Proopiomelanocortin gene expression and obesity

描述(申请人提供)：肥胖和相关的代谢紊乱，包括糖尿病，已经在全球流行，并威胁到数百万人的健康。最近，来自人类群体遗传学研究、小鼠转基因实验和神经肽药理学的数据融合表明，前阿片黑素皮质素(POMC)基因、其编码的多肽及其同源的G蛋白偶联受体是调节食物摄入量和能量消耗的神经回路的重要组成部分。进化导致了功能和形态上不同的DNA调控元件，这些调控元件控制着神经元或垂体内分泌细胞中POMC的转录，并且在哺乳动物中高度保守。这些细胞类型特异性POMC启动子和增强子元件的差异反映在转录因子的独特互补中，这些转录因子差异调节POMC基因在脑和脑垂体中的表达。POMC在下丘脑神经元中的表达与小鼠一生中的脂肪质量定量相关，我们假设在人类中，神经元POMC基因表达的类似控制是决定脂肪储存或利用的最终平衡的关键调控开关。该项目的第一个目标是利用体内转基因和体外原代神经元培养表达系统的组合，在额外的跨物种比较测序的指导下，进一步表征POMC基因的神经元特异性增强子元件。第二个目标将专门确定POMC基因调控元件与瘦素受体信号通路组件的相互作用，以导致脂肪细胞来源的激素激活神经元POMC基因转录。第三，在内源性小鼠POMC基因的背景下，选择性神经增强子突变的数量和质量效应将通过基因敲入策略进行严格测试。第四，通过激光捕获显微镜分离POMC神经元的mRNA“转录组”，并将利用计算基因组学和从扩增的POMC神经元特异性RNA构建的酵母单杂交cDNA文库的筛选，鉴定和克隆负责神经元特异性POMC基因表达的同源DNA结合转录因子。在这些研究中，我们将与我们的临床同事合作，他们参与了人类全基因组筛查和索引基于病例的基因测序项目，以翻译努力识别和功能表征POMC基因本身或参与体重调节改变的候选神经元POMC基因转录因子的其他多态等位基因。

DESCRIPTION (provided by applicant): Obesity and associated metabolic disorders including diabetes have become epidemic worldwide and threaten the health of millions. A recent confluence of data from human population genetics studies, mouse transgenic experiments, and neuropeptide pharmacology has implicated the proopiomelanocortin (POMC) gene, its encoded peptides, and their cognate G-protein coupled receptors as essential components of the neural circuits that regulate food intake and energy expenditure. Evolution has resulted in functionally and topographically distinct DNA regulatory elements that control POMC transcription in either neurons or pituitary endocrine cells, and which are highly conserved across mammalian species. These distinctions in cell-type specific POMC promoter and enhancer elements are mirrored by unique complements of transcription factors that differentially regulate POMC gene expression in the brain versus pituitary gland. POMC expression in hypothalamic neurons is quantitatively associated with adipose mass over the lifetime of a mouse and we hypothesize that in humans the analogous control of neuronal POMC gene expression is a key regulatory switch determining the ultimate balance of fat storage or utilization. The first aim of this project is to further characterize the neuronal-specific enhancer elements of the POMC gene using a combination of in vivo transgenic and in vitro primary neuronal culture expression systems, guided by additional cross-species comparative sequencing. A second aim will specifically determine the interaction of POMC gene regulatory elements with components of the leptin receptor-signaling pathway to result in activation of neuronal POMC gene transcription by the adipocyte-derived hormone. Third, the quantitative and qualitative effects of selective neural enhancer mutations in the context of the endogenous mouse POMC gene will be rigorously tested by genetic knock-in strategies. Fourth, the mRNA "transcriptome" of POMC neurons isolated by lasercapture microscopy will be defined, and the cognate DNA-binding transcription factors responsible for neuron-specific POMC gene expression will be identified and cloned using both computational genomics and screening of yeast one-hybrid cDNA libraries constructed from amplified POMC neuron-specific RNA. Throughout these studies we will collaborate with our clinical colleagues, who are involved in human genome-wide screens and index case-based gene sequencing projects, in a translational effort to identify and functionally characterize additional polymorphic alleles of either the POMC gene itself or candidate neuronal POMC gene transcription factors underlying altered weight regulation.