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：
8044167
负责人：
Kavitha Sarma
金额：
$ 5.13万
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/8044167
关键词：
Affect Alleles Amino Acids Architecture Base Sequence Binding Biological Assay C-terminal 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 MicroRNAs Modification Mus N-terminal Nature Nucleosomes Pathway interactions Phase Polycomb Proteins RNA RNA Binding Recruitment Activity Research Proposals Role S Phase 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）：我获得此奖学金的主要目标是研究 istone 变体 MacroH2A 在非编码 RNA 介导的基因沉默中的作用。 MacroH2A 富集在哺乳动物的失活 X 染色体 (Xi) 上，该染色体被 Xist 非编码 RNA 包被。首先，我将分析 MacroH2A 是否在基因组中受其他非编码 RNA 调控的区域富集。我将使用 ChlP-Seq 来识别这些域。其次，我将分析 MacroH2A 是否可以直接结合 Xist RNA，因为该变体对 Xi 的定位取决于 Xist RNA 的存在。我还将尝试使用 RNA ChlP-Seq 来鉴定与该组蛋白变体结合的其他非编码 RNA。这些实验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 期 Xi 上，macroH2A 的水平升高。 Polycomb 抑制复合物 PRC2 也被认为在同一细胞周期阶段介导组蛋白 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 MacroH2A（而不是经典组蛋白 H2A）的存在是否有助于增加 PRC2 依赖性染色质 H3K27 甲基化。这将通过体外组蛋白甲基转移酶测定以及研究 MacroH2A 敲除或缺失后 Xi 上 H3K27 甲基化的体内水平进行测定。这些实验将有助于理解 HKMT 复合物如何比其他复合物更有效地靶向基因组的特定区域。鉴于 Xi 的紧凑性质以及 MacroH2A 与失活 X 染色体的专门关联，我还想分析该组蛋白变体对染色质压缩的贡献。我将通过体外染色质重建实验以及 MacroH2A 的敲低分析来分析这一点。最后，我计划确定雄性和雌性细胞系中 MacroH2A 的特定相互作用伙伴。由于 MacroH2A 在女性的 Xi 上富集，因此该实验将使我们能够识别可能以 MacroH2A 依赖性方式定位于 Xi 的其他因素。变异组蛋白的存在是将表观遗传信息从一个细胞世代传播到下一代细胞的一种手段。抑制性变体组蛋白 MacroH2A 的作用机制尚不清楚。识别其相互作用伙伴及其对染色质结构的影响将为细胞记忆的维持提供更多见解。