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Enhancer Priming by H3K4 Methyltransferase MLL4 Controls Cell Fate Transition

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Enhancer Priming by H3K4 Methyltransferase MLL4 Controls Cell Fate Transition Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition Chaochen Wanga, Ji-Eun Leea, Binbin Laia, Todd S. Macfarlanb, Shiliyang Xua, Lenan Zhuanga, Chengyu Liuc, Weiqun Pengd,e, and Kai Gea,1 aLaboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; bDivision of Developmental Biology, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; cTransgenic Core, Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; dDepartment of Physics, The George Washington University, Washington, DC 20052; and eDepartment of Anatomy and Regenerative Biology, The George Washington University, Washington, DC 20052 Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved August 26, 2016 (received for review April 29, 2016) Transcriptional enhancers control cell-type–specific gene expres- excellent models for studying cell fate transition and the underlying sion. Primed enhancers are marked by histone H3 lysine 4 (H3K4) molecular mechanism. mono/di-methylation (H3K4me1/2). Active enhancers are further MLL4 is a major enhancer H3K4 mono- and di-methyltransfer- marked by H3K27 acetylation (H3K27ac). Mixed-lineage leukemia ase with partial functional redundancy with MLL3 in mammalian 4 (MLL4/KMT2D) is a major enhancer H3K4me1/2 methyltransferase cells (7, 8). MLL4 colocalizes with lineage-determining TFs on AEs with functional redundancy with MLL3 (KMT2C). However, its role in during adipogenesis and myogenesis. Furthermore, MLL4 is re- cell fate maintenance and transition is poorly understood. Here, we quired for enhancer activation, cell-type–specific gene expression, show in mouse embryonic stem cells (ESCs) that MLL4 associates and cell differentiation during adipogenesis and myogenesis (8). with, but is surprisingly dispensable for the maintenance of, active These observations prompted us to investigate whether MLL4 enhancers of cell-identity genes. As a result, MLL4 is dispensable for plays a more general role in the control of cell fate transition and cell-identity gene expression and self-renewal in ESCs. In contrast, how MLL4 regulates enhancer activation. In this study, we use mainly MLL4 is required for enhancer-binding of H3K27 acetyltransferase ESC differentiation and somatic cell reprogramming as model sys- p300, enhancer activation, and induction of cell-identity genes during tems to explore functions and mechanisms of MLL4 in enhancer ESC differentiation. MLL4 protein, rather than MLL4-mediated H3K4 activation, cell-identity maintenance, and cell fate transition. methylation, controls p300 recruitment to enhancers. We also show that, in somatic cells, MLL4 is dispensable for maintaining cell iden- Results tity but essential for reprogramming into induced pluripotent stem MLL4 Associates with Active Enhancers on Cell-Identity Genes in ESCs. cells. These results indicate that, although enhancer priming by MLL4 is essential for early embryonic development in mice MLL4 is dispensable for cell-identity maintenance, it controls cell fate whereas MLL3 is dispensable. In cultured cells, MLL3 partially transition by orchestrating p300-mediated enhancer activation. compensates for the loss of MLL4 (8). To investigate the role of MLL4 in ESC self-renewal and differentiation, we derived Mll3 − − enhancer | MLL4/KMT2D | H3K4 methyltransferase | cell fate transition | KO and Mll4 conditional KO (Mll3 / Mll4f/f, hereafter referred p300 to as f/f) ESCs from blastocysts. By transfecting these cells with a Cre-expressing plasmid to delete the Mll4 gene, we generated n mammalian cells, enhancers coordinate with promoters to Iprecisely control cell-type–specific gene transcription, which Significance determines the cell identity (1, 2). Comprehensive genome-wide studies have provided insights into the chromatin signatures of Transcriptional enhancers control cell-identity gene expression enhancers. Primed enhancers are marked by H3K4me1/2. Active and thus determine cell identity. Enhancers are primed by enhancers (AEs) are further marked by the histone acetyl- histone H3K4 mono-/di-methyltransferase MLL4 before they transferases CBP/p300-mediated H3K27ac (3). Recent studies are activated by histone H3K27 acetyltransferase p300. Here, classify AEs into typical enhancers and superenhancers. Super- we show that MLL4 is dispensable for cell-identity mainte- enhancers are clusters of AEs bound by lineage-determining nance but essential for cell fate transition using several model transcription factors (TFs). Compared with typical enhancers, – systems including