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• W2108453860 abstract "•The MLL1 SET domain is not necessary for hematopoietic target gene regulation•The endogenous MLL1 HMT activity is not needed for MLL-AF9 transformation•MOF-mediated H4K16Ac at hematopoietic MLL1 target genes is critical for expression•SIRT1 is a specific antagonist for MLL1 gene activation in hematopoietic cells Despite correlations between histone methyltransferase (HMT) activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4) methyltransferase critical for maintaining hematopoietic stem cells (HSCs). Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16) acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function. Despite correlations between histone methyltransferase (HMT) activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4) methyltransferase critical for maintaining hematopoietic stem cells (HSCs). Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16) acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function. Understanding the contribution of chromatin-modifying complexes to precise and heritable patterns of gene regulation in mammals has been a challenging task due to the complexity, redundancy, and, often, ubiquitous expression of such complexes. Among the first factors shown to influence the heritable transmission of gene expression states were trithorax Group (trxG) and Polycomb group (PcG) proteins (Cavalli and Paro, 1999Cavalli G. Paro R. Epigenetic inheritance of active chromatin after removal of the main transactivator.Science. 1999; 286: 955-958Crossref PubMed Scopus (211) Google Scholar, Schuettengruber et al., 2011Schuettengruber B. Martinez A.M. Iovino N. Cavalli G. Trithorax group proteins: switching genes on and keeping them active.Nat. Rev. Mol. Cell Biol. 2011; 12: 799-814Crossref PubMed Scopus (339) Google Scholar). Following their genetic identification in Drosophila melanogaster, a common protein domain defined by Su(var)3-9, Ezh2, Trithorax (SET) homology was subsequently demonstrated to possess histone methylation activity. More recently, it has become clear that histone methylation enzymes beyond the trxG/PcG homologs function in many aspects of epigenetic gene regulation from yeast to human (Shilatifard, 2012Shilatifard A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.Annu. Rev. Biochem. 2012; 81: 65-95Crossref PubMed Scopus (714) Google Scholar). The first identified mammalian member of the trxG family, MLL1, was initially noted as a gene frequently rearranged in poor-prognosis leukemia (Grossmann et al., 2012Grossmann V. Schnittger S. Kohlmann A. Eder C. Roller A. Dicker F. Schmid C. Wendtner C.M. Staib P. Serve H. et al.A novel hierarchical prognostic model of AML solely based on molecular mutations.Blood. 2012; 120: 2963-2972Crossref PubMed Scopus (210) Google Scholar, Krivtsov and Armstrong, 2007Krivtsov A.V. Armstrong S.A. MLL translocations, histone modifications and leukaemia stem-cell development.Nat. Rev. Cancer. 2007; 7: 823-833Crossref PubMed Scopus (917) Google Scholar). Mammals possess three pairs of related SET-domain containing histone methyltransferase (HMT) proteins, MLL1/2, SET1A/B, MLL3/4, and a divergent MLL5 protein. In addition to MLL1 translocations in leukemia, mutations in several trxG/MLL family members are associated with developmental disorders and cancer (Jones et al., 2012Jones W.D. Dafou D. McEntagart M. Woollard W.J. Elmslie F.V. Holder-Espinasse M. Irving M. Saggar A.K. Smithson S. Trembath R.C. et al.De novo mutations in MLL cause Wiedemann-Steiner syndrome.Am. J. Hum. Genet. 2012; 91: 358-364Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, Morin et al., 2011Morin R.D. Mendez-Lago M. Mungall A.J. Goya R. Mungall K.L. Corbett R.D. Johnson N.A. Severson T.M. Chiu R. Field M. et al.Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.Nature. 