Regulation of PPARgamma and Adipogenesis by MLL3/MLL4 complex

我的实验室之前发现了H3K4甲基转移酶MLL3和MLL4(Cho Yw，JBC 2007)和H3K27去甲基酶UTx和JMJD3(洪S，PNAS 2007)。以核蛋白PTIP为诱饵，我们从细胞核中分离出一个含有H3K4甲基转移酶ML13/MLL4、H3K27去甲基酶UTX、PTIP和一种新的蛋白PA1的蛋白质复合体(Cho yw，JBC 2007)。此外，我们发现PTIP是PPAR和C/EBP表达和脂肪形成所必需的(CHO，YW，Cell Metab 2009)。为了了解MLL3/MLL4的生理作用及其相关因素，我们在小鼠中敲除了ML3、MLL4、UTX和PA1。由于它们的胚胎致死性，我们产生了MLL4、UTX和PA1的条件敲除(KO)。通过将这些条件KO与Myf5-Cre小鼠杂交，我们发现MLL4、PTIP和PA1是脂肪形成所必需的，而UTX是必不可少的。为了研究UTX的功能，我们转向小鼠胚胎干细胞。我们发现UTX独立于H3K27去甲基酶活性控制ES细胞分化和早期胚胎发育(Wang C，PNAS 2012)。为了了解UTX的H3K27去甲基酶活性的生物学作用，我们产生了酶死亡的UTX敲击小鼠。我们最近发现MLL3/MLL4是H3K4单甲基和双甲基转移酶，在细胞分化过程中对增强子激活是必不可少的(Lee Je，eLife 2013)。增强子在细胞类型特异性基因表达中起核心作用，标记为H3K4me1/2。活性增强子进一步标记为H3K27ac。然而，负责增强剂上H3K4me1/2的甲基转移酶仍然难以捉摸。此外，这些酶如何在增强剂上发挥作用以调节特定细胞类型的基因表达尚不清楚。我们发现MLL4是一种主要的哺乳动物H3K4单甲基和双甲基转移酶，与MLL3具有部分功能冗余。以脂肪生成和肌肉生成为模型系统，我们发现MLL4表现出细胞类型和分化阶段特异性的基因组结合，并主要定位于增强剂。在分化过程中，MLL4与谱系决定转录因子(TF)共定位于活性增强子上。MLL4的缺失显著降低了H3K4me1/2、H3K27ac、增强子上的介体和聚合酶II(POL II)的水平，并导致细胞类型特异性基因表达和细胞分化的严重缺陷。综上所述，这些发现确认MLL3/MLL4是主要的哺乳动物H3K4单甲基和双甲基转移酶，在细胞分化过程中对增强子激活是必不可少的。

My lab previously identified H3K4 methyltransferases MLL3 and MLL4 (Cho YW, JBC 2007) and H3K27 demethylases UTX and JMJD3 (Hong S, PNAS 2007). Using a nuclear protein PTIP as the bait, we isolated from cell nuclei a protein complex that contains H3K4 methyltransferases MLL3/MLL4, H3K27 demethylase UTX, PTIP and a novel protein PA1 (Cho YW, JBC 2007). Further, we show that PTIP is required for PPARγ and C/EBPα expression and adipogenesis (Cho, YW, Cell Metab 2009). To understand the physiological roles of MLL3/MLL4 and associated factors, we have knocked out MLL3, MLL4, UTX and PA1 in mice. Because of their embryonic lethality, we have generated conditional knockout (KO) of MLL4, UTX and PA1. By crossing these conditional KO with Myf5-Cre mice, we found that MLL4, PTIP and PA1 are essential for adipogenesis while UTX is dispensable. To investigate UTX function, we turned to mouse embryonic stem (ES) cells. We found that UTX controls ES cell differentiation and early embryonic development independent of H3K27 demethylase activity (Wang C, PNAS 2012). To understand the biological role of the H3K27 demethylase activity of UTX, we have generated enzyme-dead UTX knockin mice. We recently identify MLL3/MLL4 as H3K4 mono- and di-methyltransferases that are essential for enhancer activation during cell differentiation (Lee JE, eLife 2013). Enhancers play a central role in cell-type-specific gene expression and are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. However, the methyltransferases responsible for H3K4me1/2 on enhancers remain elusive. Furthermore, how these enzymes function on enhancers to regulate cell-type-specific gene expression is unclear. We identify MLL4 as a major mammalian H3K4 mono- and di-methyltransferase with partial functional redundancy with MLL3. Using adipogenesis and myogenesis as model systems, we show that MLL4 exhibits cell-type- and differentiation-stage-specific genomic binding and is predominantly localized on enhancers. MLL4 co-localizes with lineage-determining transcription factors (TFs) on active enhancers during differentiation. Deletion of MLL4 markedly decreases H3K4me1/2, H3K27ac, Mediator and Polymerase II (Pol II) levels on enhancers and leads to severe defects in cell-type-specific gene expression and cell differentiation. Together, these findings identify MLL3/MLL4 as major mammalian H3K4 mono- and di-methyltransferases essential for enhancer activation during cell differentiation.