Proopiomelanocortin gene expression and obesity

项目摘要 肥胖使人类容易患上糖尿病和心血管疾病，是对健康的普遍威胁。胖的 大脑和大脑之间协调的荷尔蒙、神经和新陈代谢信号动态控制存储 外围设备。合成黑素皮质素多肽的前阿片黑素皮质素(POMC)神经元是一种初级整合 这些与能量动态平衡相关的不同信号的位置。在过去的资金周期中，我们做出了重大贡献 解释Pomc转录如何局限于下丘脑神经元亚群的研究进展 弓形核。由两个进化上截然不同的增强子nPE1和nPE2组成的模块化基因座 神经元特异性POMC表达。对单个增强子或增强子组合的靶向删除揭示了 它们在下丘脑发育期间协同作用，并在成人生活中相加，以保持足够的健壮 POMC转录，避免肥胖。人类基因组核心核苷酸序列基序的生物信息学分析 增强子结合候选同源框转录因子的解剖和功能询问 (TFS)在动物模型中发现Isl1和Nkx2.1在指导独特的时间和 弓状核中Pomc表达的空间分布。RAX TF也有助于POMC的身份 发育中的神经元，但间接作用于Is11的上游。尽管这些因素是必要的，但仅有这些因素并不是 足以充分解释神经元POMC调节的复杂性。翻译核糖体亲和力 POMC神经元的纯化(TRAP)序列已鉴定出额外的高度差异表达的TF基因 可能参与了Pomc基因表达的调控。因此，我们提出了以下具体目标 本项目更新：1)发现控制下丘脑Pomc表达的完整基因程序 通过确定POMC神经元和它们的早期身份的候选TF的功能特征 使用小鼠分子遗传学和人类多潜能干细胞，在整个生命周期内保持 (HPSCs)可在体外分化为POMC神经元；2)确定定义 通过在体功能分析下丘脑Pomc的全套神经元特异性表达 增强子，它们的关键结合基序和局部绝缘体，以组装一个完全功能的转录基因座； 3)剖析了激素相关的个体增强子和关键基序的生理意义。 瘦素对Pomc表达的调节以及对改变的饮食条件和热量需求的反应。 这些研究将提供对调节体重至关重要的基因的基础知识，并可能 确定可用于治疗目的的新的遗传或信号通路。

Project Summary Obesity predisposes humans to diabetes and cardiovascular disease and is a universal threat to health. Fat storage is dynamically controlled by orchestrated hormonal, neural and metabolic signals between the brain and periphery. Proopiomelanocortin (POMC) neurons that synthesize melanocortin peptides are a primary integrative site for these diverse signals related to energy homeostasis. In the past funding cycles, we made significant progress towards explaining how Pomc transcription is restricted to a subset of hypothalamic neurons in the arcuate nucleus. A modular locus comprised of two evolutionarily distinct enhancers, nPE1 and nPE2, directs neuron-specific Pomc expression. Targeted deletion of the individual or combination of enhancers revealed that they act synergistically during hypothalamic development and additively in adult life to maintain sufficiently robust Pomc transcription to avoid obesity. A bioinformatic analysis of core nucleotide sequence motifs within the enhancers combined with anatomic and functional interrogation of candidate homeobox transcription factors (TFs) in animal models identified a major contribution of Isl1 and Nkx2.1 in directing the unique temporal and spatial patterns of Pomc expression in the arcuate nucleus. The Rax TF also contributes to the identity of POMC neurons developmentally, but acts indirectly upstream of Isl1. Although necessary, these factors alone are not sufficient to fully account for the complexities of neuronal Pomc regulation. Translating Ribosome Affinity Purification (TRAP) seq of POMC neurons has identified additional, highly differentially expressed TF genes that are putatively involved in the control of Pomc expression. Therefore, we propose the following specific aims for this project renewal: 1) Uncover the complete genetic program controlling hypothalamic Pomc expression through the functional characterization of candidate TFs that define the early identity of POMC neurons and their maintenance throughout the entire lifetime, using mouse molecular genetics and human pluripotential stem cells (hPSCs) that can be differentiated into POMC neurons in vitro; 2) Determine the cis-acting code that defines hypothalamic Pomc expression through the in vivo functional analysis of the entire set of neuron-specific enhancers, their critical binding motifs and local insulators to assemble a fully functional transcriptional locus; and 3) Dissect the physiological significance of individual enhancers and critical motifs involved in the hormonal regulation of Pomc expression by leptin and in response to altered dietary conditions and caloric demands. These studies will provide fundamental knowledge about a gene essential for regulating body mass and possibly identify novel genetic or signaling pathways that can be exploited for therapeutic purposes.