title: The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells.
abstract: X-chromosome inactivation (XCI) is initiated by the long noncoding RNA Xist, which coats the inactive X (Xi) and targets Polycomb repressive complex 2 (PRC2) in cis. Epigenomic analyses have provided significant insight into Xist binding patterns and chromatin organization of the Xi. However, such epigenomic analyses are limited by averaging of population-wide dynamics and do not inform behavior of single cells. Here we view Xist RNA and the Xi at 20-nm resolution using STochastic Optical Reconstruction Microscopy (STORM) in mouse cells. We observe dynamics at the single-cell level not predicted by epigenomic analysis. Only ∼50 hubs of Xist RNA occur on the Xi in the maintenance phase, corresponding to 50-100 Xist molecules per Xi and contrasting with the chromosome-wide "coat" observed by deep sequencing and conventional microscopy. Likewise, only ∼50 hubs PRC2 are observed. PRC2 and Xist foci are not randomly distributed but showed statistically significant spatial association. Knock-off experiments enable visualization of the dynamics of dissociation and relocalization onto the Xi and support a functional tethering of Xist and PRC2. Our analysis reveals that Xist-PRC2 complexes are less numerous than expected and suggests methylation of nucleosomes in a hit-and-run model.
authors.affiliation: Howard Hughes Medical Institute; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; Department of Genetics, Harvard Medical School, Boston, MA 02115;
authors.forename: Hongjae
authors.initials: H
authors.lastname: Sunwoo
authors.affiliation: Howard Hughes Medical Institute; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
authors.forename: John Y
authors.initials: JY
authors.lastname: Wu
authors.affiliation: Howard Hughes Medical Institute; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; Department of Genetics, Harvard Medical School, Boston, MA 02115; lee@molbio.mgh.harvard.edu.
authors.forename: Jeannie T
authors.initials: JT
authors.lastname: Lee
mesh_terms: D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology; D000071221:Enhancer of Zeste Homolog 2 Protein; D000939:Epitopes / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D017404:In Situ Hybridization, Fluorescence; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D051379:Mice; D063151:Polycomb Repressive Complex 2 / Q000737:chemistry / Q000378:metabolism*; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000378:metabolism*; D014960:X Chromosome / Q000378:metabolism; D049951:X Chromosome Inactivation
chemical_list: D000939:Epitopes; D006657:Histones; D062085:RNA, Long Noncoding; C494163:Suz12 protein, mouse; C077885:XIST non-coding RNA; D000071221:Enhancer of Zeste Homolog 2 Protein; C498410:Ezh2 protein, mouse; D063151:Polycomb Repressive Complex 2; D008239:Lysine
keywords: Polycomb; X inactivation; Xist RNA; chromosome; super resolution microscopy