Epigenomic regulation of adipogenesis and adipose expansion

I. 我们研究了组蛋白H3K4、H3K9、H3K27和H3K36甲基转移酶在脂肪形成和PPARgamma调节中的作用。我们报道了H3K4me1甲基转移酶MLL3/MLL4和相关的PTIP控制PPARgamma和C/EBPa的诱导，并且是脂肪形成所必需的（Cell Metab 2009; eLife 2013; NAR 2019），H3K9甲基转移酶G9a抑制PPARgamma表达和脂肪生成（EMBO J 2013），H3K27甲基转移酶Ezh2组成型抑制Wnt基因以促进脂肪形成（PNAS 2010），组蛋白H3.3突变体H3.3K36M消耗Nsd2介导的H3K36甲基化会损害脂肪组织发育和功能（Nat Comm 2018）。我们还表明，表观基因组阅读器Brd4控制细胞身份基因诱导，并且对于体内脂肪形成至关重要（Nat Comm 2017）。总之，这些研究结果表明，表观基因组因子在脂肪形成的调控中起着关键作用（在MCB 2019中进行了综述）。 二. 使用条件性基因敲除小鼠和前脂肪细胞，我们惊讶地发现，尽管糖皮质激素受体（GR）加速培养物中的脂肪生成，但内源性GR在培养物和小鼠中不促进脂肪生成（MCB 2017 a）。 我们还纠正了文献，并证明转录因子（TF）KLF4和Krox20在培养物和小鼠中对脂肪形成有抑制作用（MCB 2017 b）。 三. 我们分析了增强子表观基因组写入器（MLL4，CBP），脂肪形成TF（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），转录组的基因组结合，以及来源于小鼠棕色脂肪组织的永生化前脂肪细胞脂肪形成过程中的染色质开放。我们的数据为理解培养中棕色脂肪形成的表观基因组调控提供了丰富的资源（NAR 2017）。 四. C/EBPb和C/EBPd在培养物中脂肪形成开始后数小时内被诱导。它们直接促进主成脂TF PPARgamma和C/EBPa的表达，并且是体内脂肪形成所需的。我们报道了MLL3/MLL4/PTIP相关蛋白PAGR 1与磷酸化CREB和配体活化GR合作，在脂肪形成的早期阶段直接控制C/EBPb和C/EBPd的诱导（MCB 2020）。 诉 我们发现BAF是SWI/SNF染色质重塑复合物，与MLL4和活性增强子上的谱系决定性TF（LDTF）共定位。BAF是脂肪生成所需的，而富含启动子的SWI/SNF复合物PBAF是必需的（Nat Comm 2021）。 六. 我们确定了MED1作为出生后脂肪扩张所需的脂肪生成辅激活剂的基因特异性调节作用（Genes Dev 2021）。 七.我们已经发现，在小鼠中，在谱系特异性前体细胞中染色质重塑复合物ISWI的酶亚基的缺失损害脂肪和肌肉发育。 在培养中，ISWI酶亚基的KO不影响细胞生长，但导致脂肪生成和肌生成的严重缺陷。 八.我们正在产生H3K4甲基转移酶MLL1和MLL2的敲除和敲入小鼠，以研究它们在脂肪组织发育和扩张中的作用。

I. We have investigated the roles of histone H3K4, H3K9, H3K27 and H3K36 methyltransferases in regulation of adipogenesis and PPARgamma. We reported that H3K4me1 methyltransferases MLL3/MLL4 and associated PTIP control the induction of PPARgamma and C/EBPa and are essential for adipogenesis (Cell Metab 2009; eLife 2013; NAR 2019), that H3K9 methyltransferase G9a represses PPARgamma expression and adipogenesis (EMBO J 2013), that H3K27 methyltransferase Ezh2 constitutively represses Wnt genes to facilitate adipogenesis (PNAS 2010), and that depletion of Nsd2-mediated H3K36 methylation by histone H3.3 mutant H3.3K36M impairs adipose tissue development and function (Nat Comm 2018). We also showed that the epigenomic reader Brd4 controls cell identity gene induction and is essential for adipogenesis in vivo (Nat Comm 2017). Together, these findings indicate that epigenomic factors play critical roles in the regulation of adipogenesis (reviewed in MCB 2019). II. Using conditional knockout mice and preadipocytes, we found surprisingly that although glucocorticoid receptor (GR) accelerates adipogenesis in culture, endogenous GR is dispensable for adipogenesis in culture and in mice (MCB 2017a). We also corrected the literature and demonstrated that transcription factors (TFs) KLF4 and Krox20 are dispensable for adipogenesis in culture and in mice (MCB 2017b). III. 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 brown adipose tissue. Our data provide a rich resource for understanding epigenomic regulation of brown adipogenesis in culture (NAR 2017). IV. 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. We reported that MLL3/MLL4/PTIP-associated protein PAGR1 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 (MCB 2020). V. We found that BAF is the SWI/SNF chromatin remodeling complex that colocalizes with MLL4 and lineage-determining TFs (LDTFs) on active enhancers. BAF is required for adipogenesis while the promoter enriched SWI/SNF complex PBAF is dispensable (Nat Comm 2021). VI. We identified a gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion (Genes Dev 2021). VII. We have found that in mice, deletion of enzymatic subunits of the chromatin remodeling complex ISWI in lineage-specific precursor cells impairs adipose and muscle development. In culture, KO of ISWI enzymatic subunits does not affect cell growth but leads to severe defects in adipogenesis and myogenesis. VIII. We are generating knockout and knockin mice for H3K4 methyltransferases MLL1 and MLL2 to investigate their roles in adipose tissue development and expansion.