LSD1-Mediated Epigenetic Modification Is Required for TAL1 Function and Hematopoiesis

LSD1-mediated epigenetic modification is required for TAL1 function and hematopoiesis

Xin Hua,b, Xingguo Lia, Kristell Valverdea, Xueqi Fub, Constance Noguchic, Yi Qiud,e, and Suming Huanga,e,f,1

Departments of aBiochemistry and Molecular Biology and dAnatomy and Cell Biology, fGenetics Institute, and eShands Cancer Center, University of Florida College of Medicine, Gainesville, FL 32610; bCollege of Life Science, Jilin University, Changchun 130023, China; and cMolecular Medicine Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Edited by Gary Felsenfeld, National Institutes of Health, Bethesda, MD, and approved April 30, 2009 (received for review January 14, 2009) TAL1 is a critical transcription factor required for hematopoiesis. catalyzed by oncogenic mixed lineage leukemia; (MLL), whose However, perturbation of its activity often leads to T cell leukemia. gene is found to be frequently rearranged in leukemia. In this Whether and how its transcriptional activities are regulated during regard, LSD1 may be potentially involved in cancer development hematopoiesis remains to be addressed. Here, we show that TAL1 is such as leukemogenesis. Furthermore, siRNA-mediated down- associated with histone demethylase complexes containing lysine- regulation of LSD1 in hematopoietic progenitors perturbs devel- speciﬁc demethylase 1 (LSD1), RE1 silencing transcription factor core- opment of several hematopoietic lineages (22), suggesting that pressor (CoREST), histone deacetylase 1 (HDAC1), and histone LSD1-mediated epigenetic modification may play an important deacetylase 2 in erythroleukemia and T cell leukemia cells. The role in hematopoietic development. enzymatic domain of LSD1 plays an important role in repressing the To further understand the mechanism by which TAL1-associated TAL1-directed transcription of GAL4 reporter linked to a thymidine complexes influence hematopoietic cell growth and differentiation, kniase minimal promoter. Furthermore, we demonstrate that the and how they contribute to T cell leukemogenesis, we have purified TAL1-associated LSD1, HDAC1, and their enzymatic activities are TAL1-containing protein complexes from TAL1-expressing mul- coordinately down-regulated during the early phases of erythroid tipotent erythroleukemia K562 and T-ALL Jurkat cells. We found differentiation. Consistent with the rapid changes of TAL1–corepres- that TAL1 recruits LSD1 complexes and HDM activity to a TAL1 sor complex during differentiation, TAL1 recruits LSD1 to the silenced target promoter. It represses transcription of the target gene by

p4.2 promoter in undifferentiated, but not in differentiated, murine altering the histone H3K4 methylation pattern on the promoter, BIOCHEMISTRY erythroleukemia (MEL) cells. Finally, shRNA-mediated knockdown of which suggests that TAL1 might regulate transcription by manipulating LSD1 in MEL cells resulted in derepression of the TAL1 target gene the histone code. In support of this view, down-regulation of LSD1 accompanied by increasing dimeH3K4 at the promoter region. Thus, specifically derepresses the TAL1 target gene by increasing H3K4 our data revealed that histone lysine demethylase LSD1 may nega- dimethylation and inhibits the erythroid differentiation program. tively regulate TAL1-mediated transcription and suggest that the The results suggest that the recruitment of epigenetic modifier dynamic regulation of TAL1-associated LSD1/HDAC1 complex may LSD1 complexes controls TAL1 function in hematopoiesis. determine the onset of erythroid differentiation programs. Results H3K4 methylation ͉ histone demethylation ͉ transcriptional regulation ͉ Purification of TAL1-Associated LSD1 Complex. To understand the erythroid differentiation ͉ stem cell leukemia (SCL) action of TAL1 in normal erythroid differentiation and leukemogenesis, we isolated TAL1-associated protein complexes through a he transcription factor TAL1 is a member of the basic helix– Flag epitope-specific immunoaffinity column followed by anti- Tloop–helix (bHLH) family of transcription factors that is re- TAL1 immunopurification from Flag-TAL1-expressing K562 cells. quired for the development of all hematopoietic lineages (1, 2). The purified complexes were resolved by SDS/PAGE and identified Aberrant activation of this transcription factor is involved in up to by liquid chromatography tandem mass spectrometry (LC-MS/MS) 60% of T cell acute lymphoblastic leukemia (T-ALL) cases, indi- (Fig. 1A). The majority of the polypeptides identified are involved cating that misregulation of TAL1’s activity may lead to the in pathways that regulate chromatin structure and transcription. development of T cell leukemia (3–5). Its transcription activity is They fall into the following categories (Fig. 1A): (i) E12/E47 and linked to either repression or activation of target genes during HEB, known to heterodimerize with TAL1, (ii) LSD1 complex, normal and malignant hematopoiesis (6). Importantly, TAL1 as- possessing H3K4 HDM activity, (iii) transcription factors, some of sociates with many binding partners including E2A, GATA1, them are known TAL1 binding partners, and (iv) ssDNA binding LMO2, LDB1, and ETO2, all of which are involved in modulating proteins, known as cofactors for LDB1 (23). We repeated the hematopoietic cell growth and differentiation (7–12). In particular, complex purification in Jurkat cells. Many of the TAL1-associated TAL1 differentially interacts with histone deacetylase 1/2 proteins identified match those purified from K562 cells (Fig. S1), (HDAC1/2) and histone acetyltransferases p300 and p300/CBP- suggesting that the complexes purified specifically interacted with associated factor (PCAF), which can alter TAL1 function by TAL1. Among them, the most notable complex is the LSD1 activating or repressing the transcription of downstream target complex that has not yet been reported as a TAL1-interacting A genes (13–15). Therefore, the data raise the possibility that TAL1 partner (Fig. 1 ). To validate TAL1 complexes containing both H3K4 demethylase LSD1 and HDAC1/2 (Fig. 1A and Fig. S1), we might regulate transcription by manipulating the histone code and performed a WB using the Flag immunoaffinity purification ma- local chromatin structure. Methylation of Lys-4 of histone H3 tails (H3K4) correlates with

