HNF3-mediated chromatin remodeling: A role in liver gene regulation

描述（由申请人提供）：在早期发育期间，分化细胞必须激活以前未表达的基因，这些基因是细胞类型规范和器官发生所必需的。为了使这些基因被激活，染色质必须被分解，以允许转录因子接近DNA模板。本研究的目的是研究参与染色质重塑的潜在机制。具体来说，我们将在肝脏发育模型中通过肝脏富集转录因子HNF3检查白蛋白增强子处的染色质打开。HNF3是一种先驱转录因子，在体外没有染色质重塑复合物的情况下，可以结合并打开白蛋白增强子处的致密染色质。为了确定这一现象的生理相关性，我们计划利用F9畸胎瘤细胞系研究HNF3在体内染色质重塑中的作用。这些细胞在维甲酸的作用下分化为内脏内胚层，并在分化过程中开启白蛋白基因表达。因为我们对检查白蛋白增强子的激活感兴趣，这些细胞将作为一个很好的模型来询问有关HNF3在染色质打开中的作用的问题。为了确定染色质开放的变化，我们将使用dna酶I、微球菌核酸酶（MNase）和染色质免疫沉淀（ChIP）测定，并比较染色质对白蛋白基因上调的可及性。通过突变白蛋白增强子上的HNF3结合位点，我们可以确定体内白蛋白增强子上的染色质重塑是否需要HNF3结合。用短发夹rna （shrna）对抗HNF3的进一步实验将确定HNF3的转录活性是否需要打开白蛋白增强子上的染色质。除了确定HNF3在白蛋白增强子重塑中的作用外，我们还将研究HNF3在其他肝脏特异性基因激活中的潜在作用，包括转甲状腺素和甲胎蛋白。通过充分了解正常发育过程中基因激活的机制，我们有望更好地了解这一过程中可能导致发育缺陷和癌症的畸变。

DESCRIPTION (provided by applicant): During early development, differentiating cells must activate genes previously not expressed that are required for cell type specification and organogenesis. For these genes to be turned on, chromatin must be de-compacted to allow transcription factors access to the DNA template. The goal of this research is to study the underlying mechanisms involved in chromatin remodeling. Specifically, we will examine chromatin opening at the albumin enhancer by the liver enriched transcription factor, HNF3, in a model of liver development. HNF3 is a pioneer transcription factor that can bind to and open compacted chromatin at the albumin enhancer in vitro in the absence of chromatin remodeling complexes. In order to establish the physiological relevance of this phenomenon, we plan to examine the role of HNF3 in chromatin remodeling in vivo using the F9 teratocarcinoma cell line. These cells, in the presence of retinoic acid, differentiate into visceral endoderm and turn on albumin gene expression as they differentiate. Because we are interested in examining the activation of the albumin enhancer, these cells will serve as a good model to ask questions about the role of HNF3 in chromatin opening. In order to determine changes in chromatin opening, we will use DNase I, micrococcal nuclease (MNase) and chromatin immunoprecipitation (ChIP) assays and compare chromatin accessibility to albumin gene upregulation. By mutating HNF3 binding sites on the albumin enhancer, we can determine whether HNF3 binding is required for chromatin remodeling at the albumin enhancer in vivo. Additional experiments with short hairpin RNAs (shRNAs) against HNF3 will determine whether HNF3 transcriptional activity is required to open chromatin at the albumin enhancer. In addition to determining the role of HNF3 in albumin enhancer remodeling, we will also examine a potential role for HNF3 in the activation of other liver specific genes, including transthyretin and alpha-fetoprotein. By fully understanding the mechanisms involved in gene activation during normal development, we can hopefully better understand aberrations in this process that may lead to developmental defects and cancer.