2011; 476: 298-303Crossref PubMed Scopus (1244) Google Scholar, Ng et al., 2010Ng S.B. Bigham A.W. Buckingham K.J. Hannibal M.C. McMillin M.J. Gildersleeve H.I. Beck A.E. Tabor H.K. Cooper G.M. Mefford H.C. et al.Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome.Nat. Genet. 2010; 42: 790-793Crossref PubMed Scopus (993) Google Scholar, Parsons et al., 2011Parsons D.W. Li M. Zhang X. Jones S. Leary R.J. Lin J.C. Boca S.M. Carter H. Samayoa J. Bettegowda C. et al.The genetic landscape of the childhood cancer medulloblastoma.Science. 2011; 331: 435-439Crossref PubMed Scopus (585) Google Scholar, Pasqualucci et al., 2011Pasqualucci L. Trifonov V. Fabbri G. Ma J. Rossi D. Chiarenza A. Wells V.A. Grunn A. Messina M. Elliot O. et al.Analysis of the coding genome of diffuse large B-cell lymphoma.Nat. Genet. 2011; 43: 830-837Crossref PubMed Scopus (791) Google Scholar). How these broadly expressed proteins influence particular physiologic and pathologic processes in specific cell types is largely unknown but, due to their enzymatic activities, represent attractive drug targets. Tissue-specific loss-of-function models together with biochemical approaches have delineated biologically important target genes and candidate mechanisms by which mammalian trxG/MLL family proteins function. The fact that these large proteins function within multiprotein complexes and frequently lack satisfactory in vivo structure-function assays has limited our understanding of gene regulatory mechanisms used in the tissues in which they function. To determine MLL1 function in vivo, we employed inducible Mll1 loss-of-function alleles and demonstrated that Mll1 plays a unique and essential role in HSCs and developing B cells (Artinger et al., 2013Artinger E.L. Mishra B.P. Zaffuto K.M. Li B.E. Chung E.K. Moore A.W. Chen Y. Cheng C. Ernst P. An MLL-dependent network sustains hematopoiesis.Proc. Natl. Acad. Sci. USA. 2013; 110: 12000-12005Crossref PubMed Scopus (58) Google Scholar, Jude et al., 2007Jude C.D. Climer L. Xu D. Artinger E. Fisher J.K. Ernst P. Unique and independent roles for MLL in adult hematopoietic stem cells and progenitors.Cell Stem Cell. 2007; 1: 324-337Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, Li et al., 2013Li B.E. Gan T. Meyerson M. Rabbitts T.H. Ernst P. Distinct pathways regulated by menin and by MLL1 in hematopoietic stem cells and developing B cells.Blood. 2013; 122: 2039-2046Crossref PubMed Scopus (48) Google Scholar). These studies also revealed MLL1 target genes that were MLL1 dependent only in hematopoietic cells, presumably due to tissue-specific mechanisms of recruitment (Artinger et al., 2013Artinger E.L. Mishra B.P. Zaffuto K.M. Li B.E. Chung E.K. Moore A.W. Chen Y. Cheng C. Ernst P. An MLL-dependent network sustains hematopoiesis.Proc. Natl. Acad. Sci. USA. 2013; 110: 12000-12005Crossref PubMed Scopus (58) Google Scholar). The native MLL1 complex purified from cell lines revealed several stoichiometric components, particularly a C-terminal subcomplex that is critical for HMT activity and specificity (Dou et al., 2005Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF.Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, Dou et al., 2006Dou Y. Milne T.A. Ruthenburg A.J. Lee S. Lee J.W. Verdine G.L. Allis C.D. Roeder R.G. Regulation of MLL1 H3K4 methyltransferase activity by its core components.Nat. Struct. Mol. Biol. 2006; 13: 713-719Crossref PubMed Scopus (572) Google Scholar, Nakamura et al., 2002Nakamura T. Mori T. Tada S. Krajewski W. Rozovskaia T. Wassell R. Dubois G. Mazo A. Croce C.M. Canaani E. ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation.Mol. Cell. 2002; 10: 1119-1128Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, Patel et al., 2011Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.J. Biol. Chem. 2011; 286: 3359-3369Crossref PubMed Scopus (56) Google Scholar, Steward et al., 2006Steward M.M. Lee J.S. O’Donovan A. Wyatt M. Bernstein B.E. Shilatifard A. Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL complexes.Nat. Struct. Mol. Biol. 2006; 13: 852-854Crossref PubMed Scopus (263) Google Scholar, Yokoyama et al., 2004Yokoyama A. Wang Z. Wysocka J. Sanyal M. Aufiero D.J. Kitabayashi I. Herr W. Cleary M.L. Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression.Mol. Cell. Biol. 2004; 24: 5639-5649Crossref PubMed Scopus (537) Google Scholar). Detailed mechanistic studies show that the MLL1 SET domain, in conjunction with the Wdr5/RbBP5/Ash2L/Dumpy-30 (WRAD) subcomplex, acts as a H3K4 mono- and dimethylation, or in some conditions, a trimethylation enzyme (Dou et al., 2006Dou Y. Milne T.A. Ruthenburg A.J. Lee S. Lee J.W. Verdine G.L. Allis C.D. Roeder R.G. Regulation of MLL1 H3K4 methyltransferase activity by its core components.Nat. Struct. Mol. Biol. 2006; 13: 713-719Crossref PubMed Scopus (572) Google Scholar, Patel et al., 2011Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.J. Biol. Chem. 2011; 286: 3359-3369Crossref PubMed Scopus (56) Google Scholar, Steward et al., 2006Steward M.M. Lee J.S. O’Donovan A. Wyatt M. Bernstein B.E. Shilatifard A. Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL complexes.Nat. Struct. Mol. Biol. 2006; 13: 852-854Crossref PubMed Scopus (263) Google Scholar). Given the connection between H3K4me1 and active enhancer regions as well as H3K4me3 and active/poised transcriptional start sites (reviewed in Maunakea et al., 2010Maunakea A.K. Chepelev I. Zhao K. Epigenome mapping in normal and disease States.Circ. Res. 2010; 107: 327-339Crossref PubMed Scopus (124) Google Scholar), investigators have naturally hypothesized that MLL1 family HMTs regulate transcription via promoter or enhancer targeted H3K4 methylation. To determine the mechanisms by which MLL1 maintains expression of its target genes in hematopoietic cells, we investigated the requirement for HMT activity using domain-specific deletion and conditional knockout mouse models. Surprisingly, we found that the SET domain of MLL1 was not necessary for maintaining the expression of direct target genes in hematopoietic populations, or for facilitating MLL-AF9-mediated leukemogenesis. Through acute deletion of Mll1, we identified a histone H4 modification that was rapidly and synchronously reduced as the expression of MLL1 target genes ceased. This modification implicates the MLL1-associated MOF histone acetyltransferase as the dominant activity maintaining transcription of MLL1 target genes. Remarkably, pharmacologic inhibition of H4K16Ac deacetylases was sufficient to restore MLL1-dependent gene expression in Mll1Δ/Δ cells. Collectively, our data implicate MLL1-recruited H4K16Ac activity, and not HMT activity, as the major mechanism by which gene expression is maintained in hematopoietic stem and progenitor cells (HSPCs). Furthermore, our data implicate Sirtuins as opposing factors in the transcriptional network maintained by MLL1 in HSPCs. All MLL family members harbor a SET domain with specificity for H3K4 methylation, dependent upon associated cofactors or posttranslational modifications (Patel et al., 2014Patel A. Vought V.E. Swatkoski S. Viggiano S. Howard B. Dharmarajan V. Monteith K.E. Kupakuwana G. Namitz K.E. Shinsky S.A. et al.Automethylation activities within the mixed lineage leukemia-1 (MLL1) core complex reveal evidence supporting a “two-active site” model for multiple histone H3 lysine 4 methylation.J. Biol. Chem. 2014; 289: 868-884Crossref PubMed Scopus (22) Google Scholar, Schuettengruber et al., 2011Schuettengruber B. Martinez A.M. Iovino N. Cavalli G. Trithorax group proteins: switching genes on and keeping them active.Nat. Rev. Mol. Cell Biol. 2011; 12: 799-814Crossref PubMed Scopus (339) Google Scholar). Most Mll1 disruption alleles are embryonic lethal; however, homozygotes for a germline deletion of the SET domain of MLL1 (hereafter, ΔSET) survive into adulthood, providing an opportunity to assess the role of the SET domain, thus HMT