transcriptionally-active euchromatin (16), and this methyl mark can Author contributions: Y.Q. and S.H. designed research; X.H., X.L., and K.V. performed be enzymatically removed by a newly-discovered histone demeth- research; C.N. contributed new reagents/analytic tools; X.H., X.L., X.F., Y.Q., and S.H. ylase (HDM), lysine-specific demethylase 1 (LSD1) (17). The core analyzed data; and S.H. wrote the paper. LSD1 complex consists of 4 subunits: LSD1, HDAC1/2, and RE1 The authors declare no conﬂict of interest. silencing transcription factor corepressor (CoREST) (18, 19). CoR- This article is a PNAS Direct Submission. EST and HDACs cooperatively stimulate LSD1 HDM activity by 1To whom correspondence should be addressed. E-mail: sumingh@uﬂ.edu. enhancing the binding of LSD1 to hypoacetylated nucleosomes This article contains supporting information online at www.pnas.org/cgi/content/full/ (19–21). Interestingly, LSD1 can demethylate H3K4 methylation 0900437106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900437106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 B C A B Peak fractions from MEL NE I, II, III and IV A LSD1complex Void 17 KDa 158 KDa 44 KDa

670 KDa Flag Ab IP

LC-MS/MS identification from Flag IP Frac: 2 4 6 8101214161820222426283032343638404244 46 48 50 5254 56 TAL1 expressed K562 cells: Flag peptide elute Flag-Tal1: IP: TAL1 -+ Input R-IgG anti-TAL1 WB No. of Protein ID Size (KDa) LSD1 Peptide LSD1 LSD1 CoREST I II III IV LSD1 110 2 HDAC1 Input HEB 73 11 HD1 IgG HDAC1 E12/E47 68 5 HDAC2 wB: anti-LSD1 I II III IV LSD1 ETO-2 67 4 RBBP7 HDAC1 64 3 HDAC1 CoREST HDAC2 62 1 2000 CoREST 50 1 D C 1800 D LDB1 43 3 1600 1.6 K562 Jurkat IP: 1400 SCL/TAL1 38 4 1200 α-LSD1: - ++ 1.2 SSBP3 40 2 1000

Input +-- SSBP2 37 1 R-IgG: 800 0.8 TAL1 600 36 kDa 400 0.4

wB: anti-TAL1 Reporter Luciferase 200 [3H] acetate release (CPM) E 0 Relative Fold activation of F 0 1 GST BG BG Gal4-Tal1: -++++++ HD2 1234567 1-330 2 bHLH GST-Tal1 Input 10% pcDNA-LSD1: -- I (1.8MDa)

