Transcriptional Regulation of Hepatocyte Differentiation and Function

描述（由申请人提供）：基因调控中一个关键的、未解决的问题是访问和重塑包装发育调控基因的染色质以协调细胞类型特异性基因表达程序（告知组织分化和功能）的方法。这给发育中的胚胎细胞， 其中大部分染色质仍然是致密的，以及成年生物体，其中响应于指导基本生理功能的信号级联的基因表达的改变需要染色质景观的快速改变。未能正确应对这些生物挑战中的任何一个都可能以人类先天性畸形、疾病和肿瘤的形式产生严重的生物医学后果。基于最初的“先锋”染色质结合转录因子FoxO 1重塑组蛋白的能力：体外致密染色质内的DNA接触，其将肝脏富集的调节因子和RNA pol II募集到肝细胞中的调节靶点，以及通过与信号传导途径相关的翻译后修饰调节这些活性，这些信号传导途径在维持葡萄糖中具有已证实的作用，我们假设FoxO 1与紧密并列的肝脏协同作用，丰富的转录因子进行必要的染色质结构改变的发育激活肝脏基因的关键人类肝细胞功能我们认为，翻译后修饰的FoxO 1 DNA结合域，使激素调节FoxO 1靶基因在人类肝脏中破坏这种关系。该提案解决了源于该假设的两个关键问题：1）FoxO 1如何与其他肝脏富集的调节因子合作，进行肝脏基因发育激活所需的染色质重塑和修饰？和2）这种合作是必要的激素调节转录活性染色质编码人类肝脏中的基本代谢功能？为了实现这一目标，我们将使用一种新的hiPS（人诱导多能干细胞）细胞人肝细胞分化系统，以揭示基本的分子机制，告知功能和调节FoxO 1的条件下，概括肝细胞的规范，分化和功能在人类。我们还将使用最先进的分子技术，使组装和分析定义的核蛋白结构，以测试我们的观察所涉及的机制。FoxO 1是整合肝脏葡萄糖、胆固醇、胆汁酸、 和脂质代谢;突变FoxO 1等位基因最近在人类患者的糖尿病和肝脂肪变性和纤维化中得到证实。我们的假设假设，染色质结合和重塑能力归因于FoxO 1是必不可少的核蛋白结构的装配与激活FoxO 1靶基因和介导他们的反应，以不同的信号级联。这些基因调控事件的颠覆可能是代谢紊乱和肝脏疾病的一个贡献者，这使得我们发现关键机制和参与者至关重要。 公共卫生相关性：本提案的总体目标是确定“先锋”初始染色质结合转录因子FoxO 1如何利用其重塑染色质的能力，并与紧密并列的肝脏富集转录因子合作，以启动人类肝脏中靶基因激活和激素调节所需的染色质结构变化。FoxO 1现在被确定为整合人类肝脏葡萄糖、胆固醇、胆汁酸和脂质代谢的关键调节蛋白，FoxO 1突变/不足与人类糖尿病和肝脂肪变性/纤维化有关。如果成功，拟议的实验将揭示FoxO 1调节的新机制，从而能够靶向干预肝细胞分化和功能，以治疗糖尿病和肝病。

DESCRIPTION (provided by applicant): A critical, unresolved issue in gene regulation is the means by which chromatin that packages developmentally regulated genes is accessed and remodeled to orchestrate cell-type specific gene expression programs informing tissue differentiation and function. This poses a critical dilemma in cells of both the developing embryo, in which much of the chromatin is still compacted, and the adult organism, where alterations in gene expression in response to signaling cascades directing essential physiological functions necessitate rapid alterations in the chromatin landscape. Failure to properly navigate either of these biological challenges can have grave biomedical consequences in the form of congenital malformations, disease, and neoplasia in humans. Based on the ability of the initial "pioneer" chromatin binding transcription factor FoxO1 to remodel histone:DNA contacts within compacted chromatin in vitro, its recruitment of liver-enriched regulatory factors and RNA pol II to regulatory targets in hepatocytes, and regulation of these activities by posttranslational modifications linked to signaling pathways with proven roles in maintenance of glucose, bile acid and cholesterol homeostasis in humans we hypothesize that FoxO1 cooperates with closely juxtaposed liver- enriched transcription factors to carry out chromatin structural alterations necessary for developmental activation of hepatic genes crucial to human hepatocyte function. We argue that posttranslational modification of the FoxO1 DNA binding domain enables hormonal regulation of FoxO1 target genes in the human liver by disrupting this relationship. This proposal addresses two key questions that stem from this hypothesis: 1) How does FoxO1 cooperate with other liver-enriched regulatory factors to carry out chromatin remodeling and modification necessary for developmental activation of hepatic genes? and 2) Is this cooperation necessary for hormonal regulation of transcriptionally active chromatin encoding essential metabolic functions in the human liver? To accomplish this goal, we will use a novel hiPS (human induced pluripotent stem) cell human hepatocyte differentiation system to uncover basic molecular mechanisms informing the function and regulation of FoxO1 under conditions that recapitulate hepatocyte specification, differentiation, and function in humans. We will also use state of the art molecular techniques that enable the assembly and analysis of defined nucleoprotein structures to test mechanisms implicated by our observations. FoxO1 is now established as a key transcriptional regulator integrating hepatic glucose, cholesterol, bile acid, and lipid metabolism in humans; mutant FoxO1 alleles are recently demonstrated in diabetes and hepatic steatosis and fibrosis in human patients. Our hypothesis assumes that the chromatin binding and remodeling capabilities attributed to FoxO1 are essential for the assembly of the nucleoprotein structures tasked with activating FoxO1 target genes and mediating their response to diverse signaling cascades. Subversion of these gene regulatory events is a likely contributor to metabolic derangements and hepatic disease, making it vital that we uncover the key mechanisms and players. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to determine how the "pioneer" initial chromatin binding transcription factor FoxO1 uses its abilities to remodel chromatin and cooperate with closely juxtaposed liver-enriched transcription factors to initiate changes in chromatin structure necessary for activation and hormonal modulation of target genes in the human liver. FoxO1 is now established as a key regulatory protein integrating hepatic glucose, cholesterol, bile acid, and lipid metabolism in humans and FoxO1 mutation/insufficiency is linked to diabetes and hepatic steatosis/fibrosis in humans. If successful, the proposed experiments will reveal novel mechanisms for FoxO1 regulation, enabling targeted intervention in hepatocyte differentiation and function for the treatment of diabetes and liver disease.