Enhancer regulation by H3K4 methyltransferase MLL4/KMT2D and associated factors

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

I. In search for novel coactivators for the master adipogenic transcription factor (TF) PPARgamma, we identified a nuclear protein complex that contains H3K4me1 methyltransferase MLL3 or MLL4, H3K27 demethylase UTX (KDM6A), PTIP, PA1 (PAGR1), NCOA6, and the WRAD subcomplex consisting of WDR5, RbBP5, Ash2L and DPY30 (JBC 2007; PNAS 2007). Using adipogenesis and myogenesis as models, we showed that MLL3/MLL4 co-localize with lineage-determining TFs (LDTFs) on active enhancers. MLL3/MLL4 are required for enhancer activation, cell-type-specific gene induction, and cell differentiation (eLife 2013). Using conditional knockout mice, we showed that MLL3/MLL4 are essential for the development of various tissues including adipose, muscle, mammary gland, B cells, T cells, and heart (summarized in Gene 2017). Using adipogenesis, ES cell differentiation and somatic cell reprogramming as model systems, we found that enhancer priming by MLL3/MLL4 controls cell fate transition by orchestrating H3K27 acetyltransferases CBP/p300-mediated enhancer activation (PNAS 2016; NAR 2017). II. We found that UTX protein, but not its H3K27 demethylase activity, is required for cell differentiation and mouse development (PNAS 2012; MCB 2016; JCI 2016). Our data suggest that UTX functions through MLL3/MLL4 to regulate enhancer activation in differentiation and development (reviewed in MCB 2020). Interestingly, UTX demethylase activity is required for satellite cell-mediated muscle regeneration (JCI 2016). MLL3/MLL4 and UTX are frequently mutated in many types of cancers and developmental diseases. Our findings suggest that mutations in MLL3/MLL4 and UTX would lead to defects in enhancer activation, cell-type-specific gene expression and cell differentiation. Such a mechanism may contribute to the pathogenesis of these cancers and developmental diseases. III. We reported that the epigenomic reader Brd4 co-localizes with LDTFs on active enhancers during cell differentiation. Brd4 controls cell identity gene induction and is essential for adipogenesis and myogenesis (Nat Comm 2017). Together, our data suggest a model of sequential actions of epigenomic regulators on enhancers: 1) pioneer TFs and LDTFs recruit MLL3/MLL4 to prime enhancer regions and label them with H3K4me1; 2) MLL3/MLL4 facilitate the binding of CBP/p300, which activate enhancers and label them with H3K27ac; 3) H3K27ac and acetylated TFs are recognized by Brd4, which recruits Mediator and RNA Polymerase II to activate cell type-specific gene expression (Nat Comm 2017). IV. Our previous findings suggest that MLL4 regulates enhancer activation independent of H3K4me1 (PNAS 2016). To investigate the roles of MLL3/4 enzymatic activities and H3K4me1 in enhancer activation, cell differentiation and development, we generated enzyme dead MLL3/4 knockin mice and embryonic stem cells (ESCs). Constitutive elimination of both MLL3 and MLL4 enzymatic activities leads to gastrulation failure and early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistently, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate towards the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm and trophectoderm. The failure in extraembryonic endoderm differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and embryonic stem cell differentiation. V. Using adipogenesis as a model system, we identify BAF as the major SWI/SNF complex that colocalizes with MLL4 and LDTFs on active enhancers and is required for cell differentiation. In contrast, the promoter enriched SWI/SNF complex PBAF is dispensable for adipogenesis. By depleting BAF subunits SMARCA4 (BRG1) and SMARCB1 (SNF5) as well as MLL4 in cells, we show that BAF and MLL4 reciprocally regulate each other's binding on active enhancers before and during adipogenesis. By focusing on enhancer activation by the adipogenic pioneer TF C/EBP without inducing cell differentiation, we provide direct evidence for an interdependent relationship between BAF and MLL4 in activating cell type-specific enhancers. Together, these findings reveal a positive feedback between BAF and MLL4 in promoting LDTF-dependent activation of cell type-specific enhancers (Nat Comm 2021). VI. MED1 often serves as a surrogate of the general transcription coactivator complex Mediator for identifying active enhancers. MED1 is required for phenotypic conversion of fibroblasts to adipocytes in vitro, but its role in adipose development and expansion in vivo has not been reported. Here, we show that MED1 is not generally required for transcription during adipogenesis in culture and that MED1 is dispensable for adipose development in mice. Instead, MED1 is required for postnatal adipose expansion. Our findings identify a gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion (Genes Dev 2021).