bHLH II (650 KDa) - III (300 KDa) III (300 KDa) IV (<50 KDa) 3 GST-Tal1 1-144 IV (<50 KDa) pcDNA-LSD1∆C: 35S-LSD1 4 bHLH GST-Tal1 142-330 Fig. 2. TAL1 forms 2 distinct complexes with LSD1. (A) Nuclear extracts from 185-330 Jurkat cells expressing Flag-Tal1 were fractionated through the Sephacryl 5 bHLH GST-Tal1 * * * * S-300 HR column. The fractions were collected and analyzed by WB. (B) The 6 GST-Tal1 142-185 * peak fractions from 1.8 MDa (I), 650 kDa (II), 300 kDa (III), and Ͻ50 kDa (IV) * 7 GST-Tal1 242-330 * were collected. The pooled fractions were precipitated with Flag antibody and analyzed by WB. (C) The peak fractions from 1.8 MDa (I), 650 kDa (II), 300 kDa Ͻ Coomassie Stain (III), and 50 kDa (IV) were incubated with radiolabeled hyperacetylated histone. The amount of [3H]acetate released was quantitated by liquid scin- Fig. 1. TAL1 associates with LSD1 complex in erythroid and T cell leukemia tillation counting. (D) Demethylase activity of LSD1 is required to repress cells. (A) A partial list of TAL1-associated polypeptides identified by LC-MS/MS TAL1-mediated transcription. HeLa cells were transfected with a Gal4-TK-luc is indicated. (B) The Flag-purified complexes pulled down from Flag-Tal1 and reporter, an expression vector for GAL4-TAL1, and increased amounts of LSD1 mock-transduced Jurkat cells were analyzed by WB. (C) Nuclear extract from or LSD1⌬C. A CMV-driven renilla luciferase plasmid was used as a transfection MEL cells was precipitated with TAL1 antibody and analyzed by WB using LSD1 control. Transfected cells were cultured for 48 h and lysed for measurement of and HDAC1 antibodies. (D) Nuclear extracts from Jurkat and K562 cells were luciferase activity. precipitated with LSD1 antibody and analyzed by WB using TAL1 antibody. (E) Schematic representation of GST-TAL1 fusion proteins used in a GST pull- down assay. (F) 35S-labeled LSD1 was incubated with GST and GST-TAL1 fusion (Fig. 1F, compare lanes 4 and 6 with 5) (24). Whether or not proteins preabsorbed to glutathione-Sepharose beads. (Upper) Bound LSD1 phosphorylation affects TAL1 and LSD1 interaction remains to be was visualized by fluorography after SDS/PAGE. (Lower) Coomassie-stained determined. Furthermore, the C-terminal amine oxidase domain of gel shows the relative protein loading. LSD1 required for HDM activity and transcription repression (17) is not essential for LSD1 to interact with TAL1 (Fig. S2A). terial from the Jurkat nuclear extract. TAL1 specifically associates Therefore, the in vitro pull-down assay confirmed that TAL1 with the components of the core LSD1 complex, LSD1, HDAC1, directly interacts with LSD1. HDAC2, and RBBP7 (Fig. 1B). To further address whether endogenous TAL1 interacts with the TAL1 Recruits 2 Distinct LSD1 Complexes. Given that the TAL1- LSD1 complex, we performed immunoprecipitation assays in which associated LSD1 complex comprises multiple structural compo- murine erythroleukemia (MEL) nuclear extract was precipitated nents for its enzymatic activities (19–21), we tested whether TAL1 with TAL1 antibody and sequentially blotted with antibodies recruits the entire LSD1 complex by using gel filtration analysis specific to LSD1 and HDAC1. Endogenous TAL1 interacts spe- (Fig. 2A). TAL1, LSD1, CoREST, and HDAC1 comigrate in the cifically with LSD1 and HDAC1 in MEL cells (Fig. 1C). In addition, Ϸ650- and Ϸ300-kDa fractions (Fig. 2A), suggesting that TAL1 and the reverse immunoprecipitation reactions showed that the LSD1 LSD1 form 2 complexes of 650 and 300 kDa. To confirm this antibody sufficiently pulled down TAL1 in both human erythro- finding, the peak fractions corresponding to the sizes of 1.8 MDa leukemia K562 cells and human T-ALL Jurkat cells (Fig. 1D). Thus, (I), 650 kDa (II), 300 kDa (III), and Ͻ50 kDa (IV) were collected TAL1 associates with LSD1 complex in both T cell leukemia and separately, precipitated with anti-Flag conjugated agarose beads, erythroid progenitor cells. and analyzed for the presence of LSD1 complex components, CoREST, LSD1, and HDAC1, by WB analysis. CoREST, LSD1, TAL1 Physically Interacts with LSD1. Next, we sought to determine and HDAC1 are solely present in the 650 and 300-kDa fractions whether TAL1 directly interacts with LSD1 and to characterize the (Fig. 2B). These fractions also contain significant HDAC activity, domains involved in this interaction, TAL1 and its truncated whereas the 1.8-MDa (I) or 50-kDa (IV) size fractions have only mutants were expressed as GST fusion proteins (Fig. 1E), conju- minimum HDAC activity (Fig. 2C). Taken together, the data gated with glutathione-Sepharose beads, and incubated with suggest that TAL1 forms at least 2 distinct LSD1 complexes. [35S]methionine-labeled LSD1. Tal1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142–185 LSD1 Is Involved in TAL1-Mediated Transcriptional Repression. LSD1 proximal to the bHLH domain, which contains a Ser-172 site that is a putative transcriptional repressor. We further tested whether becomes phosphorylated upon erythropoietin (EPO) induction LSD1 is required for TAL1-mediated transcriptional repression.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900437106 Hu et al. Downloaded by guest on September 25, 2021 A Time in DMSO (days) C Time in DMSO (days) (Fig. 3A), suggesting that the LSD1 complex and hSET1 may differentially regulate hematopoietic cell development and that 01 3 5 IP: 0135 LSD1 plays a negative role in erythroid differentiation. R-IgG: + ---- LSD1 Given that the LSD1 complex interacts with and confers repres- Anti-TAL1: - + +++ sor activity to TAL1, and because its levels are greatly reduced during differentiation, we asked whether TAL1-associated LSD1 CoREST 98 LSD1 and its HDM activity are also changed throughout erythroid differentiation. To investigate this possibility, MEL nuclear extracts