embryonic stem cell (ESC) differentiation superenhancers are more cell-lineage specific and control cell toward somatic cells and somatic cell reprogramming into ESC- identity (4). Enhancer activation is orchestrated through a regu- like cells. Mechanistically, MLL4 is dispensable for maintaining latory network involving lineage-determining TFs and chromatin- p300 binding on active enhancers of cell-identity genes but is modifying complexes (2). However, how chromatin-modifying required for p300 binding on enhancers activated during cell complexes regulate enhancer activation and cell fate transition is fate transition. These results indicate that, although enhancer poorly understood. priming by MLL4 is dispensable for cell-identity maintenance, it Embryonic stem cells (ESCs) derived from blastocysts are ca- controls cell fate transition by orchestrating p300-mediated pable of unlimited replication in vitro, a property known as ESC enhancer activation. self-renewal. ESC identity is maintained during self-renewal. ESC self-renewal is controlled by a core circuitry of TFs including Oct4, Author contributions: C.W. and K.G. designed research; C.W., J.-E.L., L.Z., and K.G. per- Sox2, and Nanog (4). ESCs can rapidly respond to environmental formed research; C.W., T.S.M., and C.L. contributed new reagents/analytic tools; C.W., J.-E.L., cues and differentiate into three germ layers—ectoderm, endo- B.L., S.X., W.P., and K.G. analyzed data; and C.W., W.P., and K.G. wrote the paper. derm, and mesoderm—which consist of all cell lineages within The authors declare no conflict of interest. days (5). Differentiated somatic cells can also be converted back to This article is a PNAS Direct Submission. the pluripotent stage by ectopic expression of Oct4 and Sox2 Data deposition: The sequence reported in this paper has been deposited in the Gene together with Klf4 and c-Myc, a process known as somatic cell Expression Omnibus database (accession no. GSE50534). reprogramming into induced pluripotent stem cells (iPSCs) (6). The 1To whom correspondence should be addressed. Email: [email protected]. dramatic changes of cell identity that accompany ESC differentia- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY tion and somatic cell reprogramming make these two processes 1073/pnas.1606857113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1606857113 PNAS | October 18, 2016 | vol. 113 | no. 42 | 11871–11876 Downloaded by guest on September 24, 2021 + Mll3/Mll4 double KO (DKO) ESCs. Deletion of Mll4 from the f/f Motif analysis showed that MLL4 AEs were enriched with ESCs resulted in an approximately twofold decrease of global motifs of major ESC TFs including Sox2, Oct4, Smad2/3, Nanog, H3K4me1 and ∼30% decreases of global H3K4me2 and H3K27ac and Klf4 (Fig. 1D). By comparing the genomic localization of with little effect on H3K4me3 and H3K9ac levels (Fig. 1A and SI MLL4 with those of Oct4, Sox2, and Nanog (O/S/N) and the Appendix, Fig. S1A). Thus, MLL3 and MLL4 are H3K4me1/2 H3K27 acetyltransferase p300 that marks AEs (4, 9), we found methyltransferases in ESCs. that MLL4 colocalized with O/S/N and p300 on AEs (Fig. 1E). A Next we performed ChIP-Seq (chromatin immunoprecipitation physical interaction between MLL4 and Oct4 was also observed in coupled with DNA sequencing) of MLL4 in both f/f and DKO ESCs (SI Appendix,Fig.S1B), consistent with the previous report ESCs. After filtering out nonspecific signals observed in the DKO that the MLL4 complex physically interacts with pluripotency cells (8), 12,383 high-confidence MLL4 binding sites were identi- TFs (10). Thus, MLL4 colocalizes with pluripotency TFs on AEs fied in f/f ESCs, the vast majority of which were marked by in ESCs. H3K4me1 and/or H3K4me2 (Fig. 1B). Consistent with our pre- Superenhancers (SEs), densely occupied by master TFs, are lineage-specific and associate with highly expressed cell-identity vious findings in adipocytes and myocytes (8), ChIP-Seq revealed genes (4, 11). We found that ∼90% of ESC SEs were occupied by that MLL4 was preferentially enriched on AEs in ESCs (Fig. 1C). MLL4. In contrast, only ∼30% of typical enhancers (TEs) were + MLL4 (SI Appendix, Fig. S1C). SEs had higher levels of MLL4- binding density comparing to TEs in ESCs (SI Appendix, Fig. + S1D). Moreover, genes associated with MLL4 SEs were expressed at significantly higher levels than those with TEs (SI Appendix, Fig. S1E). Consistently, gene ontology (GO) analysis of genes + associated with the 5,918 MLL4 AEs identified stem cell development, maintenance, and differentiation as the top func- tional categories (SI Appendix, Fig. S1F). For example, MLL4 colocalized with Oct4 and p300 on AEs of the ESC identity
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