Epigenomic Regulation of PPARgamma and Adipogenesis

我们研究了组蛋白H3(分别为H3K4、H3K9、H3K27、H3K36和H3K79)上K4、K9、K27、K36和K79上的位点特异性甲基转移酶在脂肪形成和PPAR-γ调控中的作用。我们以前曾报道过H3K4me1甲基转移酶ML3/MLL4和相关的PTIP控制PPARγ和C/EBPA的诱导，并且对于脂肪形成是必不可少的(Cho yw，Cell Metab 2009；Lee Je，eLife 2013；Jang Y，NAR 2019)，H3K9甲基转移酶G9a抑制PPARγ的表达和脂肪形成(王L，EMBO J 2013)，H3K27甲基转移酶Ezh2结构性抑制WNT基因促进脂肪形成(王L，PNAS 2010)，以及Nsd2介导的H3K36甲基化通过组蛋白突变体H3.3破坏前体细胞脂肪组织的发育和功能(L，Nat Comm 2018)。我们还表明，表观基因组阅读器Brd4控制细胞识别基因的诱导，并且对于体内脂肪形成是必不可少的(Lee Je，Nat Comm 2017)。综上所述，这些发现表明表观基因组因子在脂肪形成的调控中发挥着关键作用(见Lee Je，MCB 2019)。 利用条件基因敲除小鼠和前脂肪细胞，我们令人惊讶地发现，尽管配体结合的糖皮质激素受体(GR)在培养中加速脂肪生成，但内源性GR对于培养和小鼠的脂肪生成是必不可少的(Park Y，MCB 2017a)。我们还发现，KLF4和Krox20对于培养和小鼠的脂肪生成是必不可少的(Park Y，MCB 2007b)。 我们分析了增强子表观编写体(MLL4，CBP)，成脂转录因子(Ebf2，C/EBPA，C/EBpb，PPARGamma)，共激活因子MED1，RNA聚合酶II，以及表观基因组(H3K4me1/2/3，H3K9me2，H3K27me3，H3K36me3，H3K27ac)，转录组和染色质开放在小鼠BAT永生化脂肪细胞成脂过程中的基因组结合。我们的数据为理解培养中棕色脂肪生成的表观基因组调控提供了丰富的资源(LAI B，NAR 2017)。 转录因子(TF)C/EBpb和C/EBPd在培养的脂肪形成启动后数小时内被诱导。它们直接促进主要的成脂因子PPARGamma和C/EBPA的表达，是体内脂肪形成所必需的，然而，控制C/EBPB和C/EBPd诱导的机制仍然不清楚。我们先前的研究表明，组蛋白甲基转移酶MLL3/MLL4及其相关的PTIP是脂肪形成过程中诱导PPAR、GAMMA和C/EBPA所必需的。在这里，我们展示了ML3/MLL4/PTIP相关蛋白PAGR1(也称为PA1)与磷酸化的CREB和配体激活的GR协同作用，在脂肪形成的早期阶段直接控制C/EBPB和C/EBPd的诱导。白色和棕色前脂肪细胞中Pagr1的缺失阻止了C/EBPB和C/EBPd的诱导，并导致严重的脂肪生成缺陷。PAGR1缺陷细胞的成脂缺陷可以通过异位表达C/EBPB或PPARγ来挽救。最后，Myf5+前体细胞中Pagr1的缺失会损害棕色脂肪组织和肌肉的发育。因此，通过控制C/EBPB和C/EBPd的诱导，PAGR1在脂肪形成中起着关键作用(Lee Je，MCB 2020)。

We have investigated the roles of site-specific methyltransferases on K4, K9, K27, K36 and K79 of histone H3 (H3K4, H3K9, H3K27, H3K36 and H3K79, respectively) in regulation of adipogenesis and PPARgamma. We reported previously that H3K4me1 methyltransferases MLL3/MLL4 and associated PTIP control the induction of PPARgamma and C/EBPa and are essential for adipogenesis (Cho YW, Cell Metab 2009; Lee JE, eLife 2013; Jang Y, NAR 2019), that H3K9 methyltransferase G9a represses PPARgamma expression and adipogenesis (Wang L, EMBO J 2013), that H3K27 methyltransferase Ezh2 constitutively represses Wnt genes to facilitate adipogenesis (Wang L, PNAS 2010), and that depletion of Nsd2-mediated H3K36 methylation by histone H3.3 mutant H3.3K36M in progenitor cells impairs adipose tissue development and function (Zhuang L, Nat Comm 2018). We also showed that the epigenomic reader Brd4 controls cell identity gene induction and is essential for adipogenesis in vivo (Lee JE, Nat Comm 2017). Together, these findings indicate that epigenomic factors play critical roles in the regulation of adipogenesis (reviewed in Lee JE, MCB 2019). Using conditional knockout mice and preadipocytes, we found surprisingly that although ligand-bound glucocorticoid receptor (GR) accelerates adipogenesis in culture, endogenous GR is dispensable for adipogenesis in culture and in mice (Park Y, MCB 2017a). We also found that KLF4 and Krox20 are dispensable for adipogenesis in culture and in mice (Park Y, MCB 2017b). We profiled genomic binding of enhancer epigenomic writers (MLL4, CBP), adipogenic TFs (EBF2, C/EBPa, C/EBPb, PPARgamma), coactivator MED1, RNA Polymerase II, as well as epigenome (H3K4me1/2/3, H3K9me2, H3K27me3, H3K36me3, H3K27ac), transcriptome, and chromatin opening during adipogenesis of immortalized preadipocytes derived from mouse BAT. Our data provide a rich resource for understanding epigenomic regulation of brown adipogenesis in culture (Lai B, NAR 2017). Transcription factors (TFs) C/EBPb and C/EBPd are induced within hours after initiation of adipogenesis in culture. They directly promote expression of master adipogenic TFs PPARgamma and C/EBPa and are required for adipogenesis in vivo However, the mechanism that controls the induction of C/EBPb and C/EBPd remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARgamma and C/EBPa during adipogenesis. Here we show MLL3/MLL4/PTIP-associated protein PAGR1 (also known as PA1) cooperates with phosphorylated CREB and ligand-activated GR to directly control the induction of C/EBPb and C/EBPd in the early phase of adipogenesis. Deletion of Pagr1 in white and brown preadipocytes prevents the induction of C/EBPb and C/EBPd and leads to severe defects in adipogenesis. Adipogenesis defect in PAGR1-deficient cells can be rescued by ectopic expression of C/EBPb or PPARgamma. Finally, deletion of Pagr1 in Myf5+ precursor cells impairs brown adipose tissue and muscle development. Thus, by controlling the induction of C/EBPb and C/EBPd, PAGR1 plays a critical role in adipogenesis(Lee JE, MCB 2020).