Enhancer regulation by H3K4 methyltransferase MLL4/KMT2D and associated factors

I.在寻找主要的成脂转录因子(TF)PPARGamma的新的共激活因子时，我们发现了一个核蛋白复合体，它包含H3K4me1甲基转移酶ML3或MLL4，H3K27去甲基酶UTX(KDM6A)，PTIP，PA1(PAGR1)，NCOA6，以及由WDR5，RbBP5，Ash2L和DPY30组成的WRAD亚复合体(JBC 2007；PNAS 2007)。以脂肪生成和肌肉生成为模型，我们发现MLL3/MLL4与谱系决定因子(LDTF)共定位于活性增强剂上。MLL3/MLL4是增强子激活、细胞类型特异性基因诱导和细胞分化所必需的(ELife 2013)。使用条件基因敲除小鼠，我们表明MLL3/MLL4对包括脂肪、肌肉、乳腺、B细胞、T细胞和心脏在内的各种组织的发育是必不可少的(总结在基因2017中)。以脂肪形成、ES细胞分化和体细胞重编程为模型系统，我们发现由MLL3/MLL4激活的增强子通过协调H3K27乙酰基转移酶CBP/p300介导的增强子激活来控制细胞命运的转变(PNAS 2016；NAR 2017)。 我们发现，细胞分化和小鼠发育需要UTX蛋白，而不是其H3K27去甲基酶活性(PNAS 2012；MCB 2016；JCI 2016)。我们的数据表明，UTX通过ML3/MLL4调节分化和发育中的增强子激活(在MCB 2020中回顾)。有趣的是，UTX脱甲基酶活性是卫星细胞介导的肌肉再生所必需的(JCI 2016)。ML3/MLL4和UTX在多种类型的癌症和发育疾病中频繁发生突变。我们的发现表明，MLL3/MLL4和UTX突变会导致增强子激活、细胞类型特异性基因表达和细胞分化方面的缺陷。这种机制可能在这些癌症和发育疾病的发病机制中起作用。 我们报道了表观基因组阅读器Brd4在细胞分化过程中与LDTFS共定位于活性增强子上。BRD4控制细胞识别基因的诱导，对于脂肪生成和肌肉生成是必不可少的(NAT Comm 2017)。综上所述，我们的数据建议了一个表观基因组调控因子对增强子的顺序作用模型：1)先驱TF和LDTF将ML3/MLL4招募到主要增强子区域并用H3K4me1标记它们；2)MLL3/MLL4促进CBP/p300的结合，后者激活增强子并用H3K27ac标记；3)H3K27ac和乙酰化的TF被Brd4识别，它招募其介体和RNA聚合酶II来激活细胞类型特异的基因表达(NAT Comm 2017)。 IV.我们先前的发现表明，MLL4独立于H3K4me1调节增强子激活(PNAS 2016)。为了研究MLL3/4酶活性和H3K4me1在增强子激活、细胞分化和发育中的作用，我们建立了酶死亡的MLL3/4敲门小鼠和胚胎干细胞(ESCs)。MLL3和MLL4酶活性的结构性消除会导致小鼠原肠形成失败和早期胚胎死亡。然而，选择性地消除MLL3/4酶活性在胚芽中，而不是胚外，叶原肠发育基本完好。一直以来，缺乏MLL3/4酶活性的胚胎干细胞可以向三层胚胎胚层分化，但表现出向胚外内胚层和滋养外胚层异常分化。胚胎外内胚层分化的失败可以归因于谱系决定转录因子GATA6的增强子结合显著减少。此外，我们还表明，在胚胎干细胞分化过程中，MLL3/4催化的H3K4me1在很大程度上是增强子激活所必需的。综上所述，我们的发现表明，在早期胚胎发育和胚胎干细胞分化中，MLL3/4甲基转移酶活性具有谱系选择性，但不依赖于增强子激活。 V.以脂肪生成为模型系统，我们确定BAF是主要的SWI/SNF复合体，与MLL4和LDTF共定位于活性增强剂上，是细胞分化所必需的。相反，启动子丰富的SWI/SNF复合体PBAF对于脂肪的形成是必不可少的。通过消耗细胞中的BAF亚基SMARCA4(BRG1)和SMARCB1(SNF5)以及MLL4，我们证明了BAF和MLL4在脂肪形成之前和过程中相互调节彼此对活性增强剂的结合。通过关注成脂先驱TFC/EBP在不诱导细胞分化的情况下激活增强子，我们提供了BAF和MLL4在激活细胞类型特异性增强子方面相互依赖的直接证据。综上所述，这些发现揭示了BAF和MLL4在促进LDTF依赖的细胞类型特定增强子的激活方面的积极反馈(NAT Comm 2021)。 6.MED1通常作为一般转录共激活因子复合体介体的替代物，用于识别活性增强子。MED1在体外是成纤维细胞向脂肪细胞表型转化所必需的，但其在体内脂肪发育和扩张中的作用尚未见报道。在这里，我们证明了在培养的脂肪形成过程中，MED1通常不是转录所必需的，并且MED1对于小鼠的脂肪发育是必不可少的。相反，MED1是出生后脂肪扩张所必需的。我们的发现确定了MED1作为出生后脂肪扩张所需的脂肪生成辅助激活因子的基因特异性调节作用(基因Dev 2021)。

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).