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Proc Natl Acad Sci U S A. 2015 Aug 4;112(31):E4216-25. doi: 10.1073/pnas.1503690112. Epub 2015 Jul 20.

The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells.

Author information

1
Howard Hughes Medical Institute; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; Department of Genetics, Harvard Medical School, Boston, MA 02115;
2
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.
3
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.

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.

KEYWORDS:

Polycomb; X inactivation; Xist RNA; chromosome; super resolution microscopy

PMID:
26195790
PMCID:
PMC4534268
DOI:
10.1073/pnas.1503690112
[Indexed for MEDLINE]
Free PMC Article

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