全基因组DNA去甲基化和甲基化重编程在哺乳动物早期胚胎发育过程中的机制及作用

DNA methylation is a fundamentally epigenetic modification which is important for both fertility and viability of offspring. The current model postulates that following fertilization, methylation is comprehensively reprogrammed （except for imprinted genes）, while the maternal genome is passively demethylated between the zygote and pre-implantation embryos, and the paternal genome is actively demethylated in the zygote, which is followed by the establishment of new methylation landscapes. Recently, 5-hydroxymethylcytosine （5hmC） has been shown as a stable epigenetic mark which could be converted from 5-methylcytosine （5mC） by TET family enzymes（Tet1,Tet2, Tet3）. Further studies indicated that 5hmC may involve in regulating embryogenesis after fertilization, loss of DNA methylation in the male pronucleus coincides with oxidation of 5mC to 5hmC and loss of Tet3 in mice germ cells leads to defects in embryogenesis. However,the biological significance and mechanisms of this epigenetic mark during embryogenesis have remained unclear. An understanding of the roles of 5hmC in embryonic development will require analysis of the genome-wide localization of 5mC and 5hmC in gametes and early embryos. Herein, we will use mice as model organisms and cross two distantly related strains. Taking advantage of single nucleotide polymorphisms （SNPs） present in these two strains, we were able to identify allelic specific 5mC and 5hmC modifications in the hybrid embryos. Therefore, we will generate genome-wide,base-resolution, allele specific 5mC and 5hmC map in the sperm, unfertilized eggs, 2 cell embryos and ICM（inner cell mass） by 5-hmC DNA sequencing and MethylC-Seq, respectively. Our results should help with understanding the epigenetic differences between two parental genomes after fertilization and the molecular mechanism of 5hmC in regulating parental specific 5mC reprogramming. Moreover, we will integrate with corresponding small RNA data which will further elucidate the biological significance of 5mC in small RNA regulation, such as piRNA. Taken together, we will get insight into how 5mC and 5hmC are dynamically regulated during embryogenesis, and which will broaden our understanding of epigenetic reprogramming and have great implications in somatic cell reprogramming and regenerative medicine.

DNA甲基化5mC重编程对早期发育至关重要，目前认为哺乳动物受精后发生全基因组水平去甲基化。因为没有单碱基分辨率、高覆盖度全基因组5mC图谱支持，至今对全基因组DNA去甲基化的机制仍没定论。本课题以小鼠为模型，利用129品系和PWK品系SNP差异，获得能区分父源和母源序列的全基因组、单碱基分辨率的生殖细胞和早期胚胎DNA甲基化图谱和羟甲基（5hmC）图谱，分别揭示父源和母源早期胚胎发育DNA去甲基化和随后de novo加甲基化以及5hmC动态变化规律。通过整合分析5mC图谱、5hmC图谱和small RNA图谱，揭示5hmC和small RNA对甲基化重编程过程中作用机制。并深入分析基因组选择性去甲基和加甲基化的序列保守性规律，探索甲基化重编程是否有可参照的模板。最后比较不同发育时期5mC图谱，揭示DNA甲基化在早期发育中的调控作用。本课题将有助于揭示表观信息如何从父母遗传到后代的规律。

DNA甲基化的调控在发育中起着非常重要的作用。哺乳动物的早期胚胎发育中发生大规模DNA去甲基化。DNA去甲基化可以通过主动和被动方式进行。根据免疫荧光染色和RRBS测序得到的结果，之前的假说认为受精后的父源DNA甲基化修饰被主动氧化后，依赖其氧化产物的被动稀释进行去甲基化，而母源DNA不被氧化，只依赖DNA复制及甲基化维持机制缺失而被动去甲基化。但是，由于实验精度有限，并且无法区分父源和母源DNA，所以这些结果可能并不能全面而精确地反映出早期胚胎发育中DNA去甲基化是如何进行的。.通过对小鼠配子及杂交胚胎的全基因组甲基化测序，我们得到能够区分DNA亲本来源的单碱基分辨率的甲基化图谱、二细胞时期的5hmC、5fC图谱，以及E13.5时期的原始生殖细胞的甲基化图谱。分析发现在二细胞时期5hmC和5fC存在于母源和父源DNA，而且从配子到四细胞时期，在大多数去甲基化CpG位点，5mC及其氧化产物被转化为无修饰的胞嘧啶，且不依赖被动稀释。因此，在小鼠早期胚胎发育过程中，父源DNA以及绝大多数的母源DNA是通过主动的方式实现地去甲基化。同时，所有已知的印记调控区域被归类为生殖细胞印记调控区或体细胞印记调控区。.除此之外，利用本课题资金的支持，我们还研究了DNA甲基化如何在斑马鱼中从亲代传递到子代的过程。我们的工作揭示子代DNA可以完全继承父代精子的甲基化图谱，而来自母源卵子的DNA抛弃卵子的甲基化图谱，完全重编程为精子甲基化图谱。子代继承精子的甲基化图谱，可以帮助指导胚胎的早期发育。.相关的研究结果分别于2013年和2014年发表在Cell杂志上，并被Cell、Nature Genetics Review、Cell Stem Cell、Faculty 1000等广泛评叙。