activity of MLL1, in adult tissues (Terranova et al., 2006Terranova R. Agherbi H. Boned A. Meresse S. Djabali M. Histone and DNA methylation defects at Hox genes in mice expressing a SET domain-truncated form of Mll.Proc. Natl. Acad. Sci. USA. 2006; 103: 6629-6634Crossref PubMed Scopus (159) Google Scholar). Homozygous ΔSET mutants were generated, and western blotting of thymocyte extracts confirmed the expression of the predicted size MLL1 C-terminal band (Figure S1A). Mature lymphoid and myeloid populations in the bone marrow and blood of ΔSET animals were present at normal frequencies (P.E., data not shown). Because HSPCs are extremely sensitive to loss of Mll1 (Gan et al., 2010Gan T. Jude C.D. Zaffuto K. Ernst P. Developmentally induced Mll1 loss reveals defects in postnatal haematopoiesis.Leukemia. 2010; 24: 1732-1741Crossref PubMed Scopus (53) Google Scholar, Jude et al., 2007Jude C.D. Climer L. Xu D. Artinger E. Fisher J.K. Ernst P. Unique and independent roles for MLL in adult hematopoietic stem cells and progenitors.Cell Stem Cell. 2007; 1: 324-337Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, McMahon et al., 2007McMahon K.A. Hiew S.Y. Hadjur S. Veiga-Fernandes H. Menzel U. Price A.J. Kioussis D. Williams O. Brady H.J. Mll has a critical role in fetal and adult hematopoietic stem cell self-renewal.Cell Stem Cell. 2007; 1: 338-345Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), we carefully assessed the phenotype and function of HSPCs in ΔSET mutant mice. Steady-state HSPCs from wild-type (WT), heterozygotes (ΔSET/+), and homozygous ΔSET mice were quantified by determining the total lineage-negative (linneg), Sca-1-positive, c-Kit-positive (LSK) cells in animals of several age ranges (Figures 1A–1C). Given that there were no significant differences in this population, we examined the more HSC-enriched LSK/CD150+/CD48neg population and found the number of these cells was indistinguishable between WT and ΔSET animals (Figures 1D and 1E). For functional quantification of HSCs, we performed competitive repopulation unit (CRU) assays using unfractionated bone marrow cells. Consistent with the phenotypic data, WT and ΔSET bone marrow exhibited overlapping CRU frequencies (Figure 1F). To identify potentially subtle gene expression defects in ΔSET cells, we performed real-time quantitative PCR (qPCR) analyses using sorted LSK cells. We found that the strongly MLL1-dependent target genes Hoxa9, Mecom (locus encoding MDS-Evi1 and Evi1 transcripts), and Prdm16 (Artinger et al., 2013Artinger E.L. Mishra B.P. Zaffuto K.M. Li B.E. Chung E.K. Moore A.W. Chen Y. Cheng C. Ernst P. An MLL-dependent network sustains hematopoiesis.Proc. Natl. Acad. Sci. USA. 2013; 110: 12000-12005Crossref PubMed Scopus (58) Google Scholar) were expressed at normal levels in ΔSET cells (Figure 1G). Similarly, H3K4me3 and H3K4me1 enrichment around the transcription start site (TSS) of these loci was also unchanged in ΔSET HSPCs (Figure S1B). These surprising findings suggested that the MLL1 HMT activity is dispensable for regulating target genes and therefore HSPC function. To assess potential compensation by members of the WRAD subcomplex (Cao et al., 2010Cao F. Chen Y. Cierpicki T. Liu Y. Basrur V. Lei M. Dou Y. An Ash2L/RbBP5 heterodimer stimulates the MLL1 methyltransferase activity through coordinated substrate interactions with the MLL1 SET domain.PLoS ONE. 2010; 5: e14102Crossref PubMed Scopus (87) Google Scholar, Patel et al., 2011Patel A. Vought V.E. Dharmarajan V. Cosgrove M.S. A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.J. Biol. Chem. 2011; 286: 3359-3369Crossref PubMed Scopus (56) Google Scholar) or other HMTs, we knocked down components of HMT complexes in WT and ΔSET in murine embryo fibroblasts (MEFs) and linneg bone marrow cells. Depletion of Ash2L had no effect on Hoxa9 expression in either WT or ΔSET MEFs, whereas depletion of Wdr5 reduced expression of Hoxa9 