Contains source data for graphs in Fig 4.

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.HIRA复合物（HIRA complex）可介导不依赖于DNA复制的组蛋白H3.3沉积。其在小鼠基因调控中的功能仍有待全面阐明。本研究针对HIRA复合物的编码基因Ubn1与Ubn2的突变体展开分析。实验结果显示，雌雄两性的Ubn1突变小鼠均能存活且可育。与之形成鲜明对比的是，Ubn2突变会引发胚胎致死，且外显率存在差异，同时子代性别比例偏向雄性。Ubn1与Ubn2的双突变则会导致完全外显的胚胎致死，表现为胚胎在扭体期前出现发育停滞且停滞程度不一，回收获得的雌性胚胎数量显著减少。与HIRA复合物具备雌性特异性功能的推论相符，对胚胎发生过程中Hira突变的重新分析表明，此前观测到的严重与轻微表型类别分别对应雌性与雄性个体。机制层面的实验证实，小鼠胚胎干细胞中Ubn1、Ubn2及Hira的突变会干扰X染色体失活的启动过程。Xist介导的基因沉默抑制效果随Ubn1、Ubn2、Hira单突变以及Ubn1与Ubn2双突变的顺序逐步增强。研究团队进一步发现，在诱导Xist表达后，X连锁基因区域的组蛋白H3赖氨酸27三甲基化建立失败，这是最早出现的分子缺陷；而Ubinuclein（Ubn）的缺失基本不会影响Xist介导的赖氨酸27去乙酰化过程。综上，本研究揭示了X染色体失活启动阶段，组蛋白H3赖氨酸27从乙酰化修饰向三甲基化修饰的转变依赖于HIRA复合物的正常功能。