Ring1b-mediated H2A ubiquitination associates with inactive X chromosomes and is involved in initiation of X inactivation.

Histone modifications are thought to serve as epigenetic markers that mediate dynamic changes in chromatin structure and regulation of gene expression. As a model system for understanding epigenetic silencing, X chromosome inactivation has been previously linked to a number of histone modifications including methylation and hypoacetylation. In this study, we provide evidence that supports H2A ubiquitination as a novel epigenetic marker for the inactive X chromosome (Xi) and links H2A ubiquitination to initiation of X inactivation. We found that the H2A-K119 ubiquitin E3 ligase Ring1b, a Polycomb group protein, is enriched on Xi in female trophoblast stem (TS) cells as well as differentiating embryonic stem (ES) cells. Consistent with Ring1b mediating H2A ubiquitination, ubiquitinated H2A (ubH2A) is also enriched on the Xi of both TS and ES cells. We demonstrate that the enrichment of Ring1b and ubH2A on Xi is transient during TS and ES cell differentiation, suggesting that the Ring1b and ubH2A are involved in the initiation of both imprinted and random X inactivation. Furthermore, we showed that the association of Ring1b and ubH2A with Xi is mitotically stable in non-differentiated TS cells.