeutics to enhance HbF production (2). fi Recent genomewide association studies (GWAS) led to the recognized partners, we also identi ed a panel of previously un- identification of a new HbF-associated locus on chromosome 2, discovered interacting complexes, including the hematopoietic reg- located within the gene BCL11A (3). Subsequent studies demon- ulators RUNX1 and IKZF1 (or IKAROS), several transcriptional fi corepressor complexes, and the SWI/SNF chromatin remodeling strated that BCL11A, a zinc- nger transcription factor, is a bona B fide repressor of HbF expression (4–7). BCL11A protein is deve- complex (Fig. 1 ). The BCL11A-interacting corepressors include lopmentally 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 Author contributions: J.X. and S.H.O. designed research; J.X., D.E.B., M.A.K., T.D.V., S.H., globin switching and impaired HbF silencing in adult erythroid Y.-J.H., and J.J.T. performed research; H.Y. and G.M. contributed new reagents/analytic cells (5, 8). Previously silenced γ-globin genes can also be reac- tools; J.X. and S.H.O. analyzed data; and J.X. and S.H.O. wrote the paper. tivated by loss of BCL11A in adult animals (8). Most importantly, The authors declare no conflict of interest. inactivation of BCL11A alone is sufficient to ameliorate the he- Freely available online through the PNAS open access option. matologic and pathologic defects associated with SCD through 1To whom correspondence should be addressed. E-mail: [email protected]. high-level HbF induction in humanized mouse models (8). These This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genetic studies provide persuasive evidence that BCL11A functions 1073/pnas.1303976110/-/DCSupplemental. 6518–6523 | PNAS | April 16, 2013 | vol. 110 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1303976110 Downloaded by guest on September 29, 2021 # of Peptides targeting BCL11A or its interacting proteins during an expansion A B Protein Identity Cells Expressing MEL K562 phase, followed by erythroid differentiation for 5–7d.Knockdown BirA + FB-BCL11A BCL11A 13a, 11a, 12b 17a, 14a, 11a, 12b fi Transcription factors of each gene was con rmed by qRT-PCR, and shRNAs that re- Nuclear Extract FOG1 (ZFPM1) 1, 2, 0 0, 0, 0, 0 sulted in >75% decrease of target mRNA expression were selected RUNX1 1, 4, 0 0, 0, 0, 1 IKAROS (IKZF1) 1, 3, 0 0, 0, 0, 0 for subsequent analyses (Table S1; Figs. S2 and S3). KLF1 (or NuRD complex EKLF) is a critical erythroid regulator required for adult β-globin β BCL11A α Mi-2 (CHD4) 8, 8, 1 21, 10, 21, 10 -FLAG Mi-2α (CHD3) 0, 0, 0 4, 4, 9, 2 transcription (11). Recently it was shown that KLF1 occupies the MTA1 0, 0, 0 6, 5, 2, 1 BCL11A promoter and activates its expression (12, 13). Consistent MTA2 0, 0, 1 23, 16, 13, 2 MTA3 0, 0, 0 2, 2, 0, 0 with previous analysis, knockdown of BCL11A or KLF1 results FLAG Peptide Elution HDAC1 7, 7, 5 19, 16, 8, 4 in marked increase in γ-globin expression and serves as positive HDAC2 5, 4, 2 12, 15, 6, 2 RBBP4 3, 3, 1 4, 5, 5, 1 controls for the RNAi screen. We first ranked the genes according RBBP7 2, 1, 1 7, 6, 6, 1 to the level of human γ-globin expression (Fig. 2B). Notably, the BCL11A Streptavidin MBD3 0, 0, 0 7, 6, 0, 0 P66α (GATAD2A) 0, 0, 0 29, 10, 4, 1 top of the list consists of subunits of several transcriptional co- P66β (GATAD2B) 2, 1, 0 12, 12, 5, 2 LSD1/CoREST complex repressor complexes, including the NuRD complex (CHD4, SDS-PAGE Fractionation LSD1 (KDM1A) 15, 27, 8 1, 5, 12, 5 HDAC1, HDAC2, and MBD2), DNMT1, SIN3A, NCOR1, and CoREST (RCOR1) 8, 9, 3 8, 8, 3, 1 NCoR/SMRT complex the polycomb repressive complex 2 (PRC2; EZH2, EED, and NCoR (NCOR1) 6, 14, 3 0, 0, 2, 0 LC-MS/MS SUZ12). shRNA-mediated depletion of BCL11A-interacting pro- SMRT (NCOR2) 13, 16, 2 0, 0, 8, 0 e TBLR1 0, 2, 0 4, 0, 2, 0 teins also results in derepression of the human embryonic -gene. TBL1 0, 0, 0 3, 0, 0, 0 We next ranked the genes according to the level of e-globin ex- CORO2A 2, 1, 0 0, 0, 0, 0 C KAISO (ZBTB33) 7, 7, 6 26, 21, 18, 8 pression (Fig.