equivalently in both genotypes (Figures S1C and S1D). Because Wdr5 is also required for SET1A/B HMT complexes (Lee and Skalnik, 2008Lee J.H. Skalnik D.G. Wdr82 is a C-terminal domain-binding protein that recruits the Setd1A Histone H3-Lys4 methyltransferase complex to transcription start sites of transcribed human genes.Mol. Cell. Biol. 2008; 28: 609-618Crossref PubMed Scopus (149) Google Scholar), we knocked down Wdr82, a subunit uniquely required for SET1A/B complex function (Lee and Skalnik, 2008Lee J.H. Skalnik D.G. Wdr82 is a C-terminal domain-binding protein that recruits the Setd1A Histone H3-Lys4 methyltransferase complex to transcription start sites of transcribed human genes.Mol. Cell. Biol. 2008; 28: 609-618Crossref PubMed Scopus (149) Google Scholar, Austenaa et al., 2012Austenaa L. Barozzi I. Chronowska A. Termanini A. Ostuni R. Prosperini E. Stewart A.F. Testa G. Natoli G. The histone methyltransferase Wbp7 controls macrophage function through GPI glycolipid anchor synthesis.Immunity. 2012; 36: 572-585Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Depletion of Wdr82 reduced Hoxa9 expression slightly in both WT and ΔSET MEFs (Figure S1E). Similar trends were observed with the hematopoietic-specific (Artinger et al., 2013Artinger E.L. Mishra B.P. Zaffuto K.M. Li B.E. Chung E.K. Moore A.W. Chen Y. Cheng C. Ernst P. An MLL-dependent network sustains hematopoiesis.Proc. Natl. Acad. Sci. USA. 2013; 110: 12000-12005Crossref PubMed Scopus (58) Google Scholar) target gene Mecom in linneg bone marrow cells (Figures S1F and S1G), although less effective knockdown in this cell type reduced the statistical significance. Thus, ΔSET cells do not exhibit an increased dependence on potential compensatory HMTs or C-terminal subcomplex components. Several lines of evidence suggested that endogenous MLL1 is required for transformation by MLL1 fusion oncoproteins (Milne et al., 2010Milne T.A. Kim J. Wang G.G. Stadler S.C. Basrur V. Whitcomb S.J. Wang Z. Ruthenburg A.J. Elenitoba-Johnson K.S. Roeder R.G. Allis C.D. Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis.Mol. Cell. 2010; 38: 853-863Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, Thiel et al., 2010Thiel A.T. Blessington P. Zou T. Feather D. Wu X. Yan J. Zhang H. Liu Z. Ernst P. Koretzky G.A. Hua X. MLL-AF9-induced leukemogenesis requires coexpression of the wild-type Mll allele.Cancer Cell. 2010; 17: 148-159Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). For example, in Mll1−/− MEFs MLL-AF9 cannot localize to the Hoxa9 gene, but reintroduction of a human MLL1 cDNA restored Hoxa9 promoter localization (Milne et al., 2010Milne T.A. Kim J. Wang G.G. Stadler S.C. Basrur V. Whitcomb S.J. Wang Z. Ruthenburg A.J. Elenitoba-Johnson K.S. Roeder R.G. Allis C.D. Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis.Mol. Cell. 2010; 38: 853-863Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). To test whether histone methylation by endogenous MLL1 is required for MLL-AF9-mediated transformation, we initiated leukemia in both WT and ΔSET HSPCs and followed quantitative and qualitative features of the resulting acute myelogenous leukemia (AML). As shown in Figure 2A, the latency of MLL-AF9 initiated AML in the ΔSET background was indistinguishable from that initiated in WT cells. Leukemia initiated from either WT or ΔSET cells was characterized by similar high white blood cell (WBC) counts, percentages of Gr-1 ± /Mac-1+ cells, suppression of erythropoiesis, and splenomegaly (Figures 2B–2E). Qualitatively, animals receiving either WT or ΔSET MLL-AF9-transformed cells exhibited similar histopathology (Figure S2A) and comparable levels of Hoxa9 expression and histone modifications at the Hoxa9 promoter, including the H3K79Me2 modification recruited by the AF9 fusion partner (Figures S2B–S2E). Furthermore, secondary transplantation of leukemia cells (Figure 2F) and serial replating assays (Figure 2G) demonstrated