STRUCTURE OF NUCLEOSOMES CONTAINING VARIANT HISTONES

描述（改编自申请人的摘要）：单个真核生物的DNA 细胞长度超过两米，但在细胞核中由 包装成核小体和随后组织的分级方案 转化为更高级的染色质结构。基因在特定时间的激活 需要在高度致密的染色质中进行鉴定。局部解包 然后通过重塑因子重塑染色质， 转录机器 该实验室研究的中心目标是了解， 结构水平，以何种方式改变压实状态，以适应 转录机制。这是通过确定 在2A分辨率下的核小体核心颗粒的三维结构。 这里提出的实验将提供有关变化的信息， 赋予核小体结构使其具有“转录能力”。的 具体的目标是 (1)研究核小体核心颗粒的结构和稳定性 含有优先与以下相关的必需组蛋白变体： 转录活性染色质，通过确定高分辨率X射线 含有H2A.Z的核小体核心颗粒的结构，并通过测量 这些核小体的体外稳定性使用荧光能量转移。 (2)研究组蛋白的结构和功能 突变（SIN-变体），其绕过ATP依赖性 染色质重塑复合物在酵母中，通过确定的X射线结构， 含有组蛋白SIN变体的酵母核小体核心颗粒。的 酵母核小体核心颗粒的稳定性和动力学 SIN-变体将使用光谱方法进行研究， 野生型和突变核小体在体外结合转录因子将是 使用多种生物化学和光谱学方法测定。 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修饰来调节。 由于基因表达的调控在细胞调控中起着至关重要的作用， 生长、分化以及组织的建立和维持， 这些研究将直接有助于了解 与异常基因相关的许多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.