Transcriptional role of TLE3 in brown adipose tissue development and metabolism

描述(由申请人提供)：肥胖会增加患慢性疾病的风险，如2型糖尿病、心血管疾病和癌症。肥胖是由于能量摄入和能量消耗之间的不平衡造成的，多余的能量以甘油三酯的形式储存在脂肪细胞中。PPAR？是脂肪生成的关键调节因子，调节脂肪细胞系特有基因的表达。在脂肪形成过程中促进PPAR依赖的基因表达的机制还不完全清楚。先前我们曾报道TLE3是PPAR？的转录辅助调节因子。并参与一个前馈转录程序，以推动脂肪生成。在拟议的研究中，我将检验TLE3是驱动白色脂肪与棕色脂肪选择性基因表达的关键决定因素的假设。初步数据表明，在棕色脂肪组织(BAT)中过度表达TLE3的小鼠有从棕色脂肪组织(WAT)到白色脂肪组织(WAT)的表型转换。因此，TLE3转基因小鼠在受到冷暴露的挑战时，生热反应受到损害。机制研究表明，TLE3与Prdm16相反，Prdm16是一种棕色脂肪基因表达的转录辅助调节因子。在具体目标1中，我将使用体外模型来研究TLE3在执行WAT和BAT转录程序中的功能和作用机制。我将利用体外功能得失研究来确定TLE3是否影响白色脂肪与棕色脂肪的基因表达。获得功能的方法将包括逆转录病毒和腺病毒表达TLE3和/或Prdm16。功能丧失研究将利用感染对照或Cre-腺病毒的白色和棕色TLE3F/F前脂肪细胞在体外产生基因敲除。我将通过检测TLE3引导染色质重塑和组蛋白修饰酶向脂肪细胞启动子募集的能力来探索其作用机制。在具体目标2中，我将使用活体模型来确定TLE3在白色和棕色脂肪组织中的功能以及全身脂质代谢。我已经培育了在脂肪细胞中表达TLE3的转基因动物，以及在脂肪细胞中有条件缺失TLE3的小鼠。我将使用这些模型来检测TLE3影响脂肪细胞基因表达、产热以及脂肪和葡萄糖代谢的能力。拟议的研究将在加州大学洛杉矶分校Peter Tontonoz博士的实验室完成，他提供了一个丰富的环境，将促进向独立调查员职位的过渡。职业发展奖将提供受保护的时间来发展写作、网络和沟通方面的关键技能。除了……之外 参加加州大学洛杉矶分校的研讨会和参加会议，我将参加一些课程，这些课程将加强使用放射性同位素研究代谢途径、蛋白质纯化和表征以及用于表观遗传学研究的下一代测序技术方面的技术培训。建议的研究是从我的脂肪生成博士后研究到新兴的棕色脂肪细胞生物学领域的合乎逻辑的过渡。

DESCRIPTION (provided by applicant): Obesity increases the risk for chronic diseases such as type 2 diabetes, cardiovascular disease, and cancer. Obesity results from the imbalance between energy intake and energy expenditure, where excess energy is stored in adipocytes as triglycerides. PPAR? is a critical regulator of adipogenesis, regulating the expression of genes that are characteristic of the adipocyte lineage. The mechanisms that facilitate PPAR?- dependent gene expression during the course of adipogenesis are incompletely understood. Previously we reported that TLE3 is a transcriptional coregulator of PPAR? and is involved in a feed-forward transcriptional program to drive adipogenesis. In the proposed studies I will test the hypothesis that TLE3 is a key determinant in driving white versus brown fat selective gene expression. Preliminary data indicates that mice overexpressing TLE3 in brown adipose tissue (BAT) have a phenotypic switch from brown to white adipose tissue (WAT). As a result, TLE3 transgenic mice have an impaired thermogenic response when challenged with cold exposure. Mechanistic studies suggest that TLE3 counters Prdm16, a transcriptional coregulator of brown fat gene expression. In specific Aim 1 I will use in vitro models to investigate the function and mechanism of action of TLE3 in executing the WAT and BAT transcriptional programs. I will utilize in vitro gain and loss of function studies to determine whether TLE3 affects white versus brown fat gene expression. Gain of function approaches will include retroviral and adenoviral expression of TLE3 and/or Prdm16. Loss of function studies will utilize white and brown TLE3F/F preadipocytes infected with control or Cre-adenovirus to generate in vitro knockouts. I will explore the mechanism of action by examining the ability of TLE3 to direct chromatin remodeling and recruitment of histone modifying enzymes to adipocyte promoters. In specific Aim 2 I will use in vivo models to define the function of TLE3 in white and brown adipose tissue and systemic lipid metabolism. I have already generated transgenic animals expressing TLE3 in adipocytes, as well as mice with conditional deletion of TLE3 in adipocytes. I will use these models to examine the ability of TLE3 to affect adipocyte gene expression, thermogenesis and lipid and glucose metabolism. The proposed studies will be completed in the laboratory of Dr. Peter Tontonoz at UCLA, who has provided an enriching environment that will facilitate the transition to an independent investigator position. The career development award will provide protected time to develop critical skills in writing, networking, and communication. In addition to attending seminars at UCLA and attending conferences, I will take courses that will enhance technical training in the use of radioisotopes for the study of metabolic pathways, protein purification and characterization, and next generation sequencing technologies for the study of epigenetics. The proposed studies are a logical transition from my postdoctoral studies in adipogenesis to the burgeoning field of brown adipocyte biology.