Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date, 263 imprinted genes have been identified among all investigated mammalian species. However, only 21 have been described in sheep, of which 11 are annotated in the current ovine genome. Here, we aim to i) use DNA/RNA high throughput sequencing to identify new monoallelically expressed and imprinted genes in day 135 ovine fetuses and ii) determine whether maternal diet (100%, 60%, or 140% of National Research Council Total Digestible Nutrients) influences expression of imprinted genes. We also reported strategies to solve technical challenges in the data analysis pipeline. We identified 80 monoallelically expressed, 13 new putative imprinted genes, and five known imprinted genes in sheep using the 263 genes stated above as a guide. Sanger sequencing confirmed allelic expression of seven genes, CASD1, COPG2, DIRAS3, INPP5F, PLAGL1, PPP1R9A, and SLC22A18. Among the 13 putative imprinted genes, five were localized in the known sheep imprinting domains of MEST on chromosome 4, DLK1/GTL2 on chromosome 18 and KCNQ1 on chromosome 21, and three were in a novel sheep imprinted cluster on chromosome 4, known in other species as PEG10/SGCE. The expression of DIRAS3, IGF2, PHLDA2, and SLC22A18 was altered by maternal diet, albeit without allelic expression reversal. Together, our results expanded the list of sheep imprinted genes to 34 and demonstrated that while the expression levels of four imprinted genes were changed by maternal diet, the allelic expression patterns were un-changed for all imprinted genes studied.

基因组印记（Genomic imprinting）是一种基于亲本来源的等位基因差异化表达表观遗传（epigenetic）现象。截至目前，在所有已研究的哺乳动物物种中，已鉴定出263个印记基因。然而在绵羊中仅报道了21个印记基因，其中11个已在当前绵羊基因组中完成注释。本研究旨在：其一，利用DNA/RNA高通量测序技术，在妊娠135天的绵羊胎儿中鉴定新的单等位基因表达基因与印记基因；其二，探究母体日粮（分别为美国国家研究委员会（National Research Council）推荐总可消化营养物的100%、60%与140%）是否会影响印记基因的表达水平。本研究同时报道了用于解决数据分析流程中技术难题的策略。本研究以上述263个基因为参照，在绵羊中鉴定出80个单等位基因表达基因、13个新的候选印记基因以及5个已知印记基因。通过桑格测序（Sanger sequencing）验证了7个基因的等位基因表达特性，分别为CASD1、COPG2、DIRAS3、INPP5F、PLAGL1、PPP1R9A与SLC22A18。在这13个候选印记基因中，有5个定位于绵羊已报道的印记区域：4号染色体上的MEST、18号染色体上的DLK1/GTL2以及21号染色体上的KCNQ1；另有3个定位于绵羊4号染色体上的一个新印记基因簇，该基因簇在其他物种中被称为PEG10/SGCE。DIRAS3、IGF2、PHLDA2与SLC22A18的表达水平受母体日粮调控发生改变，但未出现等位基因表达模式的逆转。综上，本研究将绵羊印记基因的总数拓展至34个，并证实：尽管4个印记基因的表达水平受母体日粮影响发生改变，但所有被研究的印记基因的等位基因表达模式均未发生变化。