Investigation of genomic imprinting in cattle

Genomic imprinting is an epigenetic mechanism whereby a subset of genes, called imprinted genes, show parent-of-origin-dependent monoallelic expression. Most imprinted genes are regulated by allele-specific DNA methylation at differentially methylated regions (DMRs). Although major imprinted genes and DMRs have already been identified in human and mouse, information on genomic imprinting is sporadic and limited in other mammalian species due to the cost and technical difficulties of genome-wide allele-specific gene expression and DNA methylation analyses. Here we propose two strategies to predict DMRs from whole-genome bisulfite sequencing (WGBS) data: one is based on conservation and the other is on germline DNA methylation patterns. Both strategies do not require genotype information and predict DMRs with high sensitivity and low background. We applied these strategies to human, rhesus, mouse and cow and estimated the evolutionary trajectory of individual DMRs. These analyses suggest that various factors, including CpG density, germline methylation and sequence-specific DNA binding proteins, may contribute to species-specific differences in genomic imprinting. Our strategies will facilitate cross-species comparison of genomic imprinting and identification of novel DMRs. Genomic DNA was purified with phenol/chloroform extraction and ethanol precipitation. WGBS libraries were constructed using the post-bisulfite adapter-tagging (PBAT) method. The PBAT libraries were sequenced on the HiSeq 2500 platform (Illumina, CA, USA) with 101-bp single-end reads.

基因组印记（Genomic imprinting）是一种表观遗传机制，指一类被称为印记基因（imprinted genes）的特定基因，表现出亲本来源依赖性的单等位基因表达。多数印记基因受差异甲基化区域（differentially methylated regions, DMRs）处的等位基因特异性DNA甲基化调控。尽管人类与小鼠中已鉴定出主要的印记基因及DMRs，但由于全基因组等位基因特异性基因表达与DNA甲基化分析存在成本与技术难点，其他哺乳动物物种中关于基因组印记的信息仍零散且有限。本文提出两种从全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing, WGBS）数据中预测DMRs的策略：其一基于序列保守性，其二基于生殖系DNA甲基化模式。两种策略均无需基因型信息，且可高灵敏度、低背景地预测DMRs。我们将上述策略应用于人类、恒河猴、小鼠与牛，并对单个DMR的进化轨迹进行了评估。这些分析表明，CpG密度、生殖系甲基化以及序列特异性DNA结合蛋白等多种因素，或可导致不同物种间基因组印记的物种特异性差异。本研究策略将助力跨物种基因组印记比较研究与新型DMRs的鉴定。实验中采用酚氯仿抽提与乙醇沉淀法纯化基因组DNA。使用亚硫酸氢盐后接头标记法（post-bisulfite adapter-tagging, PBAT）构建WGBS文库，并在HiSeq 2500平台（Illumina，美国加利福尼亚州）上以101 bp单端读取模式进行测序。