Analysis of developmental imprinting dynamics in primates using SNP-free methods to identify imprinting defects in cloned placenta

Our knowledge of genomic imprinting in primates is lagging behand that of mice largely due to the difficulties of allelic analyses in outbred animals. To understand imprinting dynamics in primates, we profiled transcriptomes, DNA methylomes and H3K27me3 in uniparental monkey embryos. We further developed single-nucleotide polymorphisms (SNP)-free methods, TARSII and CARSII, to identify germline differentially methylated regions (DMRs) in somatic tissues. Our comprehensive analyses showed that allelic DNA methylation, but not H3K27me3, is a major mark that correlates with paternal-biasedly expressed genes (PEGs) in uniparental monkey embryos. Interestingly, primate germline DMRs are different from PEG-associated DMRs in early embryos and are enriched in placenta. Strikingly, most placenta-specific germline DMRs are lost in placenta of cloned monkey. Collectively, our study establishes SNP-free germline DMR identification methods, defines developmental imprinting dynamics in primates and demonstrates imprinting defects in cloned monkey placenta, which provides important clues for improving primate cloning. Overall design: We conducted RNA-seq, H3K27me3 CUT&RUN and WGBS in monkey uniparental 16-cell embryos. Also we have profiled WGBS in monkey adult tissues, WGBS and RNA-seq in monkey wild-type and SCNT placenta.

我们对灵长类动物基因组印记（genomic imprinting）的认知远滞后于小鼠，这主要源于远交动物的等位基因分析存在诸多难点。为阐明灵长类的印记调控动态，我们对单亲本猴胚胎的转录组、DNA甲基化组以及H3K27me3修饰进行了测序分析。我们进一步开发了无需单核苷酸多态性（SNP）的TARSII与CARSII方法，用于在体细胞组织中鉴定生殖系差异甲基化区域（DMRs）。我们的全面分析显示，等位基因DNA甲基化而非H3K27me3修饰，是单亲本猴胚胎中与父本偏好表达基因（PEGs）相关的主要表观遗传标记。值得注意的是，灵长类生殖系DMRs与早期胚胎中与PEG相关的DMRs存在显著差异，且在胎盘中富集。尤为惊人的是，绝大多数胎盘特异性生殖系DMRs在克隆猴胎盘中出现了丢失现象。综上，本研究建立了无需SNP的生殖系DMR鉴定方法，明确了灵长类发育过程中的印记调控动态，并证实了克隆猴胎盘中存在印记缺陷，这为优化灵长类克隆技术提供了重要线索。实验设计概述：我们对猴单亲本16细胞胚胎开展了RNA测序（RNA-seq）、H3K27me3 CUT&RUN以及全基因组亚硫酸氢盐测序（WGBS）实验。此外，我们还对猴成体组织进行了WGBS分析，并对猴野生型与体细胞核移植（SCNT）胎盘开展了WGBS与RNA-seq测序。