Transcription Factor Interactions with Nucleosomes

描述（由申请人提供）：染色质首先由Flemming于1882年定义为“细胞核中易于染色的物质”，“对消化无反应”。这个词被卡住了，现在被用来指DNA（容易染色的成分）和相关的蛋白质，使其对消化不敏感。在过去的20年里，人们已经清楚地认识到，染色质结构比以前想象的要动态得多。染色质结构的调节经常调节蛋白质接近DNA的能力，并且以这种方式在调节基因组的转录、复制和重组中起关键作用。染色质的基本结构成分核小体在这种调节中起着核心作用。核小体在DNA上的位置可以改变，核小体可以被驱逐，核小体可以包含各种组蛋白变体，并且可以以不同的方式进行共价修饰，使它们具有与调控机制相互作用的不同能力。在过去的十五年里，染色质的动态性质得到了深入的研究，其中大部分信息与核小体共价修饰的类型和程度有关。关于核小体位置和可能参与调节特定基因组位点的蛋白质的全谱的信息已经落后于关于染色质共价改变的大量信息。本申请中描述的实验的目的是研究如何在体外和体内改变核小体的位置，并确定可能参与给定基因组基因座上的这些类型的调节的蛋白质的全谱。提出了三个目标：目标1将通过检查单个重塑产物和通过结构方法来表征体外核小体重塑;目标2将表征培养的人类细胞中一组基因调控过程中染色质的结构变化;目标3将开发技术来编目与特定位点结合的蛋白质和RNA。公共卫生相关性：表观遗传机制通过改变个体基因在不同细胞类型中表达的能力来影响人类基因组的表达，并且已知其对正常发育和包括癌症在内的许多疾病过程有很大贡献。这些机制在很大程度上依赖于修改染色质结构的机制，这是本资助申请的重点。本申请提出了理解人类染色质结构如何被调节以影响基因表达变化的实验，因此与理解从癌症到感染性疾病的许多疾病过程直接相关。

DESCRIPTION (provided by applicant): Chromatin was first defined by Flemming in 1882 as the `substance in the cell nucleus which is readily stained' that `is refractile to digestion'. The term stuck, and now is taken to refer to DNA (the readily stained component) and the associated proteins that make it refractile to digestion. It has become clear over the past twenty years that chromatin structure is vastly more dynamic than previously suspected. Regulation of chromatin structure frequently regulates the ability of proteins to access DNA, and in this manner plays a key role in regulating transcription, replication and recombination of the genome. The basic structural component of chromatin, the nucleosome, plays a central role in this regulation. The position of nucleosomes on the DNA can be changed, nucleosomes can be evicted, and nucleosomes can contain various histone variants and can be covalently modified in different ways, allowing them to have differential abilities to interact with the regulatory machinery. The dynamic nature of chromatin has been intensively studied for the past fifteen years, with much of the resultant information pertaining to the type and extent of covalent modification of the nucleosome. Information on the location of nucleosomes and on the full spectrum of proteins that might be involved in regulating a specific genomic locus has lagged behind this flood of information on covalent alterations to chromatin. The purpose of the experiments described in this application is to investigate how the location of nucleosomes can be altered in vitro and in vivo and to determine the full spectrum of proteins that might be involved in these types of regulation on a given genomic locus. Three Aims are proposed: Aim 1 will characterize nucleosome remodeling in vitro by examining individual remodeled products and through structural approaches; Aim 2 will characterize structural changes to chromatin during regulation of a set of genes in cultured human cells; and Aim 3 will develop technology to catalog proteins and RNA that bind to specific loci. Public Health Relevance: Epigenetic mechanisms impact the expression of the human genome by altering the ability of individual genes to be expressed in different cell types, and are known to contribute strongly to normal development and to many disease processes including cancer. These mechanisms rely in large part upon mechanisms to modify chromatin structure, the focus of this grant application. This application proposes experiments to understand how human chromatin structure might be regulated to effect changes in gene expression and as such is directly relevant to an understanding of numerous disease processes from cancer to infectious disease.