Role of USF methylation and epigenetic modifications in lipogenic gene repression during starvation (PI: Dr. Roger Wong)
Background Mammals employ intricate and precise endocrine control to maintain a constant supply of metabolic energy. Transcription of genes encoding enzymes involved in fatty acid and triacylglycerol synthesis is coordinately regulated in lipogenic tissues liver and adipose tissue by fasting/feeding and glucagon/insulin treatment. Dysregulation of lipogenesis often contributes to metabolic diseases such as obesity, fatty liver, diabetes, and cardiovascular diseases. Transcription factors, coregulators and signaling molecules involved in transcriptional regulation of lipogenesis in lipogenic tissues represent attractive targets for the treatment of metabolic diseases. In transcriptional regulation of lipogenesis by fasting/feeding and starvation/insulin, USF-1 functions as a molecular switch to recruit four distinct families of proteins to the lipogenic and glycolytic promoters: 1) coregulators including P/CAF and HDAC9, 2) DNA break/repair machinery including, 3) signaling molecules including PP1 and DNA-PK, and 4) chromatin remodeling complex lipoBAF. During feeding/insulin, USF-1 is phosphorylated at S262 by DNA-PK, which is first dephosphorylated/activated by PP1. Phosphorylation of USF-1 allows recruitment of and acetylation by P/CAF, resulting in the lipogenesis activation. In this regard, DNA-PK deficient mice SCID mice also have a lower adipose tissue mass and body weight, indicative of a long-term defect in feeding induced lipogenesis. In parallel to the DNA-PK insulin signaling, our study links BAF60c to the aPKC insulin signaling. In response to insulin, BAF60c is phosphorylated by aPKC, which causes translocation of BAF60c to the nucleus and allows a direct interaction of BAF60c with acetylated USF-1. Thus, BAF60c is recruited to form the lipoBAF complex to remodel chromatin structure and to activate lipogenic genes. Consequently, BAF60c promotes lipogenesis in vivo and increases triglyceride levels. We, for the first time, demonstrate that USF plays a critical role in mediating the transcriptional regulation of lipid metabolism in response to fasting/feeding and starvation/insulin and converging the two insulin signaling, DNA-PK and aPKC.
Rationale So far, we have shown that USF plays an indispensable role in insulin action, lipid and glucose metabolism and that the interaction between USF and its interacting proteins including P/CAF, HDAC9, SREBP-1c and BAF60c is dependent on USF post-translational modifications (phosphorylation and acetylation) mediated by novel insulin signaling via DNA-PK and aPKC. These lessons point to us that USF modifications are the key in searching for the metabolic state specific coregulators.
Methodology and Objectives Utilizing the phosphorylation mutants of USF-1 (both phospho mimicking and blocking mutants) as baits, we have recently identified MEP50 as a USF interacting protein and we detected MEP50 in the unphosphorylated USF complex that is only present in fasting condition. MEP50 is always found bound to PRMT5 in which is a Type II protein arginine methyltransferase. In the same experiment, we detected that residue R214 of USF is methylated only when DNA-PK regulated S262 is unphosphorylated. Possibly, in fasted state whereas USF-1 is unphosphorylated at S262 leads to recruitment of HDAC9 and MEP50-PRMT5 complex to deacetylate, and methylate USF-1 at R214 and subsequently deacetylate and methylate histones locally to repress transcription via epigenetic mechanism. In this proposal, 1) we will first validate the interaction between PRMT5-MEP50 complex and modified USF. PRMT5 and MEP50 will be examined for their abilities to bind to lipogenic and glycolytic gene promoters. Direct interaction between PRMT5-MEP50 and modified USFs will be investigated along with the functional significance of coregulators on transcriptional repression. 2) USF methylation will be examined in different metabolic states and insulin treated condition along with the functional significance of the methylation on USF activation. We will determine whether R214 of USF is subjected to methylation by PRMT5-MEP50. Specific histone methylation events will be examined on promoters as well as chromatin accessibility. 3) By overexpressing and silencing PRMT5 and MEP50 in animals, we will examine the in vivo consequence of coregulators on glucose and lipid metabolism. The in vivo significance of USF methylation will be dissected by expressing methylation mutants. Identified PRMT5, MEP50 and USF site specific methylation are all potential pharmacological targets for treatments of metabolic diseases.