HDAC1 64 CoREST prepared 0, 1, 3, and 5 days after DMSO treatment were subjected to immunoprecipitation with TAL1 antibody. The associated pro- hSET1 64 HDAC1 teins were identified by WB analysis. TAL1-associated LSD1, CoREST, and HDAC1 were decreased to a very low level at day 1 in the initial onset of differentiation and then came back at the LSD1 D LSD1 B 1.6 TAL1 IP CoREST CoREST later differentiation stage (Fig. 3 C and D). The significant decline HDA C1 1.2 HDAC1 1.2 of the TAL1–LSD1 complex coincides with the time period during 1 which the MEL cells became committed to differentiation (25) and 0.8 0.8 0.6 suggests that TAL1 was released from LSD1-containing repressor 0.4 0.4 complexes in the early stages of erythroid differentiation. 0.2 Next, we investigated whether the HDM activity of the TAL1 0

Relative Fold Enrichment 0 0d 1d 3d 5d Relative Fold Enrichment 0d 1d 3d 5d complex correlated with the dynamic changes of association with Time in DMSO LSD1. The TAL1-associated complexes purified from Flag-tagged E TAL1-expressing MEL nuclear extracts treated with DMSO for 0, 1, and 3 days were incubated with chicken core histones as substrate, 3d 12-24 h Time in DMSO: 0 and HDM activity was analyzed by using antibodies specific to TAL1. com: - +++ diMeH3K4 and AcH3K9/K14 (Fig. 3E). The TAL1 complex pu- LSD1: - - -+ Input rified from MEL cells had a strong HDM activity toward dimeH3K4 diMeH3K4 compatible to the recombinant LSD1 protein (Fig. 3E

0 2 5 9 1 BIOCHEMISTRY .0 .1 .1 .4 .1 Top, compare lanes 2 and 3). Upon DMSO-induced MEL differ- 1 0 0 0 0 entiation, TAL1-containing complexes lost the H3K4 HDM activ- AcH3K9/K14

0 3 2 1 8 ity in the 12- to 24-h period after the addition of DMSO (Fig. 3E .0 .4 .1 .3 .2 1 0 0 0 0 H3 Top, compare lane 3 and 4), which is correlated with a dramatic decrease in TAL1 and LSD1 complexes (Fig. 3D). However, in the 1 2345 late stage of differentiation, the TAL1-containing complexes re- Fig. 3. TAL1-associated LSD1 complex, HDM, and HDAC activities were gained H3K4 HDM activity (Fig. 3E Top, compare lanes 3–5). subjected to regulation during erythroid differentiation. (A) Nuclear extracts Parallel to the early decrease of HDM activity, TAL1-associated from MEL cells treated with DMSO for the indicated periods of time were HDAC activity was also decreased upon DMSO-induced differen- analyzed by WB. (B) The fold difference of the expressed proteins in MEL cells tiation (Fig. 3E Middle, compare lanes 3–5). The HDAC activity did was determined from 4 independent experiments using image analysis of not recover by day 3 of differentiation (Fig. 3E Middle, compare exposed X-ray films. (C) The same nuclear extracts were precipitated with TAL1 lanes 4 and 5), even though the TAL1 and HDAC1 interaction had antibody and analyzed for the presence of LSD1, CoREST, and HDAC1 in the complex. (D) The fold difference of the immunoprecipitated proteins was reappeared (Fig. 3D). Interestingly, although the LSD1 level grad- determined from 3 independent experiments using image analysis of exposed ually decreased during differentiation (Fig. 3B), the TAL1- X-ray films by ImageQuant 400. (E) The TAL1 complexes purified from MEL containing LSD1 complex and HDM activity significantly increased cells treated with DMSO for the indicated times were incubated with 1 ␮gof by day 3 of differentiation compared with day 1 (Fig. 3 C and E), chicken core histone in the HDM buffer. The changes of diMeH3K4 and suggesting that the activity of TAL1 complexes may be differentially AcH3K9/K14 were analyzed by WB. regulated during differentiation.

