Higher-Order Protein-DNA Complexes in Gene Regulation

描述（由申请人提供）：本提案的目标是确定“先锋”转录因子如何在发育中首先结合基因，在沉默、致密的染色质中接合靶位点并促进后续蛋白质的进入。这些初始步骤是真核生物基因控制中最具决定性的步骤之一，但对潜在的机制知之甚少。以前，我们表明，染色质的转录因子HNF 3和加塔-4的网站从事未分化的内胚层，在激活白蛋白基因在肝脏规格。在过去的研究期间，我们发现HNF 3和加塔-4，而不是其他各种DNA结合因子，可以在体外结合高度致密的染色质内的靶位点，并使当地的核小体结构域接近其他蛋白质。这些活动不需要ATP依赖的核小体重塑复合物。我们进一步发现了HNF 3 α上的一个蛋白质结构域，它与核心组蛋白的一个子集相互作用，并且对于松散核小体间的相互作用至关重要，从而“解压缩”染色质。在下一个资助期，我们将结合联合收割机生物化学和转基因的方法来了解HNF 3和加塔因子如何在染色质中的靶位点启动调控事件，具体目标如下：1.确定HNF 3 α和加塔因子结合染色质如何破坏核小体间的相互作用并在体外确定的染色质模板中产生局部开放结构域。确定野生型和突变型HNF 3 α和加塔-4在转基因小鼠中诱导表达时如何在多种沉默染色质背景中打开染色质。为了确定HNF 3和加塔-4结合如何导致连接组蛋白置换，核小体连接DNA的变化，以及帮助激活转录的辅助蛋白的募集，了解在基因调控的最早阶段染色质结构如何被修饰，将为人类生长，发育和发病机理所必需的遗传过程提供基本的见解。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine how "pioneer" transcription factors, the first to bind genes in development, engage target sites in silent, compacted chromatin and promote entry of subsequent proteins. These initial steps are among the most decisive in eukaryotic gene control, yet little is known about the underlying mechanisms. Previously we showed that chromatin sites for the transcription factors HNF3 and GATA-4 are engaged in undifferentiated endoderm, prior to the activation of the albumin gene during liver specification. In the past grant period, we discovered that HNF3 and GATA-4, but not various other DNA binding factors, can bind their target sites within highly compacted chromatin in vitro and make the local nucleosomal domain accessible to other proteins. These activities do not require ATP-dependent nucleosome remodeling complexes. We further discovered a protein domain on HNF3alpha that interacts with a subset of core histones and is critical for loosening inter-nucleosomal interactions, thereby "decompacting" chromatin. In the next grant period, we will combine biochemical and transgenic approaches to understand how HNF3 and GATA factors initiate regulatory events at target sites in chromatin, with the following specific aims:1. To determine how chromatin binding by HNF3alpha and GATA factors disrupts inter-nucleosomal interactions and creates a locally open domain in defined chromatin templates in vitro.2. To determine how wild type and mutant HNF3alpha and GATA-4, when inducibly expressed in transgenic mice, open chromatin in a diverse array of silent chromatin contexts.3. To determine how HNF3 and GATA-4 binding leads to linker histone displacement, changes in nucleosome linker DNA, arid the recruitment of accessory proteins that help activate transcription.Understanding how chromatin structure is modified during the earliest stages of gene regulation will provide fundamental insight into genetic processes that are essential for growth, development, and pathogenesis in humans.