Histone H3.3 maintains genome integrity during mammalian development

Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans, and has been implicated in many important biological processes including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components, or in H3.3 encoding genes, have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3 null mouse models through classical genetic approaches. We found H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres and pericentromeric regions of chromosomes leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development. RNA-seq in embryos at E10.5 comparing 3 samples with the following genotype Trp53-/-; H3f3afl/-; H3f3bfl/-; Sox2-CreTg/0 to three samples with the following genotype Trp53-/-; H3f3afl/+; H3f3bfl/+; Sox2-CreTg/0组蛋白H3.3（Histone H3.3）是后生动物（metazoans）中高度保守的组蛋白H3替换变体，已被证实参与诸多关键生物学过程，包括细胞分化与细胞重编程。针对H3.3基因组整合通路组分或其编码基因的生殖系突变与体细胞突变，分别与人类先天性疾病和癌症存在关联。然而，H3.3在哺乳动物发育中的具体功能仍未明确。为解答这一科学问题，我们通过经典遗传学手段构建了H3.3完全敲除小鼠模型。研究发现，H3.3在小鼠发育过程中发挥不可或缺的核心作用：H3.3的完全缺失会导致发育迟缓与早期胚胎致死。在细胞层面，H3.3缺失会引发细胞周期阻滞与细胞死亡。出人意料的是，H3.3缺失并未显著干扰发育胚胎中的基因表达调控。与之相反，H3.3缺失会导致染色体端粒（telomeres）、着丝粒（centromeres）及着丝粒周边区域的异染色质（heterochromatin）结构功能异常，进而引发有丝分裂缺陷。由此产生的核型异常与DNA损伤会激活p53通路（p53 pathway）。综上，本研究结果揭示，H3.3的一项重要功能是维持染色体异染色质结构，从而在哺乳动物发育过程中维护基因组完整性。本研究对胚胎发育至E10.5阶段的样本开展RNA测序（RNA-seq），比较两组各3例样本：一组基因型为Trp53-/-; H3f3afl/-; H3f3bfl/-; Sox2-CreTg/0，另一组基因型为Trp53-/-; H3f3afl/+; H3f3bfl/+; Sox2-CreTg/0