that leukemia-initiating capacity and self-renewal are not affected by the loss of endogenous MLL1 HMT activity. In summary, none of the known hematopoietic functions of MLL1 appear to require the HMT activity or presence of the SET domain generally. To identify changes in chromatin that are directly influenced by the MLL1 complex, we established a system to temporally resolve gene downregulation and chromatin modifications in primitive hematopoietic cells. Using 4-hydroxytamoxifen (4-OHT) inducible cre recombinase (ERT2-cre) animals in which ex vivo deletion of Mll1 resulted in rapid target gene downregulation (Artinger et al., 2013Artinger E.L. Mishra B.P. Zaffuto K.M. Li B.E. Chung E.K. Moore A.W. Chen Y. Cheng C. Ernst P. An MLL-dependent network sustains hematopoiesis.Proc. Natl. Acad. Sci. USA. 2013; 110: 12000-12005Crossref PubMed Scopus (58) Google Scholar), we determined transcript levels and chromatin modifications over a period of 3 days, during which linneg bone marrow cells remain phenotypically undifferentiated (B.P.M., data not shown). ERT2-cre;Mll1F/+ cells were used as controls because target gene expression is indistinguishable between WT and heterozygous cells (Figures S3A and S3B) and induction of cre recombinase can affect cell viability and gene expression. Within 36 hr of Mll1 deletion, the levels of Hoxa9, Mecom, and Prdm16 transcripts were reduced (Figure 3A). An assessment of H3K4 methylation enrichment at these target genes by chromatin immunoprecipitation (ChIP)-qPCR demonstrated the stability of these histone modifications during the time course despite complete excision of Mll1 (Figures 3B–3E and S3C–S3F). Therefore, despite dramatic changes in gene expression, acute MLL1 loss does not result in H3K4 methylation differences at its target gene promoters. To test whether these observations were generalizable in a more HSC-enriched population, we performed ChIP-sequencing (ChIP-seq) with sorted LSK cells from control ERT2-cre;Mll1F/+ or ERT2-cre;Mll1F/F animals. In this system, 48 hr was sufficient to reduce MLL1 target gene expression (Figures S4A and S4B). We examined Hoxa9, Evi1, and Prdm16 TSS regions and found a remarkable consistency in H3K4me3 and H3K4me1 enrichment comparing control and Mll1Δ/Δ samples (Figure 4A), which was true genome-wide at all enriched loci (Figures 4B and 4C). Despite the downregulation of over 300 genes (Artinger et al., 2013Artinger E.L. Mishra B.P. Zaffuto K.M. Li B.E. Chung E.K. Moore A.W. Chen Y. Cheng C. Ernst P. An MLL-dependent network sustains hematopoiesis.Proc. Natl. Acad. Sci. USA. 2013; 110: 12000-12005Crossref PubMed Scopus (58) Google Scholar), the changes in H3K4me enrichment vary only within the range observed between replicate samples (Figures S4C and S4D). These data demonstrate that the HMT activity of MLL1 is not responsible for the maintenance of target gene expression in HSPCs. To identify chromatin changes that occur upon Mll1 deletion, we surveyed histone modifications at MLL1 target gene promoters throughout the time course shown in Figure 3. MLL1 interacts physically and/or genetically with p300/CBP, MOF, and MOZ histone acetyltransferases (Dou et al., 2005Dou Y. Milne T.A. Tackett A.J. Smith E.R. Fukuda A. Wysocka J. Allis C.D. Chait B.T. Hess J.L. Roeder R.G. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF.Cell. 2005; 121: 873-885Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, Ernst et al., 2001Ernst P. Wang J. Huang M. Goodman R.H. Korsmeyer S.J. MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein.Mol. Cell. Biol. 2001; 21: 2249-2258Crossref PubMed Scopus (197) Google Scholar, Paggetti et al., 2010Paggetti J. Largeot A. Aucagne R. Jacquel A. Lagrange B. Yang X.J. Solary E. Bastie J.N. Delva L. Crosstalk between leukemia-associated proteins MOZ and MLL regulates HOX gene expression in human cord blood CD34+ cells.Oncogene. 