Transcriptional Regulation of Hepatocyte Differentiation and Function

描述(申请人提供)：基因调控中一个关键的、尚未解决的问题是，包装发育调节基因的染色质被访问和重塑，以协调细胞类型的特定基因表达程序，通知组织分化和功能。这在发育中的胚胎的细胞中造成了一个关键的两难境地， 其中染色质的大部分仍被压缩，以及成年生物体，在那里，基因表达的变化响应于引导基本生理功能的信号级联，需要在染色质景观中迅速改变。如果不能正确应对这些生物挑战中的任何一项，都可能产生严重的生物医学后果，表现为人类的先天性畸形、疾病和肿瘤。基于最初的“先驱”染色质结合转录因子FoxO1重塑组蛋白的能力：在体外致密染色质内的DNA接触，它对肝脏富含调节因子和RNA PolII的招募到肝细胞的调节靶点，以及通过翻译后修饰对这些活动的调节，这些信号通路已被证明在人类维持葡萄糖、胆汁酸和胆固醇稳态中发挥了重要作用，我们假设FoxO1与紧密并列的肝脏富含转录因子合作，进行染色质结构改变，从而对对人类肝细胞功能至关重要的肝基因的发育激活起到必要的作用。我们认为，FoxO1 DNA结合域的翻译后修饰通过破坏这种关系，能够对人类肝脏中的FoxO1靶基因进行激素调节。这一建议解决了源于这一假说的两个关键问题：1)FoxO1如何与其他富含肝脏的调节因子合作，进行肝脏基因发育激活所需的染色质重塑和修饰？2)这种合作对于人类肝脏中编码基本代谢功能的转录活性染色质的激素调节是必要的吗？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来揭示在概括人类肝细胞规格、分化和功能的条件下，FoxO1功能和调控的基本分子机制。我们还将使用最先进的分子技术，使已定义的核蛋白结构的组装和分析能够测试我们观察到的机制。Foxo1现在被确定为一种关键的转录调节因子，它整合了肝脏的葡萄糖、胆固醇、胆汁酸、 突变的FoxO1等位基因最近被证明与人类患者的糖尿病、肝脏脂肪变性和纤维化有关。我们的假设认为，FoxO1的染色质结合和重塑能力对于核蛋白结构的组装是必不可少的，核蛋白结构负责激活FoxO1靶基因，并介导它们对不同信号级联反应的反应。这些基因调控事件的颠覆可能是代谢紊乱和肝病的促成因素，因此我们揭示关键机制和参与者至关重要。 与公共健康相关：这项建议的总体目标是确定“先驱”初始染色质结合转录因子FoxO1如何利用其能力重塑染色质，并与紧密并列的肝脏富含转录因子合作，启动人类肝脏靶基因激活和激素调节所需的染色质结构变化。Foxo1基因突变/缺失与人类糖尿病和肝脏脂肪变性/纤维化有关。Foxo1基因突变/缺失与人类肝脏葡萄糖、胆固醇、胆汁酸和脂肪代谢有关。如果成功，拟议的实验将揭示FoxO1调节的新机制，使有针对性地干预肝细胞分化和治疗糖尿病和肝病的功能。

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.