The Landscape of Genomic Imprinting at the Porcine SGCE/PEG10 Locus from Methylome and Transcriptome of Parthenogenetic Embryos

Abstract

In mammals, imprinted genes often exist in the form of clusters in specific chromosome regions. However, in pigs, genomic imprinting of a relatively few genes and clusters has been identified, and genes within or adjacent to putative imprinted clusters need to be investigated including those at the SGCE/PEG10 locus. The objective of this study was to, using porcine parthenogenetic embryos, investigate imprinting status of genes within the genomic region spans between the COL1A2 and ASB4 genes in chromosome 9. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were conducted with normal and parthenogenetic embryos, and methylome and transcriptome were analyzed. As a result, differentially methylated regions (DMRs) between the embryos were identified, and parental allele-specific expressions of the SGCE and PEG10 genes were verified. The pig imprinted interval was limited between SGCE and PEG10, since both the COL1A2 and CASD1 genes at the centromere-proximal region and the genes between PPP1R9A and ASB4 toward the telomere were non-imprinted and biallelically expressed. Consequently, our combining analyses of methylome, transcriptome, and informative polymorphisms revealed the boundary of imprinting cluster at the SGCE/PEG10 locus in pig chromosome 9 and consolidated the landscape of genomic imprinting in pigs.

Genomic Imprinting at the Porcine PLAGL1 Locus and the Orthologous Locus in the Human

“…Oocytes from Landrace × Yorkshire × Duroc (LYD) pigs were collected and matured in vitro. As described in our previous reports [ 5 , 9 ], parthenogenetic embryos were generated by electrical stimulation of oocytes. Those parthenogenetic embryos were developed for 21 days after placing into oviducts of surrogate gilts.…”

“…Those parthenogenetic embryos were developed for 21 days after placing into oviducts of surrogate gilts. As experimental controls, fertilized embryos were collected at day 21 from LYD gilts after natural mating as previously described in our publications [ 5 , 9 ].…”

“…Genomic DNA was isolated from control (CN, n = 3) and parthenogenetic (PA, n = 3) whole embryos and subjected to WGBS as reported previously [ 5 , 9 ]. In brief, based on the manufacturer’s instructions, bisulfite conversion of genomic DNA was performed using Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences, Inc., Ann Arbor, MI, USA).…”

“…RNA-seq was performed to produce transcriptome as described [ 5 , 9 ]. Total RNA was isolated from the whole collected CN and PA embryos ( n = 3, each) using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instruction.…”

“…Consequently, the resulting monoallelic or allele-specific expression (or, allelic imbalance) plays an important role in mammalian growth, development, tissue function, and phenotypic plasticity [ 7 , 8 ]. Compared with widely documented imprinting status in humans and mice, current understanding of genomic imprinting in pigs is relatively limited; therefore, our attempts were made to delineate imprinting in porcine loci using parthenogenetic embryos, and whole-genome bisulfite sequencing (WGBS), and RNA-sequencing (RNA-seq) [ 5 , 9 ].…”