Transcriptional enhancers control cell-type-specific gene expression. Of the two major types of enhancers in mammalian cells, primed enhancers are marked by histone H3K4 mono-methylation (H3K4me1) while active enhancers are further marked by H3K27 acetylation (H3K27ac). We identified MLL3 (KMT2C)/MLL4 (KMT2D) as the major H3K4 mono-methyltransferases and CBP/p300 as the H3K27 acetyltransferases in mammalian cells (EMBO J 2011; eLife 2013). In search for novel coactivators for the master adipogenic transcription factor (TF) PPARgamma, we identified a nuclear protein complex that contains H3K4me1 methyltransferases MLL3/MLL4, H3K27 demethylase UTX (KDM6A), PTIP, PA1, 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 tissue-specific 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). 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. 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. 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). We showed that MLL4 protein, rather than MLL4-mediated H3K4me1, controls p300 recruitment to enhancers during ES cell differentiation, suggesting that MLL4 may regulate enhancer activation independent of its enzymatic activity and H3K4me1 (PNAS 2016). Ectopic expression of H3.3K4M, an inhibitor of H3K4 methylation, or deletion of the enzymatic SET domain, in lineage-specific precursor cells destabilizes MLL3/MLL4 proteins, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2019). We have generated MLL3/MLL4 enzyme-dead mutant mice. Initial analyses indicate that MLL3/MLL4 regulate mouse development through both enzymatic activity-dependent and -independent mechanisms.
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