Regulation of Brown Fat Development and Function by Cyclin C

介体复合物是一种多亚基蛋白质复合物，通过将许多转录因子连接到RNA聚合酶II而充当转录辅因子。细胞周期蛋白C（Cyclin C，CycC）是介体复合物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。我们最近确定CycC作为棕色脂肪细胞发育和功能的调节剂。Myf 5+细胞中的CycC敲除减少但不消除棕色脂肪组织。残留的棕色脂肪细胞显示维持在标准食物饮食的小鼠中脂质积累的显著减少。基因表达分析显示，CycC基因敲除选择性地损害了ChREBP靶基因的表达，包括Fasn，从头脂肪生成的关键基因。发现CycC-介体与ChREBP物理相互作用，并且ChREBP的转录活性在CycC敲除细胞中降低。这些数据表明，在高碳水化合物饮食条件下，棕色脂肪细胞自主ChREBP依赖性从头脂肪生成可能需要脂滴形成。虽然CycC敲除对其余的介体复合物的整体完整性几乎没有影响，但在CycC敲除的棕色前脂肪细胞中分化受到抑制。这可能是由于CycC需要表达关键的脂肪形成基因，包括Zfp 423和Pparg。PPARg的过表达或PPARg配体的添加挽救了CycC敲除细胞中的缺陷，表明CycC不是PPARg转录活性所需的，而是Pparg基因表达所需的。Zfp 423和Pparg的表达受EBF转录因子的调节，并且CycC-介体也与EBF 1物理相互作用。 基于初步研究，我们假设CycC在介体复合物的背景下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-介体复合物是EBF 1介导的Zfp 423和Pparg基因激活所必需的，这是棕色/米色前脂肪细胞测定所必需的。其次，CycC-介体复合物也是ChREBP转录活性所必需的，ChREBP转录活性对于细胞自主从头脂肪生成所必需的棕色/米色脂肪细胞脂肪生成基因表达至关重要。为了验证这些假设，我们提出了两个相关但独立的具体目标。目的1将研究CycC-介体对棕色/米色前脂肪细胞中EBF 1和EBF 2的转录活性和表达的调节，介体在棕色/米色脂肪细胞体内发育中的功能，以及CycC对控制棕色/米色前脂肪细胞决定的转录程序的上下文依赖性调节。目的2将检查棕色/米色脂肪细胞中ChREBP的CycC介导调节，以及在各种饮食和温度条件下小鼠模型中CycC或ChREBP的Ucp 1+细胞特异性敲除的组织学、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定型的分子基础以及从头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.