Proopiomelanocortin gene expression and obesity

描述（由申请人提供）：肥胖和相关的代谢紊乱，包括糖尿病，已成为全球流行病，威胁着数百万人的健康。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，proopiomelanocortin （POMC）基因、其编码肽及其同源g蛋白偶联受体是调节食物摄入和能量消耗的神经回路的重要组成部分。进化导致了功能和地形上不同的DNA调控元件，这些元件控制神经元或垂体内分泌细胞的POMC转录，并且在哺乳动物物种中高度保守。细胞类型特异性POMC启动子和增强子元件的这些差异反映在独特的转录因子互补中，这些转录因子在脑和脑垂体中调节POMC基因表达的差异。下丘脑神经元中的POMC表达与小鼠一生中脂肪量的定量相关，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最终平衡的关键调节开关。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结合，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。第二个目标将具体确定POMC基因调控元件与瘦素受体信号通路组分的相互作用，从而导致脂肪细胞衍生激素激活神经元POMC基因转录。第三，将通过基因敲入策略严格测试内源性小鼠POMC基因背景下选择性神经增强子突变的定量和定性影响。第四，将定义通过激光捕获显微镜分离的POMC神经元的mRNA“转录组”，并使用计算基因组学和筛选由扩增的POMC神经元特异性RNA构建的酵母单杂交cDNA文库，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选和基于索引病例的基因测序项目，在翻译工作中识别和功能表征POMC基因本身或候选神经元POMC基因转录因子的额外多态性等位基因，这些等位基因可能是体重调节改变的基础。

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.