Epigenetic Regulation of Adipogenesis

绝大多数组蛋白甲基转移酶和去甲基酶是在21世纪发现的，但对它们的生物学功能知之甚少。我们使用脂肪生成作为模型系统来研究组蛋白甲基转移酶和去甲基酶的作用，以及特定部位的组蛋白甲基化的动态，在基因表达和细胞分化的调节中。我们对组蛋白H3(分别为H3K4、H3K9、H3K27和H3K36)的K4、K9、K27和K36上的甲基化感兴趣。 多梳抑制复合体2(PRC2)是动物发育的主要调节因子。PrC2主要通过其酶促亚单位Ezh2介导的H3K27(H3K27me3)上的三甲基化来抑制基因表达。利用Ezh2条件性KO前体脂肪细胞，我们报道了Ezh2及其H3K27甲基转移酶活性是脂肪形成所必需的，并且Ezh2结构性地抑制Wnt基因以促进脂肪形成(王L，PNAS 2010)。组蛋白甲基转移酶G9a负责H3K9二甲基化(H3K9me2)，这是基因抑制的表观遗传标记。利用G9a条件性KO前体脂肪细胞，我们最近报道了G9a抑制PPAR-γ的表达和脂肪形成。G9a调节脂肪生成的正、负主调控因子：G9a依赖于其H3K9甲基转移酶活性抑制PPAR-γ的表达，而促进Wnt的表达不依赖于其酶活性(王L，EMBO J 2013)。我们最近发现组蛋白H3赖氨酸(K)36的K-M突变强烈抑制脂肪生成。连同我们的报告，H3K4me1/2甲基转移酶ML3/MLL4是PPARGamma和C/EBPalpha表达和脂肪生成所必需的(Lee Je，eLife 2013)，这些发现提供了脂肪生成的表观遗传调控的初步观点，并暗示组蛋白甲基化控制脂肪生成的正和负主调制子的表达(在BBA 2012和细胞与生物2014中回顾)。 组蛋白乙酰转移酶(HAT)Gcn5是胚胎发育的关键基因，与其同源基因PCAF存在部分功能冗余。然而，Gcn5和PCAF的组织和细胞系特异性功能仍然没有很好的确定。在最近的一项研究中，我们探讨了Gcn5和PCAF在脂肪形成中的作用。我们发现Gcn5/PCAF的双基因敲除(DKO)抑制了主要的成脂转录因子基因PPARGamma的表达，从而阻止了脂肪细胞的分化。Gcn5/PCAF DKO细胞中的成脂缺陷可通过异位表达PPARGamma来修复，提示Gcn5/PCAF在PPARGamma的上游起促进成脂的作用。Gcn5/PCAF对PPARGamma表达的要求意外地被成脂诱导剂3-异丁基-1-甲基黄嘌呤(IBMX)延长治疗所绕过。然而，无论是PPARγ的异位表达还是延长的IBMX处理都不能挽救DKO细胞Prdm16表达的缺陷，这表明Gcn5/PCAF对于Prdm16的正常表达是必不可少的。Gcn5/PCAF在转录过程的不同阶段调节PPARGamma和Prdm16的表达，促进RNA聚合酶II募集到Prdm16和PPARGamma转录本的延伸。总体而言，我们的研究表明，Gcn5/PCAF通过调节PPARGamma的表达来促进脂肪生成，并通过影响Prdm16的表达来调节棕色脂肪的生成(金Q等人，2014)。

The vast majority of histone methyltransferases and demethylases were identified in the 21st century but their biological functions are poorly understood. We use adipogenesis as a model system to study the roles of histone methyltransferases and demethylases, and the dynamics of site-specific histone methylation, in regulation of gene expression and cell differentiation. We are interested in methylations on K4, K9, K27 and K36 of histone H3 (H3K4, H3K9, H3K27 and H3K36, respectively). The Polycomb repressive complex 2 (PRC2) is a major regulator of animal development. PRC2 represses gene expression mainly through its enzymatic subunit Ezh2-mediated tri-methylation on H3K27 (H3K27me3). Using Ezh2 conditional KO preadipocytes, we report that Ezh2 and its H3K27 methyltransferase activity are required for adipogenesis and that Ezh2 constitutively represses Wnt genes to facilitate adipogenesis (Wang L, PNAS 2010). Histone methyltransferase G9a is responsible for H3K9 di-methylation (H3K9me2), an epigenetic mark for gene repression. Using G9a conditional KO preadipocytes, we report recently that G9a represses PPARgamma expression and adipogenesis. G9a regulates both positive and negative master regulators of adipogenesis: G9a represses PPARgamma expression dependent on its H3K9 methyltransferase activity while promotes Wnt expression independent of its enzymatic activity (Wang L, EMBO J 2013). We have recently found that K-to-M mutation of histone H3 lysine (K) 36 strongly inhibits adipogenesis. Together with our reports that H3K4me1/2 methyltransferases MLL3/MLL4 are required for PPARgamma and C/EBPalpha expression and adipogenesis (Lee JE, eLife 2013), these findings provide an initial view of epigenetic regulation of adipogenesis, and suggest that histone methylations control expression of positive and negative master regulators of adipogenesis (reviewed in BBA 2012 and Cell & Biosci 2014). Histone acetyltransferase (HAT) Gcn5 is critical for embryogenesis and shows partial functional redundancy with its homolog PCAF. However, the tissue- and cell lineage-specific functions of Gcn5 and PCAF are still not well defined. In a recent study, we probe the functions of Gcn5 and PCAF in adipogenesis. We found that the double knockout (DKO) of Gcn5/PCAF inhibits expression of the master adipogenic transcription factor gene PPARgamma, thereby preventing adipocyte differentiation. The adipogenesis defects in Gcn5/PCAF DKO cells are rescued by ectopic expression of PPARgamma, suggesting Gcn5/PCAF act upstream of PPARgamma to facilitate adipogenesis. The requirement of Gcn5/PCAF for PPARgamma expression was unexpectedly bypassed by prolonged treatment with an adipogenic inducer, 3-isobutyl-1-methylxanthine (IBMX). However, neither PPARgamma ectopic expression nor prolonged IBMX treatment rescued defects in Prdm16 expression in DKO cells, indicating that Gcn5/PCAF are essential for normal Prdm16 expression. Gcn5/PCAF regulate PPARgamma and Prdm16 expression at different steps in the transcription process, facilitating RNA polymerase II recruitment to Prdm16 and elongation of PPARgamma transcripts. Overall, our study reveals that Gcn5/PCAF facilitate adipogenesis through regulation of PPARgamma expression and regulate brown adipogenesis by influencing Prdm16 expression (Jin Q. et al., 2014).