STRUCTURE OF NUCLEOSOMES CONTAINING VARIANT HISTONES

描述(摘自申请者的摘要)：单个真核生物的DNA 细胞长度超过两米，但在细胞核内以一种 包装成核小体和后续组织的分级方案 转化为更高阶的染色质结构。某一基因在给定时间的激活 需要在高度紧凑的染色质中进行鉴定。本地拆包 并通过重塑因子对染色质进行重塑，从而允许结合 转录机器。 该实验室研究的中心目标是了解，在 结构级别，以何种方式更改压缩状态以适应 转录机器。这是通过确定 核小体核心粒子在2a分辨率下的三维结构。 这里提出的实验将提供有关以下变化的信息 赋予核小体结构，使其具有转录能力。这个 具体目标是 (1)研究核小体核心颗粒的结构和稳定性 含有基本的组蛋白变体，这些变体优先与 转录活性染色质，通过测定高分辨率X射线 含H_2A.Z的核小体核心颗粒的结构和测量 用荧光能量转移法研究这些核小体的体外稳定性。 (2)研究组蛋白的结构和功能后果 绕过对ATP依赖的要求的突变(SIN变异体) 酵母染色质重塑复合体的X射线结构测定 酵母核小体核心颗粒，含有组蛋白的SIN变异体。这个 酵母核小体核心颗粒的稳定性和动力学 SIN变异体将使用光谱方法进行研究，并且能够 野生型和突变型核小体在体外结合转录因子 使用各种生化和光谱方法进行检测。 这些研究将直接有助于我们了解转录是如何 在真核细胞中通过核小体结构的改变而调节。 因为基因表达的调节在细胞控制中是至关重要的 组织的生长、分化、建立和维持 器官，这些研究将直接有助于了解 与基因异常相关的多种疾病状态的分子基础 表情。

DESCRIPTION(adapted from applicant's abstract): The DNA of a single eukaryotic cell is over two meters in length, but compacts in the cell nucleus by a hierarchical scheme of packaging into nucleosomes and subsequent organization into higher order chromatin structures. Activation of a gene at a given time requires its identification within highly compacted chromatin. Local unpacking and remodeling of chromatin by remodeling factors then allows the binding of the transcription machinery. The central goal of this laboratory's research is to understand, at the structural level, in what manner the compacted state is altered to accommodate the transcription machinery. This was initiated by determining the three-dimensional structure of the nucleosome core particle at 2A resolution. The experiments proposed here will provide information on the changes that are imparted on nucleosome structure to make it 'transcription competent'. The specific aims are (1) To study the structure and stability of nucleosome core particles containing essential histone variants that are preferentially associated with transcriptionally active chromatin, by determining the high-resolution X-ray structure of the nucleosome core particle containing H2A.Z, and by measuring the in vitro stability of these nucleosomes using fluorescence energy transfer. (2) To investigate the structural and functional consequences of histone mutations (the SIN-variants) which bypass the requirement for an ATP-dependent chromatin-remodeling complex in yeast, by determining the X-ray structure of the yeast nucleosome core particle containing the SIN-variants of histones. The stability and dynamics of the yeast nucleosome core particle containing SIN-variants will be studied using spectroscopic methods, and the ability of wild type and mutant nucleosomes to bind transcription factors in vitro will be assayed using a variety of biochemical and spectroscopic methods. These studies will contribute directly to our knowledge of how transcription is regulated in the eukaryotic cell by modification of nucleosome structure. Because the regulation of gene expression is crucial in the control of cell growth, differentiation, and the establishment and maintenance of tissues and organs, these studies will contribute directly to the goal of understanding the molecular basis of many diseased states that are associated with aberrant gene expression.