Proopiomelanocortin Gene Expression and Obesity

描述(申请人提供)：在西方世界和发展中国家，目前对健康和寿命的最大威胁之一是超重和肥胖，这是由于2型糖尿病、心血管疾病和其他代谢综合征的表现。通过荷尔蒙、神经和代谢信号在大脑和外周组织之间相互作用的复杂协调，体重通常保持在一个狭窄的范围内。肥胖症的流行是因为最近的环境、社会和遗传相互作用，不利地使平衡倾向于将过多的卡路里储存作为身体脂肪。调节卡路里摄入和消耗的神经回路的一个关键组成部分是位于下丘脑和延髓的极少量神经元，它们表达POMC基因，并从前体蛋白前阿片黑素皮质素(POMC)合成神经肽aMSH和b内啡肽。在目前的项目期间，我们在解释POMC的转录如何限于这些专门的神经元方面取得了重大进展，这些神经元是与能量流动相关的外周信号的主要门户。我们在外显子1的上游定位了一个大约10kb的模块化增强子基因，它引导POMC神经元特异性基因的表达，并独立于垂体特异性启动子元件发挥作用。进一步的生物信息学和功能询问揭示了哺乳动物谱系中两个在进化上截然不同的模块，我们将其命名为nPE1和nPE2，即神经元POMC增强器。产生了携带单个或两个NPE元件(D1、D2和D1D2)缺失的突变小鼠品系。我们对这些小鼠的初步表型分析，以及在远端增强子基因座插入PGK-neo选择盒的平行菌株，使我们假设nPE1和nPE2对大脑中POMC的表达具有不同的、非冗余的控制，此外，至少还有一个POMC的神经元增强子尚未确定。增强子内的核心核苷酸序列基序和对候选转录因子的持续测试表明，同源结构域因子的独特组合负责POMC神经元特异性表达。因此，此次计划更新的具体目标是：1)剖析POMC神经元增强子nPE1和nPE2在能量平衡调节中的生理作用；2)利用NPE突变株分析神经内分泌信号通路、细胞激活和调控神经元POMC表达的POMC增强子元件之间的相互关系；3)通过鉴定一整套增强子和调控下丘脑Arc神经元和NTS中POMC表达所需的基本基序来揭开神经元POMC转录密码；以及4)确定负责中枢神经系统中POMC基因细胞特异性反式激活的转录因子。这些目标的完成将提供有关体重的基本神经生物学控制点的基础知识，并可能为基于潜在分子机制的肥胖预防或治疗指明新的途径。

DESCRIPTION (provided by applicant): One of the greatest current threats to health and lifespan in both the Western world and developing countries is overweight and obesity due to the predisposition for type-2DM, cardiovascular disease, and other manifestations of the metabolic syndrome. Body weight is normally maintained in a narrow range through a complex orchestration of hormonal, neural, and metabolic signals reciprocating between the brain and peripheral tissues. The obesity epidemic has emerged because of recent environmental, sociological, and genetic interactions that have unfavorably tipped the balance in favor of excess caloric storage as body fat. One critical component of the neural circuitry that regulates caloric intake and expenditure is the tiny population of neurons located in the hypothalamus and medulla that express the Pomc gene and synthesize the neuropeptides aMSH and bEndorphin from the precursor protein, proopiomelanocortin (POMC). In the current project period, we made significant progress towards explaining how transcription of Pomc is restricted to these specialized neurons, which are a primary portal to the brain for peripheral signals relating energy flux. We located a modular enhancer locus about 10 kb upstream of exon 1 that directs POMC neuron-specific gene expression and functions independently of pituitary-specific promoter elements. Closer bioinformatic and functional interrogation of this locus revealed two evolutionarily distinct modules within the Mammalian lineage that we named nPE1 and nPE2 for neuronal POMC Enhancer. Mutant strains of mice were generated that carry deletions of individual or both nPE elements (D1, D2, and D1D2). Our preliminary phenotypic analyses of these mice, and parallel strains with insertions of a PGK-neo selection cassette in the distal enhancer locus, led us to the hypotheses that nPE1 and nPE2 possess distinct, nonredundant control over Pomc expression in the brain, and moreover, that at least one additional neuronal enhancer for Pomc has yet to be identified. Core nucleotide sequence motifs within the enhancers and ongoing testing of candidate transcription factors suggest that a unique combination of homeodomain factors is responsible for POMC neuron-specific expression. Therefore, the specific aims for this project renewal are to: 1) Dissect the physiological roles of the Pomc neuronal enhancers nPE1 and nPE2 on the regulation of energy balance; 2) Analyze the interrelationships among neuroendocrine signaling pathways, cellular activation, and Pomc enhancer elements that regulate neuronal Pomc expression using the nPE mutant strains; 3) Unravel the neuronal Pomc transcriptional code through the identification of the entire set of enhancers and the essential motifs necessary to control Pomc expression in hypothalamic Arc neurons and the NTS; and 4) Identify the transcription factors responsible for cell-specific transactivation of the Pomc gene in the CNS. Completion of these aims will provide fundamental knowledge about an essential neurobiological control point for body mass and may point to new avenues for the prevention or treatment of obesity based on exploitation of the underlying molecular mechanisms.