体细胞克隆牛染色质开放区域异常及其表观遗传学研究

Developmental anomalies of cloned animals are caused by epigenetic modification anomalies. It however remains unclear how abnormal epigenetic modifications regulate and influence chromatin states in cloned animals. Our previous studies have shown that anomalies of gene expression promoting epigenetic modifications (H3K4me3, 5hmC)have greater influence on cloned cattle development than anomalies of expression inhibiting modifications (H3K9me2/3, H3K27me3, 5mC).This project intends to examine anomalies in accessible chromatin of somatic cell cloned cattle by using ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing). Cloned cattle individuals showing comparatively large differentials in chromatin accessibility are selected for transcriptome analysis and Chromatin immunoprecipitation analysis, so that genome specific diagnostic loci can be identified. Correlational studies shall be conducted on epigenetic modification anomalies and chromatin acceptability differentials in order to pinpoint the more vital epigenetic modifications. In conclusion, this project shall facilitate our understanding of the negative influences exerted on cloned animals by anomalies in accessible chromatin. This study may provide important clues to solving long-standing problems such as abnormal development of somatic cell cloned animals and low efficiency in animal cloning.

克隆动物发育异常是表观遗传修饰异常造成的，但是，异常的表观修饰如何调控和影响克隆动物染色质状态还不清楚。我们前期研究发现，与抑制基因表达的表观遗传修饰（H3K9me2/3 、H3K27me3和5mC）相比，促进基因表达的表观遗传修饰（H3K4me3、5hmC）异常对克隆牛发育影响更大。本项目拟采用ATAC-seq技术研究体细胞克隆牛染色质开放区域的异常和差异，选取染色质开放区域差异较大的克隆牛样品进行转录组以及染色质免疫共沉淀实验，分析、发现基因组特异的差异位点，开展多种表观遗传修饰异常和染色质开放区域差异相关性研究，比较哪些表观遗传修饰对染色质开放区域差异起重要作用。总之，本项目研究将帮助我们理解染色质开放区域表观修饰异常对克隆动物造成的不良影响，该项研究也将为彻底解决体细胞克隆动物发育异常、克隆效率低下等瓶颈问题提供新的科学理论依据。

背景：有研究表明体细胞核移植效率与供体细胞类型有关。先前的研究发现与胎儿成纤维细胞（Fetal fibroblasts，FFB）相比，胎儿输卵管上皮细胞（Fetal oviduct epithelial cell，FOV）具有更高的囊胚形成率，但是在植入后的妊娠率、产犊率和足月率等各项指标上却低于前者，造成这种差异的原因并不清楚。我们假设不同供体核体细胞染色质的疏松程度可能会对克隆动物胚胎及其个体发育造成影响。.结果：本研究利用ATAC-seq（Assay for Transposase-Accessible Chromatin with high throughput sequencing）、RNA-seq、全基因组5-羟甲基胞嘧啶(5-hydroxymethylcytosine，5hmC)和5-甲基胞嘧啶(5-methylcytosine，5mC)含量测定，多种方法分析分别以FFB与FOV为供体核的克隆牛之间的表观遗传差异，结果发现FOV供体核来源的克隆牛在染色质开放程度、基因表达水平以及5hmC含量等方面均高于FFB，同种供体核来源的克隆牛耳组织ATAC-seq和RNA-seq出现明显的聚类倾向。本研究还发现，FFB源的存活克隆牛5hmC含量均在4‰以上，死亡克隆牛5hmC含量均不超过2‰。.结论：与自然繁殖重编程相比，体细胞核移植重编程技术，其重编程是不完全的，体细胞克隆牛即使经过体细胞重编程和分化发育过程，它们仍然会保留其供体核的表观遗传和基因表达记忆，而且这些体细胞记忆可能会影响到克隆囊胚的发育率、植入后克隆胎儿的出生率和发育状况。我们认为不同供体核体细胞存活克隆牛的表观遗传修饰存在着差异，这种差异主要分布在染色质的基因间区。