Deciphering the Role of MacroH2A in Non-coding RNA Mediated Silencing

解读 MacroH2A 在非编码 RNA 介导的沉默中的作用

基本信息

批准号：
7806710
负责人：
Kavitha Sarma
金额：
$ 4.76万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7806710
关键词：
Affect Alleles Amino Acids Architecture Base Sequence Binding Biological Assay C-terminal Cell Cycle Cell Line Cells Chromatin Chromatin Structure Chromosomes Complex DNA Methylation DNA-Directed RNA Polymerase Dosage Compensation (Genetics)Epigenetic Process Fellowship Female Fibroblasts Functional RNA Gene Expression Gene Silencing Generations Genes Genetic Transcription Genome Genomics Heterochromatin Histone H2A Histone H3 Histones Human In Vitro Lysine Maintenance Mammals Mediating Memory Methylation Modification Mus N-terminal Nature Nucleosomes Pathway interactions Phase Polycomb Proteins RNA RNA Binding RNA I Recruitment Activity Research Proposals Role Sports Staging Transcript Variant X Chromosome base chromatin remodeling embryonic stem cell gene repression genome-wide histone methyltransferase histone modification imprint in vivo insight interest knock-down macroH2A histone male reconstitution research study transmission process

项目摘要

DESCRIPTION (provided by applicant): My broad aim for this fellowship is to study the role of the istone variant MacroH2A in non-coding RNA mediated gene silencing. MacroH2A is enriched on the inactive X chromosome (Xi) in mammals, which is coated by the Xist non-coding RNA. First, I will analyze whether macroH2A is enriched at regions of the genome that are regulated by other non-coding RNAs. I will use ChlP-Seq to identify these domains. Second, I will analyze whether macroH2A can directly bind Xist RNA, since the localization ofthis variant to the Xi is dependent on the presence of Xist RNA. I will also try to identify other non-coding RNAs that bind to this histone variant using RNA ChlP-Seq. The information provided by these expriments will allow us to determine whether non-coding RNAs utilize a similar pathway/mechanism to mediate gene silencing. Interestingly, the levels of macroH2A are elevated on the Xi in the S phase of the cell cycle. The Polycomb Repressive Complex, PRC2, is also thought to mediate Histone H3 Lysine 27 (H3K27) methylation at the same cell cyle phase. Based on this, my third objective is to study whether the presence of the histone variant macroH2A instead of the canonical histone H2A contributes to increased PRC2 dependent H3K27 methylation of chromatin. This will be assayed both by in vitro histone methyltransferase assays as well as studying the in vivo levels of H3K27 methylation on the Xi upon macroH2A knock down or deletion. These experiments will facilitate the understanding of how HKMT complexes target specific regions ofthe genome more efficiently than others. Given the compact nature of the Xi and the association of macroH2A exclusively with the inactive X chromosome, I would also like to analyze the contribution ofthis histone variant to chromatin compaction. I will analyse this both by in vitro chromain reconstitution experiments as well as knock down analyses of macroH2A. Finally, I plan to identify specific interaction partners of macroH2Ain male and female cell lines. Since macroH2A is enriched on the Xi in females, this experiement will allow us to identify additional factors that might localize to the Xi in a macroH2A dependent manner. The presence of variant histones is a means of propagating epigenetic information from one cell generation to the next. The mechanism of action ofthe repressive variant histone macroH2A is not well understood. Identification of its interacting partners and its impact on chromatin structure will provide additional insights into the maintenace of cellular memory.

描述（由申请人提供）：我对该奖学金的广泛目的是研究ISTONE变体MacroH2a在非编码RNA介导的基因沉默中的作用。 MacroH2a在哺乳动物中富集了无活性X染色体（XI），该染色体被XIST非编码RNA覆盖。首先，我将分析MacroH2a是否在受其他非编码RNA调控的基因组区域富集。我将使用CHLP-Seq识别这些域。其次，我将分析MacRoH2a是否可以直接结合Xist RNA，因为该变体与XI的定位取决于Xist RNA的存在。我还将尝试识别使用RNA CHLP-SEQ与该组蛋白变体结合的其他非编码RNA。这些征收提供的信息将使我们能够确定非编码RNA是否利用类似的途径/机制来介导基因沉默。有趣的是，在细胞周期的S相中，XI上的MacroH2a水平升高。 polycomb抑制性复合物PRC2也被认为可以介导同一细胞相的组蛋白H3赖氨酸27（H3K27）甲基化。基于此，我的第三个目标是研究组蛋白变体麦克罗2a而不是规范组蛋白H2a是否有助于染色质的PRC2依赖性H3K27甲基化增加。这将通过体外组蛋白甲基转移酶测定以及研究麦克罗2A敲击或缺失时在XI上的H3K27甲基化水平来测定这一点。这些实验将促进对HKMT复合物如何比其他建筑更有效地针对基因组的特定区域的理解。鉴于XI的紧凑性和MacroH2a与非活性X染色体的关联，我还想分析这种组蛋白变体对染色质压实的贡献。我将通过体外镀铬重建实验以及对MacRoH2A进行分析来分析这一点。最后，我计划确定MacRoH2Ain男性和女性细胞系的特定相互作用伙伴。由于MacroH2a在女性的XI上富含，因此这一经验将使我们能够以依赖于MacRoH2A的方式确定可能将其定位到XI的其他因素。变体组蛋白的存在是从一个细胞产生到下一个细胞的表观遗传信息的一种手段。抑制性变体组蛋白MacroH2a的作用机理尚不清楚。识别其相互作用的伴侣及其对染色质结构的影响将为细胞记忆的维护方面提供更多见解。