TAL1 Recruits LSD1 to Regulate Target Gene Expression During Erythroid A Tal1 cDNA fused to the DNA-binding domain of the yeast Differentiation. Erythroid protein EPB4.2 (P4.2) is an important transcription factor GAL4 was cotransfected with the GAL4- component of the erythrocyte membrane and cytoskeleton struc- TK-Luc reporter and increasing amounts of an LSD1-encoded ture that regulates stability and flexibility of erythrocytes (26). plasmid into HeLa cells. The coexpression of LSD1 inhibited Within the P4.2 gene, 2 E box-GATA motifs are located at its TAL1-mediated reporter activity in a dose-dependent fashion proximal promoter (Fig. 4A). The binding of TAL1 to the promoter (Fig. 2D). In contrast, the deletion of the amine oxidase domain, depends on the integrity of these motifs and is critical for P4.2 gene which is essential for the HDM activity of LSD1, resulted in a expression (12). Fig. 4B shows that the P4.2 gene is suppressed in complete loss of repression of TAL1-directed transcription (Fig. undifferentiated MEL cells and induced upon DMSO treatment. 2D), indicating that LSD1 represses TAL1-mediated transcrip- We reasoned that TAL1 might target LSD1 to the P4.2 promoter tional activity via its HDM activity. Given the role of TAL1 in to inhibit P4.2 expression in undifferentiated MEL cells and might hematopoiesis, the data also suggest that HDM activity of LSD1 dissociate from LSD1 after differentiation. To test this possibility, may be required for TAL1-mediated transcriptional repression we carried out TAL1 ChIP and TAL1/LSD1 sequential ChIP to during hematopoiesis. determine whether TAL1 and LSD1 colocalize at the P4.2 promoter before and after differentiation and whether colocalization TAL1-Associated LSD1 and HDM Activity Are Dynamically Regulated results in the changes of promoter H3K4 methylation. As expected, During Hematopoiesis. To access the role of LSD1 in hematopoiesis, TAL1 is specifically bound to the P4.2 promoter but not to the 3Ј we determined the expression levels of LSD1 core complex and UTR (Fig. 4C). Although the binding of TAL1 alone at the P4.2 hSET1 in DMSO-induced MEL cell differentiation. The levels of promoter did not change upon DMSO induction (Fig. 4C, compare LSD1 and CoREST gradually decline during erythroid differenti- lanes 5 and 7), TAL1 and LSD1 only cooccupied the P4.2 promoter ation whereas HDAC1 remains relatively constant (Fig. 3 A and B). when P4.2 was silenced and the P4.2 promoter-bound LSD1 was In contrast, hSET1 is significantly increased upon differentiation significantly decreased upon activation of P4.2 in differentiated cells

Hu et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 A B MEL Double E box/GATA sites Double E box/GATA sites A P4.2 gene P4.2 gene DMSO: - + P4.2 Primers: bK6- P4.2P RTU’3 GAPDH Primers: bK6- P4.2P RTU’3

C α-TAL1 ChIP: D Double ChIP: 12 vect or -LSD1 C α B LSD1 kd 10 LSD1 -TAL1 -TAL1/ ∆ IP: α 8 Seq. ChIP α Input IgG IP: Input IgG - - - + - -- DMSO: DMSO: + 6 MEL-pSuper MEL- Primer set: MEL Primer set: 4 P4.2P P4.2P 3’UTR 3’UTR P4.2P P4.2P 3’UTR 3’UTR LSD1 P4.2P 3’UTR P4.2P 3’UTR P4.2P P4.2P 3’UTR 3’UTR P4.2P P4.2P 2 β-actin 3’UTR 3’UTR

12 34 5 6 78 level mRNA The relative 0 12345678

12 P4.2 E TAL1 F Day 0

10 LSD1 -globin GATA-2 Day 3 H3K4me2 GATA-1 β 8.0 β 8 7.0 D 6 6.0 H3K4me2 5.0 Ve ctor 4 4.0 8.0 3.0 7.0 KD 2 2.0 6.0 0 1.0 0.0 5.0 Relative fold enrichment

