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

描述(由申请者提供)：我这项研究的广泛目标是研究iStone变体MacroH2A在非编码RNA介导的基因沉默中的作用。在哺乳动物的非活性X染色体(XI)上富含MacroH2A，该染色体被Xist非编码RNA包被。首先，我将分析是否在基因组中受其他非编码RNA调控的区域富含宏H_2A。我将使用ChlP-Seq来标识这些域。其次，我将分析宏H_2A是否能直接与Xist RNA结合，因为这个变体对XI的定位取决于Xist RNA的存在。我还将尝试使用RNA ChlP-Seq来识别与这个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，宏H_2A的水平在XI上是升高的。多梳抑制复合体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异体宏H_2A而不是典型的组蛋白H_2A的存在是否有助于增加依赖于PRC2的染色质H3K27甲基化。这将通过体外组蛋白甲基转移酶试验以及研究H3K27甲基化水平在大分子H2A被敲除或缺失时在XI上的水平来进行检测。这些实验将有助于理解HKMT复合体如何比其他区域更有效地靶向基因组的特定区域。鉴于XI的紧凑性质以及宏H_2A仅与不活跃的X染色体相关联，我还想分析这个组蛋白变体对染色质紧凑的贡献。我将通过体外色胺重组实验以及大分子H_2A的击倒分析来分析这一点。最后，我计划在雄性和雌性细胞系中确定大分子H_2A的特定相互作用伙伴。由于大分子H_2A在雌性体内富含XI，这一经验将使我们能够识别可能以大分子H_2A依赖的方式定位于X_1的额外因素。 变异组蛋白的存在是一种将表观遗传信息从一个细胞世代传播到下一代细胞的方法。抑制变异型组蛋白大分子H_2A的作用机制尚不清楚。识别其相互作用的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的见解。

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.