Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Obstetrics and Gynecology and Center for Reproductive Sciences, University of California San Francisco, 35 Medical Center Way, San Francisco, CA 94143, USA.
Development. 2013 Sep;140(17):3624-34. doi: 10.1242/dev.095513. Epub 2013 Jul 31.
Histone variants can replace canonical histones in the nucleosome and modify chromatin structure and gene expression. The histone variant H3.3 preferentially associates with active chromatin and has been implicated in the regulation of a diverse range of developmental processes. However, the mechanisms by which H3.3 may regulate gene activity are unclear and gene duplication has hampered an analysis of H3.3 function in mouse. Here, we report that the specific knockdown of H3.3 in fertilized mouse zygotes leads to developmental arrest at the morula stage. This phenotype can be rescued by exogenous H3.3 but not by canonical H3.1 mRNA. Loss of H3.3 leads to over-condensation and mis-segregation of chromosomes as early as the two-cell stage, with corresponding high levels of aneuploidy, but does not appear to affect zygotic gene activation at the two-cell stage or lineage gene transcription at the morula stage. H3.3-deficient embryos have significantly reduced levels of markers of open chromatin, such as H3K36me2 and H4K16Ac. Importantly, a mutation in H3.3K36 that disrupts H3K36 methylation (H3.3K36R) does not rescue the H3.3 knockdown (KD) phenotype. In addition, H3.3 KD embryos have increased incorporation of linker H1. Knockdown of Mof (Kat8), an acetyltransferase specific for H4K16, similarly leads to excessive H1 incorporation. Remarkably, pan-H1 RNA interference (RNAi) partially rescues the chromosome condensation of H3.3 KD embryos and allows development to the blastocyst stage. These results reveal that H3.3 mediates a balance between open and condensed chromatin that is crucial for the fidelity of chromosome segregation during early mouse development.
组蛋白变体可以取代核小体中的经典组蛋白，并修饰染色质结构和基因表达。H3.3 组蛋白变体优先与活性染色质结合，并参与调节多种发育过程。然而，H3.3 调节基因活性的机制尚不清楚，而且基因复制阻碍了对 H3.3 在小鼠中的功能分析。在这里，我们报告说，在受精的小鼠胚胎中特异性敲低 H3.3 会导致胚胎停滞在桑葚胚阶段。这种表型可以通过外源性 H3.3 挽救，但不能通过经典的 H3.1 mRNA 挽救。H3.3 的缺失导致染色体过早地过度凝聚和错误分离，早在二细胞阶段就出现高比例的非整倍体，但似乎不会影响二细胞阶段的合子基因激活或桑葚胚阶段的谱系基因转录。H3.3 缺陷型胚胎的开放染色质标志物水平显著降低，如 H3K36me2 和 H4K16Ac。重要的是，破坏 H3K36 甲基化的 H3.3K36 突变（H3.3K36R）不能挽救 H3.3 敲低（KD）表型。此外，H3.3 KD 胚胎中连接蛋白 H1 的掺入量增加。乙酰转移酶特异性针对 H4K16 的 Mof（Kat8）的敲低也导致 H1 过度掺入。值得注意的是，泛 H1 RNA 干扰（RNAi）部分挽救了 H3.3 KD 胚胎的染色体凝聚，并允许其发育到囊胚阶段。这些结果表明，H3.3 介导了开放和凝聚染色质之间的平衡，这对于早期小鼠发育过程中染色体分离的保真度至关重要。
