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

 描述（由申请人提供）：本提案的目的是检查ATP依赖性重塑因子INO 80和组蛋白变体H2 A. Z之间的相互作用，以便更好地理解它们在激活转录和阻止启动子区域双向转录中的作用。由于相互矛盾的信息，H2A.Z在转录中的作用一直令人困惑。在体外将H2A.Z掺入核小体和核小体阵列中比典型的H2 A更稳定核小体，并促进更高阶折叠成染色质纤维，所有这些都表明H2A.Z应该是转录难治的。然而，H2A.Z是高等真核生物中活性基因的一致标记，并且被认为可以“平衡”基因以进行转录。在转录早期，H2A.Z被来自启动子区域的H2 A迅速取代，并且H2A.Z核小体比典型核小体更迅速地翻转和丢失，这导致它们可以“打开”染色质结构的暗示。虽然我们知道INO 80在重塑过程中将H2A.Z交换为H2 A，但我们对这是如何发生的以及所涉及的反应中间体是否使核小体更容易接近并易于分解知之甚少。在解决H2A.Z核小体的INO 80重塑是否可以解释体外和体内观察结果之间的差异时，这个困境的解决不仅可以回答转录中的基本问题，而且还可能回答DNA修复，与着丝粒和端粒形成相关的基因组稳定性以及复制叉稳定性;所有这些都涉及INO 80和H2A.Z。 INO 80和核小体之间的结合界面将通过确定INO 80结合时与核心组蛋白和核小体和非核小体DNA中的特定位点相关的INO 80结构域来绘制。将通过删除或突变这些结构域内的保守残基来检查INO 80中交联区域与结合和重塑的功能相关性。将使用系综和单分子技术来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H2 A. Z-H2 B与DNA和H3-H4四聚体的接触的破坏，以及其从核小体的最终损失。我们将使用所述突变体确定INO 80促进二聚体排出和进入的H2 A-H2 B二聚体的部分。将通过组蛋白和DNA交联来检测组蛋白H3的赖氨酸56（H3 K56 ac）乙酰化对INO 80与核小体接触的影响。将使用相同的系综和单分子技术来检查当核小体含有H3 K56 ac与未乙酰化的H3时H2 AZ和H2 A交换反应途径的变化，以了解乙酰化如何增强交换反应。

 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.