Relative fold enrichment 4.0 3’UTR 3’UTR IgG IgG IgG -dime -dime H3k4 H3k4 -dime P4.2 Pro. P4.2 Pro. 3.0 H3k4 α α α 2.0 DMSO: 0 Day 3 Day -6Kb P4.2P 3’UTR 1.0 0.0 Fig. 4. TAL1 recruits LSD1 to the p4.2 promoter in undifferentiated, but not differentiated, MEL cells. (A) Schematic representation of the mouse P4.2 Relative fold enrichment IgG IgG gene locus. The primers are indicated. (B) mRNAs from WT or TAL1-transduced IgG -dime -dime H3k4 H3k4 -dime H3k4

MEL cells treated with 1.5% DMSO were purified and analyzed by Northern α α α blotting using the P4.2 probe. (C and D) Cross-linked chromatin from MEL cells in the presence and absence of 1.5% DMSO was precipitated with TAL1 -6Kb P4.2P 3’UTR antibody. The TAL1-chromatin complexes were reversed and the selected DNA was analyzed by PCR (C). (D) TAL1-DNA complexes from the first TAL1 ChIP Fig. 5. The KD of LSD1 derepresses the expression of P4.2 gene. (A) Schematic were subjected to a second IP with anti-LSD1. The cross-links of these double representation of the mouse P4.2 gene locus. The primers are indicated. (B) IP protein/DNA complexes were reversed, and the DNA was analyzed by PCR. WB analysis of whole-cell extracts from WT, pSuper vector control, and LSD1 (E) Cross-linked chromatin from MEL cells in the presence and absence of 1.5% KD MEL cells. (C) The RNA from the pSuper vector or LSD1 KD (shLSD1) MEL DMSO was precipitated with TAL1 and LSD1 antibodies. The selected chro- cells was isolated and quantitative RT-PCR was performed. (D) The cross-linked matin complexes were reversed and analyzed by quantitative PCR. (F) The chromatin from the vector control and LSD1 KD MEL cells were precipitated cross-linked chromatin from MEL cells treated with or without DMSO was with dimeH3K4 antibody. The precipitated DNA fragments were amplified by precipitated with dimeH3K4 antibody. The precipitated DNA fragments were using primers specific for the Ϫ6 Kb, the p4.2 promoter, and 3Ј UTR regions. amplified by using primers specific for Ϫ6 Kb, the P4.2 promoter, and 3Ј UTR regions by quantitative PCR. retroviral-mediated expression of LSD1 shRNA. The KD MEL Ϸ (Fig. 4D, compare lanes 5 and 7). In addition, quantitative ChIP cells displayed an 70% reduction in LSD1 protein levels com- confirmed that the P4.2 promoter-bound LSD1 was markedly pared with the parental and the vector control MEL cells (Fig. 5B). We then examined the effects of LSD1 KD on terminal erythroid decreased upon differentiation, whereas TAL1 binding was in- differentiation by benzidine staining for the accumulation of he- creased (Fig. 4E). Furthermore, accompanying the loss of TAL1- moglobin. The benzidine-positive cells were significantly reduced in associated LSD1 at the P4.2 promoter upon differentiation was a Ͼ the LSD1 KD cells (Fig. S3A), reflecting the inhibition of erythroid 11-fold increase in dimeH3K4 at the same region in differentiated differentiation. Importantly, the KD of LSD1 in MEL cells resulted MEL cells (Fig. 4F). Histone H3 and H4 acetylation were also in an increased expression of p4.2 and GATA-2, whereas the up-regulated and depended on the TAL1 binding of the P4.2 expression of ␤-globin and GATA-1 was significantly inhibited (Fig. promoter (Fig. S2B). The elevated dimeH3K4 and histone acety- 5C). To further confirm the role of LSD1 in hematopoiesis, we lation at the promoter correlated with the up-regulation of P4.2 knocked down LSD1 in mouse ES cells that were differentiated into transcription (Fig. 4B). The results suggest that the recruitment of erythrocytes in the presence of EPO (Fig. S3B). The effects of LSD1 and its HDM activity by TAL1 may play a role in silencing LSD1 KD in ES cells resembled the MEL KD data (Fig. S3C). the P4.2 gene in undifferentiated MEL cells, and decreased LSD1 Interestingly, in both cells, GATA-2 is up-regulated, whereas occupancy at the promoter then allows TAL1 to activate P4.2 upon GATA-1 is repressed, which suggests that LSD1 may regulate differentiation. erythroid differentiation by controlling the interplay of GATA factors, although it is currently unclear whether LSD1 regulates Knockdown (KD) of LSD1 in Erythroid Cells Leads to the Derepression of GATA factors directly or indirectly. This should be the focus of the TAL1 Target Genes. If LSD1 is required for TAL1-mediated future studies. transcriptional repression, then the down-regulation of LSD1 by Next, we tested whether the derepression of the TAL1-targeted siRNA should derepress the TAL1 target gene. We directly ad- gene p4.2 upon the depletion of LSD1 was accompanied by an dressed this notion by knocking down LSD1 expression using increase in dimeH3K4 at the p4.2 promoter (Fig. 5A). A ChIP assay

