Proopiomelanocortin Gene Expression and Obesity

描述（由申请人提供）：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，这是由于易患2型糖尿病、心血管疾病和代谢综合征的其他表现。体重通常通过激素、神经和代谢信号在大脑和外周组织之间往复的复杂协调而维持在一个狭窄的范围内。肥胖症的流行是由于最近的环境，社会学和遗传学的相互作用，这些相互作用不利地打破了平衡，有利于多余的热量储存为身体脂肪。调节热量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髓质中的少量神经元，其表达Pomc基因并从前体蛋白质阿黑皮素原（POMC）合成神经肽aMSH和b内啡肽。在当前的项目期间，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通量相关的外周信号进入大脑的主要门户。我们位于一个模块化的增强子基因座约10 kb的外显子1，指导POMC神经元特异性基因的表达和功能独立的垂体特异性启动子元件的上游。对该基因座的更密切的生物信息学和功能询问揭示了哺乳动物谱系中的两个进化上不同的模块，我们将其命名为nPE 1和nPE 2，用于神经元POMC增强子。产生携带单个或两个nPE元件（D1、D2和D1 D2）缺失的小鼠突变株。我们对这些小鼠的初步表型分析，以及在远端增强子位点插入PGK-neo选择盒的平行品系，使我们假设nPE 1和nPE 2对大脑中的Pomc表达具有不同的非冗余控制，此外，至少还有一种额外的Pomc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正在进行的候选转录因子测试表明，同源结构域因子的独特组合是POMC神经元特异性表达的原因。因此，本项目更新的具体目的是：1）剖析Pomc神经元增强子nPE 1和nPE 2在调节能量平衡中的生理作用; 2）利用nPE突变株分析神经内分泌信号通路、细胞活化和调节神经元Pomc表达的Pomc增强子元件之间的相互关系; 3）通过鉴定下丘脑弧神经元和NTS中控制Pomc表达所必需的整组增强子和基本基序来解开神经元Pomc转录密码;和4）鉴定负责CNS中Pomc基因的细胞特异性反式激活的转录因子。这些目标的完成将提供关于身体质量的基本神经生物学控制点的基础知识，并可能指出基于开发潜在分子机制的预防或治疗肥胖的新途径。 公共卫生相关性：目前对公共健康的最大威胁是超重、肥胖、糖尿病和代谢综合征的发病率不断增加。一组复杂的神经回路将热量需求和利用之间的平衡与进食相关的行为和心理过程相结合。这个项目集中在大脑的进食回路的关键分子组成部分，proopiomelanocortin基因，以及该基因如何在下丘脑的特定神经元中响应代谢和激素信号（包括瘦素，胰岛素和葡萄糖流量）进行调节。

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 2 DM, 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. PUBLIC HEALTH RELEVANCE: Among the greatest current threats to public health are the continually increasing rates of overweight, obesity, diabetes, and the metabolic syndrome. A complex set of neural circuits integrates the balance between caloric demand and utilization with the behavioral and psychological processes related to feeding. This project centers on a key molecular component of the brain's feeding circuits, the proopiomelanocortin gene, and how the gene is regulated in specific neurons of the hypothalamus in response to metabolic and hormonal signals including leptin, insulin, and glucose flux.