Effects of Maternal Energy Intake on Genomic Imprinting in Fetal Sheep

Background: Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date a total of 255 imprinted genes have been identified or predicted among all investigated mammal species. However, only 21 have been described in sheep and 11 are annotated in current ovine genome. Results: Here we aim to use DNA/RNA throughput sequencing to identify monoallelically expressed and imprinted genes in organs of day 135 fetal sheep, and 2) to determine whether different levels of maternal energy intake (100% of NRC energy requirement or control, 140% or over-, and 60% or under-fed) influence genomic imprinting. We report strategies to solve technical challenges in the next-generation sequencing data analysis pipeline, including alignment bias of RNA sequencing reads and filtering potential false positives. We identified 80 monoallelically expressed and 18 putatively imprinted genes using the list of 255 stated above as a guide. Five (45.6% of 11) were already known imprinted genes in sheep, the other 13 were known imprinted in other mammals. Sanger sequencing confirmed four putative sheep imprinted genes INPP5F, PLAGL1, CASD1 and PPP1R9A. Among the 13 putative imprinted genes, five localized in the known sheep imprinting clusters of MEST domain on chromosome 4, DLK1/GTL2 domain on chromosome 18 and IGF2/H19 domain on chromosome 21, three were in a novel sheep imprinted cluster on chromosome 4 known in other species as PEG10/SGCE. Additionally, we found that the imprinted genes PHLDA2, SLC22A18, DIRAS3, and IGF2 were differentially expressed, albeit without allelic expression reversal, among the three maternal nutritional groups. Conclusions: Together, our results expanded the sheep imprinted gene list to 34 and demonstrated the influence of maternal diet on fetal imprinting under the conditions studied. Overall design: DNA sequencing of ram and RNA sequencing of fetal tissus was used to discover novel genomic imprinting genes in sheep day 135 fetal tissues. Then the maternal nutruition effects was determined. Please note that [1] re-sequenced Ram DNA (for the purpose of genotype individual father for each fetus) is an essential part of the study and therefore included in the records. [2] Since reads were mapped to both reference/alternative genome, there are 2 processed files for Ram genotypes, but for fetuses, the reads coverage from two genomes were averaged, therefore one 1 file for each.