Site-Specific Silencing of Regulatory Elements as a Mechanism of X Inactivation

Abstract

The inactive X chromosome’s (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequence is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to non-transcribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X-inactivation occurred within, and X-inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X-inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi.

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

“…Moreover, although biochemical analysis indicates that Xist and PRC2 directly interact (7,29,30) and it is known that they nucleate together at the Xic (8), whether Xist and PRC2 stay together as they spread along the Xi has not been answered definitively by epigenomic analyses (10,11,15). Additionally, a recent analysis using 3D-SIM with 100-nm resolution suggested that Xist RNA and PRC2 are spatially separated on the Xi, to a degree that called into question the idea that Xist recruits PRC2 to the Xi (20).…”

“…The unexpectedly low number of Xist transcripts led us to question how Xist can target PRC2 so broadly to the Xi, resulting in the chromosome-wide enrichment of H3K27me3 as demonstrated by ChIP-seq analysis (11,15) and by conventional microscopic techniques (7,9,12,13,17,28). Moreover, although biochemical analysis indicates that Xist and PRC2 directly interact (7,29,30) and it is known that they nucleate together at the Xic (8), whether Xist and PRC2 stay together as they spread along the Xi has not been answered definitively by epigenomic analyses (10,11,15).…”

“…Understanding how silencing factors spread in cis has been advanced by employment of next-generation sequencing techniques. For example, CHART-seq (capture hybridization analysis of RNA targets with deep sequencing) and ChIP-seq analyses have provided a first molecular examination of Xist binding patterns (10,14) and localization dynamics of various chromatin epitopes (11,15) at a population-wide level. Developmental profiling demonstrates that Xist RNA and PRC2 initially localize to broad gene-rich, multimegabase clusters and later spread into intervening gene-poor regions.…”

Nuclear receptors and chromatin: an inducible couple

“…Recent work pioneered by studies on X-chromosome inactivation has highlighted a central role for long non-coding RNAs (lncRNAs) in epigenetic silencing and enhancer activation (Calabrese et al 2012). Enhancer RNAs (eRNAs) are a class of lncRNAs with a potential role in the mediation of epigenetic modifications as well as interactions with TFs (Gupta et al 2010, Kogo et al 2011, Calabrese et al 2012.…”

The great escape

“…47 A recent study suggests that the number of mouse escape genes can be higher than previously thought, depending on the cell type. 48 Thus, in females, the number of escape genes (i.e., degree of escape) is variable in each tissue.…”