BRCA1 associates with the inactive X chromosome in late S-phase, coupled with transient H2AX phosphorylation

The BRCA1 tumor suppressor gene encodes an E3-ubiquitin ligase that has been implicated in several distinct biochemical processes. As the cell cycle progresses, BRCA1 proteins interact transiently with nuclear foci containing DNA replication and DNA double-strand repair machinery. A hallmark of these foci is the presence of S139 phosphorylated histone H2AX. BRCA1 was recently shown to associate with facultative heterochromatin at the inactive X chromosome (Xi), where it may play a role in maintaining gene silencing. As the kinetics of this interaction has not been described, we sought to establish whether association of BRCA1 with the Xi also correlated with replication. Here we demonstrate that the interaction of BRCA1 and the Xi is transient, occurring during late S-phase. This interaction is concomitant with the presence of distinct foci of S139 phospho-H2AX and specifically corresponds with late replication of the Xi. BRCA1 and phospho-H2AX appear on the Xi immediately adjacent to CAF-1, a known marker of replication fork activity. Taken together, these data implicate BRCA1 and the H2AX kinase in replication of facultative heterochromatin on the Xi, most likely in a fashion similar to that performed at sites of DNA replication and double-strand break repair observed on somatic chromosomes.