Zhang Kun, Wang Han, Rajput Sandeep K, Folger Joseph K, Smith George W
Laboratory of Mammalian Molecular Embryology, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
Institute of Dairy Sciences, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
Mol Reprod Dev. 2018 Feb;85(2):106-116. doi: 10.1002/mrd.22939. Epub 2018 Jan 9.
Histone variant H3.3 is encoded by two distinct genes, H3F3A and H3F3B, that are closely associated with actively transcribed genes. H3.3 replacement is continuous and essential for maintaining correct chromatin structure during mouse oogenesis. Upon fertilization, H3.3 is incorporated to parental chromatin, and is required for blastocyst formation in mice. The H3.3 exchange process is facilitated by the chaperone HIRA, particularly during zygote development. We previously demonstrated that H3.3 is required for bovine early embryonic development; here, we explored the mechanisms of its functional requirement. H3F3A mRNA abundance is stable whereas H3F3B and HIRA mRNA are relatively dynamic during early embryonic development. H3F3B mRNA quantity is also considerably higher than H3F3A. Immunofluorescence analysis revealed an even distribution of H3.3 between paternal and maternal pronuclei in zygotes, and subsequent stage-specific localization of H3.3 in early bovine embryos. Knockdown of H3.3 by targeting both H3F3A and H3F3B dramatically decreased the expression of NANOG (a pluripotency marker) and CTGF (Connective tissue growth factor; a trophectoderm marker) in bovine blastocysts. Additionally, we noted that Histone H3 lysine 36 dimethylation and linker Histone H1 abundance is reduced in H3.3-deficient embryos, which was similar to effects following knockdown of CHD1 (Chromodomain helicase DNA-binding protein 1). By contrast, no difference was observed in the abundance of Histone H3 lysine 4 trimethylation, Histone H3 lysine 9 dimethylation, or Splicing factor 3 B1. Collectively, these results established that H3.3 is required for correct epigenetic modifications and H1 deposition, dysregulation of which likely mediate the poor development in H3.3-deficient embryos.
组蛋白变体H3.3由两个不同的基因H3F3A和H3F3B编码，这两个基因与活跃转录的基因密切相关。在小鼠卵子发生过程中，H3.3的替换是持续的，并且对于维持正确的染色质结构至关重要。受精后，H3.3被整合到亲代染色质中，并且是小鼠囊胚形成所必需的。伴侣蛋白HIRA促进了H3.3的交换过程，特别是在合子发育期间。我们之前证明了H3.3是牛早期胚胎发育所必需的；在此，我们探究了其功能需求的机制。在早期胚胎发育过程中，H3F3A mRNA丰度稳定，而H3F3B和HIRA mRNA相对动态变化。H3F3B mRNA的量也显著高于H3F3A。免疫荧光分析显示，合子中父源和母源原核之间H3.3分布均匀，并且在早期牛胚胎中H3.3随后呈现阶段特异性定位。通过靶向H3F3A和H3F3B敲低H3.3显著降低了牛囊胚中NANOG（一种多能性标记物）和CTGF（结缔组织生长因子；一种滋养外胚层标记物）的表达。此外，我们注意到在H3.3缺陷胚胎中，组蛋白H3赖氨酸36二甲基化和连接组蛋白H1丰度降低，这与敲低CHD1（染色质结构域解旋酶DNA结合蛋白1）后的效果相似。相比之下，组蛋白H3赖氨酸4三甲基化、组蛋白H3赖氨酸9二甲基化或剪接因子3B1的丰度没有差异。总体而言，这些结果表明H3.3是正确的表观遗传修饰和H1沉积所必需的，其失调可能介导了H3.3缺陷胚胎发育不良。
