CHAF1b在牛早期克隆胚胎发育过程中的作用机制

During early development of cloned embryos, chromatin disaggregation was incomplete, embryonic genome activation was suppressed by persisting epigenetic modification (E.g H3K9me3) of donor cell genome, which impeded embryonic development following somatic cell nuclear transfer (SCNT). Our and other recent study revealed that the expression level of histone chaperone chromatin assembly factor 1 (CAF-1) subunit CHAF1b in bovine and human nuclear transfer embryos was significantly higher than in vitro fertilization embryos. It has been reported that histone chaperone CAF-1 involves in chromatin condensation and safeguards somatic cell identity, which is an important obstacle to somatic reprogramming. CAF-1 markedly suppressed iPS cell formation. We found that knockdown of CHAF1b could increased the abundance of histone H3.3, decreased the abundance of histone H3K9me3, and increased the development rate of bovine SCNT blastocysts. However, the molecular mechanism underlying the CHAF1b's effects during reprogramming of bovine SCNT embryo remain incompletely understood. The proposed studies will address the role of CHAF1b and the mechanism regulating in chromatin remodeling and epigenetic reprogramming, chromatin agglutination, replacement of histone variant H3.1-3.3, embryonic genome activation, and embryonic development of the bovine SCNT embryos. This proposed research will provide new insights into revealing the mechanisms of aberrant reprogramming during SCNT and the cause of low cloning efficiency.

克隆胚胎早期发育过程中，染色质去凝集不彻底，来自体细胞的表观遗传修饰H3K9me3等擦除不完全，抑制胚胎基因组激活，导致克隆胚胎发育失败。分析高通量数据发现组蛋白伴侣CAF-1的亚基CHAF1b在牛和人克隆胚胎上异常高表达。而在诱导多能性干细胞上的研究显示CAF-1调控染色质凝集并阻遏重编程。我们前期研究发现敲降CHAF1b可提高组蛋白H3.3含量，降低染色质凝集状态的标志修饰H3K9me3的丰度，并提高牛克隆胚胎发育率，但其作用机制尚不清楚。本项目将通过体细胞核移植和显微注射等技术研究CHAF1b对牛克隆胚胎染色质组分、表观遗传修饰、染色质凝集状态、组蛋白H3.1-3.3置换、胚胎基因组激活和胚胎发育的影响，来探讨CHAF1b作为潜在的重编程抑制因子在牛克隆胚胎发育过程中的作用和机制。本项目的实施将为阐明体细胞克隆胚胎异常重编程的形成机制以及提高家畜克隆效率提供依据。

克隆胚胎早期发育过程中，染色质去凝集不彻底，来自体细胞的表观遗传修饰H3K9me3等擦除不完全，抑制胚胎基因组激活，导致克隆胚胎发育失败。本项目主要研究CHAF1b作为潜在的重编程抑制因子在牛克隆胚胎发育过程中的作用和机制，获得以下结果：1.通过单细胞RNA-seq测序分析牛附植前多个时期的体细胞克隆胚胎与体外受精胚胎的差异表达基因，结果发现牛体细胞克隆胚胎在合子激活阶段存在基因表达调控和代谢等方面存在异常。2.分析高通量数据发现组蛋白伴侣CAF-1的亚基CHAF1b在牛和人克隆胚胎上异常高表达。研究发现敲降CHAF1b可上调克隆胚胎组蛋白变体H3.3的含量，降低组蛋白H3K9me3丰度，并显著提高克隆胚胎的桑椹胚和囊胚发育率。3.进一步在模式动物上通过干扰片段显微注射实验，研究敲低CHAF1b对植入前胚胎发育的影响，结果显示敲低CHAF1b并不影响囊胚发育率，但导致囊胚孵出率下降，体外生长失败以及体内胚胎移植后的出生率下降。本研究发现敲降CHAF1b导致囊胚期胚胎质量下降，表现为囊胚细胞总数、内细胞团细胞数、滋养层细胞数目减少，但囊胚细胞凋亡数和DNA损伤增加。敲降CHAF1b还导致谱系分化的关键调控因子（包括Oct4，Cdx2，Sox2和Nanog）的表达下调。进一步的分析表明，CHAF1b介导了组蛋白H3.1/3.2替代H3.3，这与抑制性组蛋白标记（H3K9me2/3和H3K27me2/3）减少和激活性组蛋白标记（H3K4me2/3）增加有关。此外，RNA测序分析显示敲降CHAF1b导致1508个基因的差异表达，包括表观遗传修饰，多个谱系分化和凋亡相关的基因。ATAC-seq测序分析的结果表明，沉默CHAF1b会改变谱系分化基因和凋亡相关基因的染色质可及性。本项目的研究将为阐明体细胞克隆胚胎异常重编程的形成机制以及提高家畜克隆效率提供重要科学依据。