Proopiomelanocortin gene expression and obesity

描述（由申请人提供）：肥胖和相关代谢紊乱（包括糖尿病）已成为全球流行病，并威胁着数百万人的健康。最近的汇合数据，从人类群体遗传学研究，小鼠转基因实验，和神经肽药理学有牵连的前阿黑皮素（POMC）基因，其编码的肽，和它们的同源G-蛋白偶联受体的神经回路，调节食物摄入和能量消耗的重要组成部分。进化导致了功能和地形不同的DNA调控元件，控制POMC转录在神经元或垂体内分泌细胞，这是高度保守的哺乳动物物种。细胞类型特异性POMC启动子和增强子元件中的这些区别通过独特的转录因子互补物反映，所述转录因子差异调节脑与垂体中的POMC基因表达。下丘脑神经元中的POMC表达与小鼠一生中的脂肪量定量相关，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节开关。该项目的第一个目的是进一步表征神经元特异性增强子元件的POMC基因使用体内转基因和体外原代神经元培养表达系统的组合，指导额外的跨物种比较测序。第二个目标将具体确定POMC基因调控元件与瘦素受体信号传导途径的组分的相互作用，从而导致脂肪细胞衍生的激素激活神经元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定量和定性影响将通过遗传敲入策略进行严格测试。第四，mRNA“转录组”的POMC神经元分离的激光捕获显微镜将被定义，同源DNA结合转录因子负责神经元特异性POMC基因表达将被确定和克隆使用计算基因组学和筛选酵母单杂交cDNA文库构建扩增POMC神经元特异性RNA。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基因转录因子的其他多态性等位基因，这些基因是改变体重调节的基础。

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.