Chromatin Remodeling During Liver Development

描述（由申请人提供）：我的目标是确定“先驱”或初始染色质结合转录因子如何参与沉默的、致密的染色质中的靶位点，并促进后续蛋白质的进入。先锋转录因子HNF3结合并激活分化和代谢所需的许多肝脏基因。先前的研究表明，在肝脏发育过程中，在白蛋白基因激活之前，HNF3参与未分化内胚层染色质的特定位点。利用体外重组核小体系统，我已经证明HNF3可以在高度致密的染色质包装白蛋白增强子内结合其靶位点。这种结合，以及随后的核小体定位，创造了一个染色质的局部开放区域，其他蛋白质可以进入。我假设由HNF3启动的染色质结合和重塑建立了一个染色质环境，使白蛋白增强子激活所需的后续结合、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。为了验证这些假设，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)在染色质打开期间，HNF3是否从白蛋白增强子上取代连接体组蛋白？2) hnf3介导的染色质重塑能否在体内激活白蛋白增强子？3) hnf3介导的转录能力是否对肝脏基因的激活至关重要？这些研究旨在确定先锋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。

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.