Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 112, Issue 31, Pages E4216-E4225Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1503690112
Keywords
super resolution microscopy; Xist RNA; Polycomb; X inactivation; chromosome
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Funding
- MGH Fund for Medical Discovery
- National Science Foundation
- NIH [RO1-GM090278]
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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 similar to 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 similar to 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.
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