2010; 29: 5019-5031Crossref PubMed Scopus (39) Google Scholar). Although the CBP/p300-mediated H3K27Ac and MOZ-mediated H3K9Ac modifications were stable throughout the time course (Figures S4E, S5A, and S5B), H4K16Ac was rapidly reduced around the TSS of MLL1 target genes in Mll1Δ/Δ cells (Figure 5A) as was MOF itself (Figure 5B). Polycomb-complex-mediated H3K27me3 modifications increased, but late in the time course, suggesting they occur as a secondary consequence of cessation of transcription (Figures S5E and S5F). Interestingly, ΔSET mutant linneg cells exhibited unchanged H4K16Ac enrichment, whether gene-specific or global levels were assessed (Figures S5G and S5H). Because MOF-mediated acetylation creates a binding site for Brd4, which recruits the PTEFb transcriptional elongation complex (Yang et al., 2005Yang Z. Yik J.H. Chen R. He N. Jang M.K. Ozato K. Zhou Q. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4.Mol. Cell. 2005; 19: 535-545Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, Zippo et al., 2009Zippo A. Serafini R. Rocchigiani M. Pennacchini S. Krepelova A. Oliviero S. Histone crosstalk between H3S10ph and H4K16ac generates a histone code that mediates transcription elongation.Cell. 2009; 138: 1122-1136Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), we assessed promoter Brd4 and Cdk9 levels (the latter representative of the PTEFb complex). As shown in Figures 5C and 5D, Brd4 and Cdk9 were also reduced at MLL1 target genes. Consistent with the reduced Cdk9, the actively elongating form of RNA polymerase II (serine 2 phosphorylated) was also decreased (Figure 5E). These data suggest that MLL1 loss results in the rapid loss of promoter H4K16Ac, and, as a consequence, reduced Brd4 and PTEFb recruitment. In addition, both serine 5 phosphorylated, paused RNA polymerase II (ser5 Pol II, Figure 5F) and nascent Hoxa9, Evi1 (specific isoform from the Mecom locus), and Prdm16 transcripts were reduced upon Mll1 deletion, demonstrating a role for MLL1 in enhancing transcriptional initiation (Figures S5C and S5D). These data show that the MLL1 complex maintains target gene expression through both transcriptional initiation and elongation, in part, through maintaining H4K16Ac levels by recruitment of MOF. If MLL1-associated MOF is required for maintaining MLL1 target genes in HSPCs, we predicted that blocking H4K16 deacetylation might enhance the expression of MLL1 target genes. Both SIRT1 and SIRT2 deacetylate H4K16Ac, the former acting constitutively in the nucleus and the latter cytoplasmic enzyme acting mainly on chromatin at the G2/M transition (Martínez-Redondo and Vaquero, 2013Martínez-Redondo P. Vaquero A. The diversity of histone versus nonhistone sirtuin substrates.Genes Cancer. 2013; 4: 148-163Crossref PubMed Scopus (110) Google Scholar). To test the role of these enzymes in MLL1-dependent gene expression, we used the class III histone deacetlylase inhibitor nicotinamide or the SIRT1-specific inhibitor Ex-527 (Napper et al., 2005Napper A.D. Hixon J. McDonagh T. Keavey K. Pons J.F. Barker J. Yau W.T. Amouzegh P. Flegg A. Hamelin E. et al.Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1.J. Med. Chem. 2005; 48: 8045-8054Crossref PubMed Scopus (425) Google Scholar) in conjunction with Mll1 deletion. Remarkably, either inhibitor could restore MLL1 target gene expression in Mll1Δ/Δ cells (Figure 6). Interestingly, treatment with Ex-527 had a negligible effect on MLL1 target gene expression in WT cells, although induction of Cdkn1 was observed (Figures S6A and S6B). Induction Cdkn1 occurs through deacetylation and thereby activation of the nonhistone SIRT1 target p53 (Yuan et al., 2013Yuan H. Su L. Chen W.Y. The emerging and diverse roles of si" @default.
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• W2108453860 title "The Histone Methyltransferase Activity of MLL1 Is Dispensable for Hematopoiesis and Leukemogenesis" @default.
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