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900437106 Hu et al. Downloaded by guest on September 25, 2021 was carried out by using antibody specific to dimeH3K4 in the with LSD1 and demethylase activity in undifferentiated MEL cells, vector control and LSD1 KD MEL cells. As we expected, the but not in day 1 differentiated cells (Fig. 3 C and E), which coincides KD of LSD1 led to a 23-fold increase in dimeH3K4 level at with the time period during which the MEL cells become commit- the P4.2 proximal promoter (Fig. 5D). In contrast, the level of ted to differentiation (25). However, association with the LSD1 dimeH3K4 was moderately elevated at the Ϫ6 Kb (3.4-fold) complex was recovered during the late phases of differentiation and 3Ј UTR (2.8-fold) regions upon LSD1 depletion (Fig. 5D). (Fig. 3C). There are 2 possible scenarios for the dynamic changes These data demonstrate that LSD1-mediated H3K4 demeth- of TAL1/LSD1 interaction during differentiation. First, in progen- ylation plays a critical role in maintaining locally-repressive itor cells, LSD1 is recruited to restrict the ability of these cells chromatin structure of the p4.2 gene in early hematopoietic responding to cellular differentiation program. After differentia- progenitors, presumably targeted by TAL1. It is possible that tion, LSD1 is then switched to repress TAL1 target genes that the ectopic activation of this late terminal differentiation mark promote cellular proliferation. In supporting this view, LSD1 may perturb erythroid differentiation. disappeared from the p4.2 promoter (Fig. 4 D and E) and the promoter became H3K4-hypermethylated and histone-hyperacety- Discussion lated during differentiation (Fig. 4F and Fig. S2B). Another pos- Although several studies have revealed that the recruitment of sibility is that the LSD1 protein complex changes its cofactor coactivators and corepressors is the likely mechanism underlying composition, which may alter its enzyme specificity. For example, TAL1-mediated transcriptional regulation (7, 9, 13–15), it remains when androgen receptor (AR) associates with LSD1, it changes the unclear how TAL1 transcriptional activity is controlled during substrate specificity of LSD1 to demethylate repressive monoH3K9 hematopoiesis. To exert transcriptional repression at certain stages or dimeH3K9 marks, which then promotes AR-dependent tran- of hematopoiesis, TAL1 can associate with several corepressors, scription (31). Therefore, LSD1 can repress or activate transcription including mSin3A, HDACs, and ETO-2 (7, 9, 14). LSD1 is a by removing specific epigenetic makers and creating a condensed lysine-specific HDM that specifically removes monoH3K4 and or relaxed chromatin environment, respectively. In supporting the dimeH3K4 (17). CoREST and HDAC1/2 can greatly potentiate possible dual functions of LSD1 during hematopoiesis, we found LSD1 HDM activity (19–21). The recruitment of LSD1 by TAL1 that TAL1 forms 2 LSD1 complexes in Jurkat cells (Fig. 2 A–C). suggests that the H3K4 HDM activity of LSD1 in part confers the Second, overexpression or down-regulation of LSD1 inhibits ery- repressive activity of TAL1 and may restrict TAL1 function in throid differentiation (Fig. S3 and Fig. S4), suggesting a dual role hematopoiesis (9, 14), therefore contributing to TAL1-induced for LSD1 in erythroid differentiation.

