Contains source data for graphs in Fig 6.

The HIRA complex mediates deposition of histone H3.3 independent of replication. Its functions in gene regulation in mice remain to be fully understood. Here we analyze mutations of the HIRA complex genes Ubn1 and Ubn2. We observe that Ubn1 mutant mice of both sexes are viable and fertile. In contrast, mutation of Ubn2 causes embryonic lethality with variable penetrance and skewed sex ratio in favor of males. Combined Ubn1 and Ubn2 mutations cause embryonic lethality with complete penetrance, variable developmental arrest before turning, and reduced recovery of female embryos. Consistent with a female specific function of the HIRA complex, reanalysis of the Hira mutation during embryogenesis reveals that previously observed severe and mild phenotypic classes correspond to female and male sex. Mechanistically, we show that mutations of Ubn1, Ubn2, and Hira in mouse embryonic stem cells affect the initiation of X inactivation. Xist mediated gene silencing is impaired to increasing extent by Ubn1, Ubn2, Hira, and combined Ubn1 and Ubn2 mutations. We identify a failure of establishing histone H3 tri-methyl lysine 27 over X-linked genes after induction of Xist expression as earliest molecular defect, whereas deacetylation of lysine 27 by Xist remains largely unaffected by the loss of Ubinucleins. Our study thereby identifies a switch from histone H3 acetyl to tri-methyl lysine 27 at the initiation of X inactivation that depends on HIRA complex function.