Proopiomelanocortin gene expression and obesity

项目摘要 肥胖使人类易患糖尿病和心血管疾病，是对健康的普遍威胁。脂肪 储存是由大脑和大脑之间协调的激素、神经和代谢信号动态控制的。 外围合成黑皮质素肽的前阿黑皮素（POMC）神经元是一种主要的整合性神经元。 这些不同的信号与能量平衡有关。在过去的融资周期中， 在解释Pomc转录如何局限于下丘脑神经元的一个子集方面取得了进展， 弓状核。一个由两个进化上不同的增强子nPE 1和nPE 2组成的模块化基因座， 神经元特异性Pomc表达。单个增强子或增强子组合的靶向缺失揭示， 它们在下丘脑发育过程中协同作用，在成年后相加作用， Pomc转录，以避免肥胖。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，确定了Isl 1和Nkx2.1在指导独特的时间和 Pomc在弓状核中表达的空间模式。Rax TF也有助于POMC的身份 神经元发育，但间接作用于Isl 1的上游。尽管这些因素是必要的，但 足以完全解释神经元Pomc调控的复杂性。翻译核糖体亲和力 POMC神经元的纯化（TRAP）序列已经鉴定了另外的高度差异表达的TF基因， 参与Pomc表达的调控。因此，我们提出以下具体目标， 本项目更新：1）揭示控制下丘脑Pomc表达的完整遗传程序 通过对候选TF的功能表征，确定POMC神经元的早期身份， 使用小鼠分子遗传学和人类多能干细胞， （hPSC），其可以在体外分化成POMC神经元; 2）确定定义POMC神经元的顺式作用密码。 下丘脑Pomc表达通过在体内功能分析的整套神经元特异性 增强子、其关键结合基序和局部绝缘子，以组装全功能转录位点; 和3）剖析参与激素调节的单个增强子和关键基序的生理意义。 Pomc表达的调节瘦素和响应改变的饮食条件和热量需求。 这些研究将提供有关调节体重所必需的基因的基础知识， 鉴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.