Chromatin Remodeling During Liver Development

描述(申请人提供)：我的目标是确定“先驱者”或初始染色质结合转录因子如何在沉默、紧凑的染色质中结合目标位置，并促进后续蛋白质的进入。先锋转录因子HNF3结合并激活许多分化和新陈代谢所需的肝脏基因。以前的研究表明，HNF3在肝脏发育过程中白蛋白基因激活之前，就与未分化内胚层染色质中的特定部位发生了接触。使用重组核小体的体外系统，我已经证明了HNF3可以结合其高度紧凑的染色质包装白蛋白增强剂的靶点。这种结合，以及随后的核小体定位，创造了一个其他蛋白质可访问的染色质的局部开放区域。我假设HNF3启动的染色质结合和重塑建立了一个染色质环境，使随后的白蛋白增强子激活所需的结合、重塑和修饰事件成为可能。我还提出，HNF3介导的转录能力的建立是肝细胞分化和功能所需基因发育激活的先决条件。为了验证这些假说，我将结合体外生化和体内遗传学方法来解决以下具体目标：1)HNF3是否在染色质开放过程中取代白蛋白增强子中的连接子组蛋白？2)HNF3介导的染色质重塑能否在体内激活白蛋白增强子？以及3)HNF3介导的转录能力对于肝脏基因的激活是必不可少的吗？这些研究旨在确定先驱DNA结合调节因子在基因激活所需的染色质重塑中所起的作用。了解染色质结构变化如何指定早期基因调控事件，将有助于深入了解人类生长、发育和疾病的分子机制。 这项资助的主要目标是确定HNF3蛋白激活肝脏基因的机制。HNF3对肝脏发育是必不可少的，在某些形式的癌症和糖尿病中会发生突变。这笔赠款中描述的研究结果将为肝脏疾病和肿瘤的研究和治疗提供新的靶点。

DESCRIPTION (provided by applicant): My goal is to determine how "pioneer" or initial chromatin binding transcription factors engage target sites in silent, compacted chromatin and promote entry of subsequent proteins. The pioneer transcription factor HNF3 binds to and activates numerous liver genes required for differentiation and metabolism. Previous studies have shown that HNF3 engages specific sites in chromatin in undifferentiated endoderm, prior to the activation of the albumin gene during liver specification. Using an in vitro system of reconstituted nucleosomes, I have demonstrated that HNF3 can bind its target sites within highly compacted chromatin packaging the albumin enhancer. This binding, and subsequent nucleosome positioning, creates a localized, open domain of chromatin accessible to other proteins. I hypothesize that chromatin binding and remodeling initiated by HNF3 establishes a chromatin environment that enables the subsequent binding, remodeling, and modification events required for albumin enhancer activation. I also propose that establishment of HNF3-mediated transcriptional competency is a prerequisite to the developmental activation of genes required for hepatocyte differentiation and function. In order to test these hypotheses, I will combine in vitro biochemical and in vivo-genetic approaches to address the following specific aims: 1) Does HNF3 displace linker histone from the albumin enhancer during chromatin opening? 2) Can HNF3-mediated chromatin remodeling activate the albumin enhancer in vivo? and 3) Is HNF3-mediated transcriptional competency essential for the activation of liver genes? These studies are designed to define the role played by pioneer DNA binding regulatory factors in the chromatin remodeling required for gene activation. Understanding how chromatin structural changes specify early gene regulatory events will provide insight into molecular mechanisms which underlie growth, development, and disease in humans. This primary goal of this grant is to determine the mechanisms used by the HNF3 protein to activate genes in the liver. HNF3 is essential for liver development, and is mutated in certain forms of cancer and diabetes. The results of the studies described in this grant will provide novel targets for the study and treatment of liver disease and neoplasia.