leukemogenesis (27). This idea is supported by the observation that What is the biological role of LSD1 in hematopoiesis? LSD1 BIOCHEMISTRY overexpression of LSD1 stimulates growth of erythroid progenitor regulates gene expression by modulating active or repressive chro- cells (Fig. S4A). matin domains (32, 33). It could potentially connect to human LSD1 associates with CoREST, HDAC1/2, and other compo- disease by perturbing normal gene expression patterns that are nents that are required for its enzymatic activity and transcriptional required for normal cell growth and differentiation. TAL1 is an repression (19–21). In T-ALL Jurkat cells, TAL1 forms 2 distinct oncoprotein whose activation is associated with a majority of complexes with LSD1 (Fig. 2 A and B). According to the predicted T-ALL patients. The repressive function of TAL1 has been linked molecular mass, the 300-kDa complex apparently consists of the to leukemogenesis (9, 28). In T cell leukemia, TAL1 inhibits LSD1 core complex, whereas the 650-kDa complex might contain E2A/HEB transcriptional activity and perturbs cell cycle progres- additional components. Although both complexes possess strong sion during the DN stage, where normal thymocytes undergo deacetylase and demethylase activities (Figs. 2C and 3E), the extensive cell division (6, 27). Thus, the possible function of difference of these complexes in regulating TAL1 and LSD1 corepressor LSD1 in TAL1-mediated transcriptional repression functions in normal or malignant hematopoiesis is currently un- (Figs. 2D and 5 B–D) suggests that LSD1 may be involved in known. Nevertheless, protein–protein interactions between LSD1 leukemogenesis by repressing E2A/HEB activity through its inter- and its associated components control its enzymatic activity and action with TAL1. substrate specificity. It is possible that the unknown components in the TAL1/LSD1 complex may be specifically required for control- Materials and Methods ling the action of TAL1 or LSD1 during hematopoiesis. Cell Lines, Transfection, and shRNA KD. K562, MEL, and Jurkat cells were main- The deletion of LSD1 perturbs hematopoietic differentiation in tained as described (14). Murine ES cells were cultured and induced to differen- both MEL and murine ES cells (Fig. 5C and Fig. S3), suggesting that tiate with EPO as described (34). Transfection, plasmid constructs, and shRNA- the ubiquitously-expressed LSD1 controls tissue-specific differen- mediated KD are described in SI Text. The primer sequences are available in SI tiation processes, perhaps by interacting with tissue-specific tran- Text. scription factors. Accordingly, LSD1 interacts with hematopoietic- specific repressor Gfi-1b to affect erythroid, megakaryocytic, and Purification of TAL1 Complexes. TAL1 complexes were purified by a Flag immunoaffinity column from retroviral vector transduced K562 or Jurkat cells express- granulocytic differentiations (22). The data from Orkin’s group ing a FLAG/HA double-tagged Tal1 by using a system developed by Nakatani and (22) and our study suggest that LSD1 exerts its repressive effects colleagues (35). The FLAG-purified materials were subsequently purified with with the guidance of hematopoietic-specific transcription factors, TAL1-specific antibodies. The purified polypeptides were identified by LC-MS/MS such as TAL1 and Gfi-1b, to control hematopoietic-restricted gene at the Harvard Medical School Taplin Protein Sequence Facility, Boston. expression. Several lines of evidence suggest that TAL1 and Gfi-1b physically interact with each other and become part of the same Gel Filtration, GST Pull-Down, and HDM Assays. Jurkat nuclear extract was frac- transcriptional pathway. For example, the deletion of TAL1 and tionated on a Sephacryl S-300HR column (GE Healthcare). The eluted fractions Gfi-1b in adult hematopoietic cells exhibits similar phenotypes were analyzed by WB and immunoprecipitation analysis. The GST pull-down (14) (28–30). In addition, corepressor ETO-2 mediates the interaction and histone demethylation (17) assays were carried out as described. Briefly, for between TAL1 and Gfi-1b in erythroid cells (9). Although ETO-2 the HDM assay, the HDM reaction mixture was analyzed by SDS/PAGE/WB by using dimeH3K4- or diacH3K9/K14-specific antibodies (Upstate). is present in the TAL1/LSD1 complexes from our biochemical purifications from K562 and Jurkat cells (Fig. 1A and Fig. S1), it RNA Isolation and Quantitative RT-PCR. Total RNA isolated with a SV total RNA remains to be determined whether TAL1, Gfi-1b, ETO-2, and isolation kit (Promega) were reverse-transcribed by using oligo(dT) and SYBR LSD1 are in the same complex. Green quantitative PCR was performed as described (36). It has been reported that TAL1 dynamically interacts with coactivators and corepressors during erythroid differentiation (9, ChIP and Double ChIP. Real-time PCR quantitative ChIP assays were carried out as 14, 15). Consistent with this view, we found that TAL1 associates described (25) with slight modifications. Double ChIP was performed as described

Hu et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 25, 2021 (37). LSD1 and CoREST antibodies were purchased from Upstate. TAL1 and London) for reagents and Jo¨rg Bungert and Lizi Wu for helpful suggestions and HDAC1 were obtained from Santa Cruz Biotechnology. comments on the manuscript. This work was supported by National Institutes of Health Grants HL090589 and HL091929 (to S.H.) and grants from the Bankhead- ACKNOWLEDGMENTS. We thank Jo¨rg Bungert (University of Florida, Gaines- Coley Cancer Research Program (to S.H. and Y.Q). C.N. is supported by the ville), Yoshihiro Nakatani (Dana-Farber Cancer Institute, Boston), Yang Shi (Har- Intramural Research Program, National Institute of Diabetes, Digestive and Kid- vard Medical School, Boston), and Eric So (The Institute of Cancer Research, ney Diseases, National Institutes of Health.

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