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.
|Sarma, Kavitha; Cifuentes-Rojas, Catherine; Ergun, Ayla et al. (2014) ATRX directs binding of PRC2 to Xist RNA and Polycomb targets. Cell 159:869-83|
|Simon, Matthew D; Pinter, Stefan F; Fang, Rui et al. (2013) High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation. Nature 504:465-469|
|Jeon, Yesu; Sarma, Kavitha; Lee, Jeannie T (2012) New and Xisting regulatory mechanisms of X chromosome inactivation. Curr Opin Genet Dev 22:62-71|
|Sarma, Kavitha; Levasseur, Pierre; Aristarkhov, Alexander et al. (2010) Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome. Proc Natl Acad Sci U S A 107:22196-201|