Modulation of nucleosome dynamics

描述(申请人提供)：核小体是染色质的组成部分，染色质是在细胞核中发现的DNA的浓缩形式。首先，组蛋白H3和H4的四聚体与DNA结合形成四体，然后四体与两个H_2A-H_2B晚餐结合形成核小体。这种结构被连接物DNA结合的连接物组蛋白(H1)进一步压缩，进一步压缩DNA并限制基因表达所需的蛋白质-DNA相互作用。染色质动力学的调节对细胞的生存至关重要。在这里，我建议研究细胞调节染色质动力学从而调节DNA可及性的两种不依赖于ATP的方式：将组蛋白变体掺入核小体，以及组蛋白伴侣蛋白在DNA可及性中的作用。特定的组蛋白二聚体变体经常在活跃转录(基因表达)的位置附近发现；因此，组蛋白变体被认为是更动态的，从而促进有效的DNA-蛋白质相互作用。此外，组蛋白二聚体变体可能形成不那么紧密或不完整的核小体。这些部分核小体(或六体)在转录过程中被观察到是中间产物。改变二聚体的自由浓度可以促进这些部分核小体的形成。因此，细胞调节染色质动力学的第二种方法是通过使用组蛋白伴侣蛋白来控制游离组蛋白的浓度。我建议使用核小体组装蛋白1(NAP1)作为一种手段来控制游离组蛋白的浓度，测量组蛋白二聚体和变异体的亲和力和协同性，并探索DNA对蛋白质的可及性。这些研究将有助于我们理解含有主要类型和变异二聚体组蛋白的核小体之间染色质动力学的差异，包括在转录过程中可能存在的亚饱和条件下这些性质的检测。此外，我还将探索连接物组蛋白H1和NAP1在染色质动力学中的作用，并使用X射线结晶学来定义NAP1-H1的相互作用。这些研究的结果将加强我们对蛋白质如何获得核小体DNA，以及细胞如何通过控制游离组蛋白浓度来调节这一点的了解。这些数据也应该有助于我们理解染色质背景下的转录。

DESCRIPTION (provided by applicant): The nucleosome is the building block of chromatin, the condensed form of DMA found in the cell nucleus. First, a tetramer of histones H3 and H4 binds to DNA to form the tetrasome, which then binds two H2A-H2B dinners to form the nucleosome. This structure is further compacted by the linker histone (H1) that binds the linker DNA, further compacting the DNA and limiting protein-DNA interactions that are necessary for gene expression. Regulation of chromatin dynamics is critical for cell survival. Herein I propose to investigate two ATP-independent ways in which the cell regulates chromatin dynamics and thereby DNA accessibility: the incorporation of histone variants into nucleosomes and the role of histone chaperones in DNA accessibility. Specific histone dimer variants are often found near locations of active transcription (gene expression); therefore, histone variants are thought to be more dynamic thereby facilitating efficient DNA-protein interactions. Furthermore, histone dimer variants may form less compact, or partial, nucleosomes. These partial nucleosomes (or hexasomes) have been observed as intermediates during transcription. Altering the free concentration of dimers could facilitate the formation of these partial nucleosomes. Therefore, a second method for the cell to regulate chromatin dynamics is through the use of histone chaperones to control the free histone concentration. I propose to use Nucleosome Assembly Protein 1 (NAP1) as a means to control free histone concentrations, to measure the affinity and cooperativity of histone dimers and variants, and to probe DNA accessibility to proteins. These studies will aid our understanding of the differences in chromatin dynamics between nucleosomes containing major type and variant dimer histones, including examination of these properties under sub-saturating conditions, which may exist during transcription. Additionally, I will probe the roles of the linker histone H1 and NAP1 in chromatin dynamics, and define the NAP1-H1 interaction using X-ray crystallography. The results from these studies will enhance our knowledge of how proteins gain access to nucleosomal DNA and how cells regulate this by controlling free histone concentrations. These data should also aid in our understanding of transcription in the context of chromatin.