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.
The epidemic of obesity has caused an alarming prevalence of type 2 diabetes and other metabolic diseases in the United States and worldwide. Millions of Americans are now diagnosed with type 2 diabetes and the health care cost of diabetes is in billions of dollars each year. However, the molecular mechanisms underlying the regulation of energy and nutrient homeostasis remain poorly understood. This proposal will study novel mechanisms underlying the regulation of brown/beige adipose tissue development, lipid biosynthesis in brown/beige adipocytes and metabolic functions of brown/beige adipose tissues. The outcome of this project will have significant and positive impacts on the field of metabolic disease and gene transcription, and may also aid the development of new approaches for the treatment or prevention of obesity and type 2 diabetes.
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