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

 描述(申请人提供)：本提案的目的是研究依赖ATP的重构体INO80和组蛋白变异体H2 A.Z之间的相互作用，以更好地了解它们在激活转录和防止启动子区域双向转录中的作用。给出了相互矛盾的信息，对于H2A.Z在转录中的作用一直是令人困惑的。在体外，将H_2A.Z掺入核小体和核小体阵列中，比典型的H_2A更能稳定核小体，并促进高阶折叠成染色质纤维，所有这些都表明H_2A.Z对转录应该是难于转录的。然而，在高等真核生物中，H_2A.Z是活性基因的一致标记，被认为是转录的“平衡”基因。在转录早期，H2A.Z从启动子区域迅速被H2A取代，并且H2A.Z核小体比规范的核小体更快地翻转和丢失，这表明它们可以“打开”染色质结构。虽然我们知道INO80在重塑过程中将H_2A.Z交换为H_2A，但我们对这一过程是如何发生的以及所涉及的反应中间体是否使核小体变得更容易接近和易于拆解知之甚少。在讨论INO80是否可以解释体外和体内观察之间的差异时，这一两难问题的解决不仅将回答转录中的基本问题，还可能回答DNA修复、与着丝粒和端粒形成有关的基因组稳定性以及复制叉稳定性；所有这些都涉及INO80和H2A.Z。当INO80结合时，将通过确定与核心组蛋白、核小体和核外DNA中特定位点相关的INO80结构域来绘制INO80与核小体之间的结合界面。INO80中的交联区与结合和重塑的功能相关性将通过删除或突变这些结构域中的保守残基来检验。将使用系综和单分子技术来研究组蛋白交换的结构动力学，以确定事件的速度和顺序，例如H_2A、Z-H_2B与DNA和H3-H4四聚体的接触中断，以及它最终从核小体中丢失。我们将使用所描述的突变体来确定INO80促进二聚体喷射和进入H_2A-H_2B二聚体的部分。组蛋白H3(H3K56ac)的赖氨酸56乙酰化对INO80与核小体接触的影响将通过组蛋白和DNA交联来检测。当核小体含有H3K56ac与未乙酰化的H3时，将使用相同的系综和单分子技术来研究H_2A.Z和H_2A交换反应途径的变化，以了解乙酰化如何促进交换反应。

 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.