The interplay between the chromatin remodeler INO80 and histone variant H2A.Z.

染色质重塑因子 INO80 和组蛋白变体 H2A.Z 之间的相互作用。

• 批准号: 9060966
• 负责人: Blaine Bartholomew
• 金额: $ 43.42万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9060966
• 关键词: Acetylation; Address; B-Lymphocytes; Binding; Cell Cycle; Cell Proliferation; Cells; Centromere; Chromatin; Chromatin Fiber; Chromatin Remodeling Factor; Chromatin Structure; Chromosomes; Complex; DNA; DNA Crosslinking; DNA Damage; DNA Repair; DNA biosynthesis; DNA replication fork; Development; Eukaryota; Event; Gene Activation; Gene Expression; Genes; Genetic Transcription; Genome; Genome Stability; Goals; Health; Histone H3; Histones; ISWI; In Vitro; Link; Lymphoma; Lysine; Maintenance; Malignant Neoplasms; Maps; Mission; Modeling; Molecular; Mutagenesis; Neoplasm Metastasis; Nucleosomes; Oncogene Activation; Pathway interactions; Patients; Process; Promoter Regions; Public Health; RNA Polymerase II; Reaction; Recruitment Activity; Refractory; Regulation; Research; Resolution; Role; Site; Specificity; Suggestion; Techniques; Transcriptional Activation; Variant; War; Work; base; cell transformation; chromatin remodeling; crosslink; dimer; embryonic stem cell; genome integrity; human disease; in vivo; mutant; prevent; promoter; repaired; single molecule; telomere; telomere loss

 DESCRIPTION (provided by applicant): The objective of this proposal is to examine the interplay between the ATP-dependent remodeler INO80 and the histone variant H2A.Z in order to better understand their roles in activating transcription and preventing bidirectional transcription from promoter regions. It has been confusing as to the role of H2A.Z in transcription given the contradictory information. Incorporation of H2A.Z in vitro into nucleosomes and nucleosomal arrays stabilizes the nucleosome more than canonical H2A and promotes higher-order folding into a chromatin fiber, all of which suggests H2A.Z should be refractory to transcription. However, H2A.Z is a consistent marker of active genes in higher eukaryotes and has been thought to "poise" genes for transcription. Early in transcription H2A.Z is rapidly replaced by H2A from promoter regions and H2A.Z nucleosomes are more rapidly turned over and lost than canonical nucleosomes leading to the suggestion that they can "open" the chromatin structure. Although we know INO80 exchanges H2A.Z for H2A during remodeling, we know little about how this happens and if the reaction intermediates involved predispose nucleosomes to become more accessible and prone to disassemble. In addressing whether INO80 remodeling of H2A.Z nucleosomes can explain the discrepancy between in vitro and in vivo observations, the resolution of this dilemma will not only answer basic question in transcription but also potentially in DNA repair, genomic stability linked to the formation of centromeres and telomeres, and replication fork stability; all of which involve INO80 and H2A.Z. The binding interface between INO80 and nucleosomes will be mapped by determining the INO80 domains associated with specific sites in core histones and nucleosomal and extranucleosomal DNA when INO80 is bound. The functional relevance of the crosslinked regions in INO80 for binding and remodeling will be examined by deletion or mutagenesis of conserved residues within these domains. The structural dynamics of histone exchange will be examined using ensemble and single molecule techniques to determine the rate and order of events such as disruption of the contacts of H2A.Z-H2B with DNA and the H3-H4 tetramer, and its eventual loss from nucleosomes. We will determine the parts of INO80 facilitating dimer ejection and the entry of the incoming H2A-H2B dimer using the described mutants. The effects of acetylation of lysine 56 of histone H3 (H3K56ac) on the contacts of INO80 with nucleosomes will be examined by histone and DNA crosslinking. Alterations in the reaction pathways of H2A.Z and H2A exchange when nucleosomes contain H3K56ac versus unacetylated H3 will be examined using the same ensemble and single molecule techniques to understand how acetylation enhances the exchange reaction.

 描述（由适用提供）：该提案的目的是检查依赖ATP的重塑器INO80与组蛋白变体H2A.Z之间的相互作用，以便更好地了解它们在激活转录和预防启动子区域的双向转录中的作用。考虑到矛盾的信息，H2A.Z在转录中的作用一直令人困惑。将H2A.Z的体外掺入核小体中，核小体阵列比规范H2A更稳定核体，并促进高阶折叠到染色质纤维中，所有这些都表明H2A.Z应折磨于转录。然而，H2A.Z是较高真核生物中活性基因的一致标记，被认为是“有利的”基因进行转录。在转录早期，H2A.Z迅速被启动子区域的H2A替换，而H2A.Z核小体比规范核糖体更快地翻转和丢失，导致他们建议它们可以“打开”染色质结构。尽管我们知道Ino80在重塑过程中将H2A.Z换成H2A，但我们对这种情况的发生却一无所知，并且反应中间体是否涉及易于易于核素，更容易访问并容易拆卸。在解决H2A.Z核骨体的INO80是否可以解释体外和体内观测之间的差异时，解决这一困境的解决不仅会回答转录中的基本问题，而且还可能在DNA修复中，基因组稳定性，与Centromeres和Centromeres和Centromeres and Telomeres和telomeres and Telomeres和Replicational和Replicational fork Storition fork Stitose;所有涉及INO80和H2A.Z。通过确定与核心组蛋白和核小体中特定位点相关的INO80结构域以及当INO80结合时，将映射INO80和核小体之间的结合界面。 INO80中交联区域在结合和重塑中的功能相关性将通过这些域内组成的残差的缺失或诱变来检查。将使用集合和单分子技术检查组蛋白交换的结构动力学，以确定事件的速率和顺序，例如H2A.Z-H2B与DNA和H3-H4 Tetramer的接触的破坏以及其最终因核组的损失。我们将使用所描述的突变体确定支持二聚体射血的INO80部分以及传入的H2A-H2B二聚体的进入。组蛋白和DNA交联检查了组蛋白H3（H3K56AC）赖氨酸56（H3K56AC）对INO80与核小体接触的影响。 H2A.Z和H2A交换的反应途径的变化将使用相同的集合和单分子技术研究，以了解乙酰化如何增强交换反应。