Higher-Order Protein-DNA Complexes in Gene Regulation

描述(申请人提供)：这项建议的目标是确定“先驱”转录因子，第一个结合发育中的基因，在沉默的，紧凑的染色质中参与目标位置，并促进后续蛋白质进入。这些最初的步骤是真核基因控制中最具决定性的步骤之一，但人们对其潜在机制知之甚少。以前，我们发现转录因子HNF3和GATA-4的染色质部位在白蛋白基因激活之前参与了未分化的内胚层。在过去的资助期间，我们发现HNF3和GATA-4，而不是其他各种DNA结合因子，可以在体外与高度致密的染色质结合其靶点，并使局部核小体区域可被其他蛋白质访问。这些活性不需要依赖于ATP的核小体重塑复合体。我们进一步发现了HNF3pha上的一个蛋白质结构域，它与核心组蛋白的子集相互作用，对于松散核小体之间的相互作用，从而对染色质进行“解压缩”是至关重要的。在下一个授权期，我们将结合生化和转基因方法来了解HNF3和GATA因子如何在染色质的目标位置启动调控事件，具体目标如下：1.确定HNF3pha和GATA因子如何结合染色质破坏核小体之间的相互作用，并在体外定义的染色质模板中创建一个局部开放的区域。确定野生型和突变型HNF3pha和GATA-4在转基因小鼠中诱导表达时，如何在不同的沉默染色质环境中打开染色质。为了确定HNF3和GATA-4结合是如何导致连接体组蛋白置换、核小体连接体DNA的变化以及帮助激活转录的辅助蛋白的招募的，了解染色质结构在基因调控的早期阶段是如何改变的，将为我们提供对人类生长、发育和发病至关重要的遗传过程的基本见解。

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.