Regulation of Brown Fat Development and Function by Cyclin C

细胞周期蛋白C(Cyclin C，CycC)是介体复合体的一个高度保守的亚基，但其在棕色/米色脂肪细胞中的作用尚不清楚。我们最近发现CycC是棕色脂肪细胞发育和功能的调节因子。Myf5+细胞中的CycC基因敲除减少了棕色脂肪组织，但不能消除棕色脂肪组织。残留的棕色脂肪细胞显示，在标准饮食维持的小鼠体内，脂肪堆积显著减少。基因表达分析表明，CycC基因敲除选择性地削弱了ChREBP靶基因的表达，包括FASN，这是从头开始脂肪生成的关键基因。在CycC基因敲除细胞中，发现CycC-Mediator与ChREBP发生物理作用，并且ChREBP的转录活性降低。这些数据表明，在高碳水化合物饮食条件下，棕色脂肪细胞可能需要依赖于ChREBP的从头脂肪生成来形成脂滴。尽管CycC基因敲除对其余介体复合体的整体完整性影响不大，但CycC基因敲除的棕色前脂肪细胞的分化受到抑制。这可能是由于CycC对包括Zfp423和PPARG在内的关键成脂基因的表达的要求。过表达PPARg或添加PPARg配体可以挽救CycC基因敲除细胞中的缺陷，表明CycC不是PPARg转录活性所必需的，而是PPARG基因表达所必需的。Zfp423和PPARG的表达受EBF转录因子的调控，CycC-Mediator也与EBF1发生物理作用。在初步研究的基础上，我们假设，在中介复合体的背景下，CycC通过两种不同的机制调节棕色/米色脂肪细胞的发育和功能。首先，CycC-Mediator复合体是EBF1介导的Zfp423和PPARG基因激活所必需的，这些基因是决定棕色/米色前脂肪细胞所必需的。其次，CycC-Mediator复合体也是ChREBP转录活性所必需的，对棕色/米色脂肪细胞脂肪生成基因的表达至关重要，这是细胞自主从头脂肪生成所必需的。为了检验这些假设，我们提出了两个相关但独立的具体目标。目的1研究CycC对棕色/米色前体脂肪细胞转录活性和EBF1和Ebf2表达的调节，在体内棕色/米色脂肪细胞发育中的中介功能，以及CycC对控制棕色/米色前体脂肪细胞决定转录程序的上下文依赖性调节。目的2将研究在棕色/米色脂肪细胞中ChREBP的CycC介体调节，以及在不同饮食和温度条件下Ucp1+细胞特异性敲除CycC或ChREBP在小鼠模型中的组织学、代谢和分子结果。长期目标是了解棕色和米色脂肪细胞谱系承诺的分子基础，以及新生脂肪生成在棕色和米色脂肪细胞功能生理学中的作用。

The Mediator complex is a multi-subunit protein complex that acts as a transcriptional cofactor by connecting a number of transcription factors to the RNA polymerase II. Cyclin C (CycC) is a highly conserved subunit of the Mediator complex, but its role in brown/beige adipocytes remains unclear. We recently identify CycC as a regulator of brown adipocyte development and function. CycC knockout in Myf5+ cells reduces but does not eliminate brown adipose tissues. The residual brown adipocytes display a marked reduction in lipid accumulation in mice maintained on the standard chow diet. Gene expression analyses revealed that CycC knockout selectively impaired the expression of ChREBP target genes including Fasn, the key gene for de novo lipogenesis. The CycC-Mediator was found to physically interact with ChREBP and the transcriptional activity of ChREBP was reduced in CycC-knockout cells. These data suggest that under high-carbohydrate dietary conditions, brown adipocyte autonomous ChREBP-dependent de novo lipogenesis may be required for lipid droplet formation. Although CycC knockout had little effect on the overall integrity of the rest of the Mediator complex, differentiation was inhibited in CycC-knockout brown preadipocytes. This was likely due to the requirement of CycC for the expression of key adipogenic genes, including Zfp423 and Pparg. Overexpression of PPARg or addition of PPARg ligands rescued the defect in CycC-knockout cells, indicating that CycC is not required for PPARg transcriptional activity, but for Pparg gene expression. The expression of Zfp423 and Pparg are regulated by the EBF transcription factors and the CycC-Mediator also physically interacts with EBF1. Based on the preliminary studies, we hypothesize that CycC in the context of the Mediator complex regulates brown/beige adipocyte development and function through two distinct mechanisms. First, the CycC-Mediator complex is required for EBF1-mediated activation of Zfp423 and Pparg genes necessary for brown/beige preadipocyte determination. Second, the CycC-Mediator complex is also required for ChREBP transcriptional activity critical for brown/beige adipocyte lipogenic gene expression necessary for cell autonomous de novo lipogenesis. To test these hypotheses, we propose two related but independent Specific Aims. Aim 1 will examine the CycC-Mediator regulation of the transcriptional activity and expression of EBF1 and EBF2 in brown/beige preadipocytes, the Mediator functions in brown/beige adipocyte development in vivo, and the context-dependent regulation of CycC on the transcription program that controls the brown/beige preadipocyte determination. Aim 2 will examine the CycC-Mediator regulation of ChREBP in brown/beige adipocytes, and histologic, metabolic and molecular outcomes of Ucp1+ cell- specific knockout of CycC or ChREBP in mouse models under various dietary and temperature conditions. The long-term objective is to understand the molecular basis for brown and beige adipocyte lineage commitment and the role of de novo lipogenesis in the physiology of brown and beige adipocyte function.