Epigenetic Regulation of Adipogenesis

具体目的1：检测脂肪形成早期PPARgamma启动子上组蛋白修饰的变化。通过ChIP检测，我们观察到在脂肪形成的早期阶段，PPARgamma启动子上的多个组蛋白乙酰化和甲基化的稳健变化。 具体目标二：我们最近发现，组蛋白H3 K27甲基转移酶Ezh 2抑制Wnt基因，促进脂肪形成。具体来说，我们显示Ezh 2和它的H3 K27甲基转移酶活性是脂肪形成所必需的。Ezh 2直接抑制前脂肪细胞和脂肪形成过程中的Wnt 1、-6、-10a和-10b基因。Ezh 2的缺失消除了Wnt启动子上的H3 K27 me 3并解抑制Wnt表达，这导致Wnt/β-连环蛋白信号传导的激活和脂肪形成的抑制。野生型（WT）Ezh 2的异位表达，而不是酶失活的F667 I突变体，防止了H3 K27 me 3的丢失和Ezh 2（-/-）前脂肪细胞中脂肪形成的缺陷。Ezh 2（-/-）细胞中的脂肪形成缺陷可以通过脂肪形成转录因子PPARgamma、C/EBPalpha或Wnt/β-连环蛋白信号传导抑制剂的表达来挽救。有趣的是，Ezh 2（-/-）细胞显示H3 K27乙酰化在整体上以及在Wnt启动子上显著增加。这些结果表明H3 K27甲基转移酶Ezh 2直接抑制Wnt基因以促进脂肪形成，并表明H3 K27上的乙酰化和三甲基化在调节Wnt表达中起相反的作用（Wang L.例如，Proc Natl Acad Sci U S A（107）：7317-22，2010）。 具体目标3：研究其他表观遗传机制的作用，特别是染色质重塑和非编码RNA，在调节PPARgamma表达和脂肪形成。

Specific Aim 1: examine the change of histone modifications on PPARgamma promoter in the early phase of adipogenesis. By ChIP assays, we have observed robust change of multiple histone acetylation and methylation on PPARgamma promoter in the early phase of adipogenesis. Specific Aim 2: investigate the functional significance of the histone modification change on PPARgamma promoter by depleting the responsible enzymes.We show recently that histone H3K27 methyltransferase Ezh2 represses Wnt genes to facilitate adipogenesis. Specifically, we show Ezh2 and its H3K27 methyltransferase activity are required for adipogenesis. Ezh2 directly represses Wnt1, -6, -10a, and -10b genes in preadipocytes and during adipogenesis. Deletion of Ezh2 eliminates H3K27me3 on Wnt promoters and derepresses Wnt expression, which leads to activation of Wnt/beta-catenin signaling and inhibition of adipogenesis. Ectopic expression of the wild-type (WT) Ezh2, but not the enzymatically inactive F667I mutant, prevents the loss of H3K27me3 and the defects in adipogenesis in Ezh2(-/-) preadipocytes. The adipogenesis defects in Ezh2(-/-) cells can be rescued by expression of adipogenic transcription factors PPARgamma, C/EBPalpha, or inhibitors of Wnt/beta-catenin signaling. Interestingly, Ezh2(-/-) cells show marked increase of H3K27 acetylation globally as well as on Wnt promoters. These results indicate that H3K27 methyltransferase Ezh2 directly represses Wnt genes to facilitate adipogenesis and suggest that acetylation and trimethylation on H3K27 play opposing roles in regulating Wnt expression (Wang L. et al., Proc Natl Acad Sci U S A (107): 7317-22, 2010). Specific Aim 3: investigate the roles of other epigenetic mechanisms, in particular chromatin remodeling and non-coding RNAs, in regulation of PPARgamma expression and adipogenesis.