采用CRISPR/Cas9 介导的表观遗传改造策略研究精子中组蛋白甲基化对表观遗传跨代遗传的影响

Postfertilization, the epigenomes of mammalian sperm and oocytes are reprogrammed during early embryogenesis. This process make parental epigenetic signatures acquired by environment factors hard be transmitted to next generation. However, a growing body of evidence indicate epigenetic modification can be transgenerational inheritance. It suggests that in some genomic loci epigenetic modification can resist to epigenetic reprogramming during early embryonic development. There are about 1% histones retained in the mouse sperm chromatin, one kind of histone modification H3K27me3 is reported enriched in gene regulating regions. H3K27me3 perhaps is the most promising epigenetic factor for paternal epigenetic inheritance was revealed upon the discovery that paternal diet could affect H3K27me3 status in sperm. In order to verify whether the histone H3K27me3 in the sperm can mediate epigenetic transgenetational inheritance, we first analysis and compare the distribution of H3K27me3 in spermatogonial stem cells, sperm and the male pronucleus of zygotes. We try to identify potential H3K27me3 reprogramming resistant sites in the genome. Further, we use the CRISPR/Cas9 system to targeting erase H3K27me3 at these potential sites specifically in the male germ cells, and then we analyze the enrichment of H3K27me3 on the male pronucleus of fertilized eggs at these sites and expression level of downstream gene. By these study, we can confirm whether histone H3K27me3 in the sperm can mediate transgenerational inheritance. Our study is critical for revealing the mechanism of epigenetic transgeneraional inheritance.

哺乳动物受精后，雌雄配子所携带的表观遗传组要发生全基因组的重编程，这使得亲本由于受环境因素造成的表观遗传改变很难传递给子代。然而越来越多的证据表明，表观遗传可以跨代遗传，这提示基因组上某些位点表观遗传的改变，能够抵抗早期胚胎的重编程。在小鼠的精子中大约有1%的组蛋白残留，其中带有H3K27me3修饰的组蛋白主要分布在基因调控区域，该修饰极可能跟父本表观遗传跨代遗传有关。为了验证精子中组蛋白H3K27me3 能否传递表观遗传信息，我们首先全基因组分析比较了H3K27me3 在精原干细胞、精子以及受精卵的雄原核上 的分布，试图找出潜在对重编程有抵抗作用的位点。然后我们利用CRISPR/Cas9 系统在雄性生殖细胞中，对这些潜在位点上的H3K27me3进行定向擦除。通过分析雄原核上该位点H3K27me3的富集以及对下游基因表达的影响，从而证实精子中的组蛋白H3K27的甲基化能否跨代遗传。