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

描述（由申请人提供）：我参加这项研究的主要目的是研究istone变体MacroH 2A在非编码RNA介导的基因沉默中的作用。MacroH 2A富集在哺乳动物的失活X染色体（Xi）上，其被Xist非编码RNA包被。首先，我将分析macroH 2A是否在基因组中由其他非编码RNA调控的区域富集。我将使用ChIP-Seq来鉴定这些域。其次，我将分析macroH 2A是否可以直接结合Xist RNA，因为这种变体在Xi上的定位依赖于Xist RNA的存在。我还将尝试使用RNA ChIP-Seq鉴定与这种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，macroH 2A的水平在细胞周期的S期的Xi上升高。多梳抑制复合物PRC 2也被认为在相同的细胞周期阶段介导组蛋白H3赖氨酸27（H3 K27）甲基化。基于此，我的第三个目标是研究组蛋白变体macroH 2A而不是典型组蛋白H2 A的存在是否有助于增加PRC 2依赖的H3 K27染色质甲基化。这将通过体外组蛋白甲基转移酶测定以及研究macroH 2A敲低或缺失后Xi上H3 K27甲基化的体内水平来测定。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。考虑到Xi的紧密性和macroH 2A只与失活的X染色体相关，我也想分析这个组蛋白变体对染色质致密化的贡献。我将通过体外染色质重建实验以及macroH 2A的敲除分析来分析这一点。最后，我计划在雄性和雌性细胞系中鉴定macroH 2A的特定相互作用伙伴。由于macroH 2A在女性的Xi上富集，该实验将使我们能够鉴定可能以macroH 2A依赖性方式定位于Xi的其他因子。 变异组蛋白的存在是将表观遗传信息从一代细胞传播到下一代细胞的一种手段。抑制性变体组蛋白macroH 2A的作用机制还不清楚。识别其相互作用的伙伴和它对染色质结构的影响将提供更多的见解维持细胞记忆。

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.