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

Abstract

Implementation of genomic imprinting in mammals often results in cis-acting silencing of a gene cluster and monoallelic expression, which are important for mammalian growth and function. Compared with widely documented imprinting status in humans and mice, current understanding of genomic imprinting in pigs is relatively limited. The objectives of this study were to identify DNA methylation status and allelic expression of alternative spliced isoforms at the porcine PLAGL1 locus and assess the conservation of the locus compared to the orthologous human locus. DNA methylome and transcriptome were constructed using porcine parthenogenetic or biparental control embryos. Using methylome, differentially methylated regions between those embryos were identified. Alternative splicing was identified by differential splicing analysis, and monoallelic expression was examined using single nucleotide polymorphism sites. Moreover, topological boundary regions were identified by analyzing CTCF binding sites and compared with the boundary of human orthologous locus. As a result, it was revealed that the monoallelic expression of the PLAGL1 gene in porcine embryos via genomic imprinting was maintained in the adult stage. The porcine PLAGL1 locus was largely conserved in regard to maternal hypermethylation, tissue distribution of mRNA expression, monoallelic expression, and biallelic CTCF-binding, with exceptions on transcript isoforms produced by alternative splicing instead of alternative promoter usage. These findings laid the groundwork for comparative studies on the imprinted PLAGL1 gene and related regulatory mechanisms across species.

Loss of Monoallelic Expression of IGF2 in the Adult Liver Via Alternative Promoter Usage and Chromatin Reorganization

“…Our analyses using pigs support the biallelic conversion and alternative promoter usage that might occur gradually at post-natal ages while ages for initiation of the conversion might vary (Figure 4). These allelic expression patterns were verified based on individual-matched genomic DNA sequence data from WGS and mRNA sequence data from RNA-seq in both pigs and humans (Figures 5, 7, and 8) using informative SNPs found on genomic DNA that served as markers to confirm allelic imbalance of mRNA expression (Castel et al, 2015;Ahn et al, 2021b;a). We primarily examined SNPs in non-overlapping exons to identify and analyze allelic expression at the isoform level while there is a previous study relied on a marker in the last overlapping exon (Braunschweig et al, 2011).…”

“…The multi-layered epigenetic regulatory machineries responsible for DNA methylation, chromatin accessibility, histone modifications, and gene expression can be investigated using integrative omics approaches. As the gold standard for DNA methylation analysis, whole-genome bisulfite sequencing (WGBS) of animal models including parthenogenetic embryos has been utilized to identify differentially methylated regions (DMRs) (Clark et al, 2006;Ahn et al, 2020a;Ahn et al, 2020c;Ahn et al, 2021b;a;Morrison et al, 2021). To assess chromatin accessibility (Thurman et al, 2012) and capture open chromatin sites, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) has been used (Buenrostro et al, 2013).…”

“…Collectively, epigenetic modulations change chromatin structure and thereby alter DNA accessibility, which affects availability of enhancers and promoters to the transcriptional machinery. These epigenetic modifications might affect tissue-specific monoallelic gene expression within the H19/IGF2 locus, which can be identified by profiling informative single nucleotide polymorphisms (SNPs) in genomic DNA and mRNA of the same individual (Ahn et al, 2021b;a). In addition, chromosomal conformation capture-based methods such as Hi-C have enabled unbiased identification of chromatin interactions across the genome (Lieberman-Aiden et al, 2009).…”

PLAGL1 Gene Demethylation Induced by Epigallocatechin Gallate Promotes Pheochromocytoma Cell Apoptosis Via Wnt/β-catenin Signaling Pathway

“…Extensive studies have suggested chromosome abnormalities, oncogene mutation or amplification activation, and tumor suppressor gene inactivation play a very important role in the pathogenesis and progress of malignant tumors Foggetti et al, 2021;Masclef et al, 2021;Alqahtani et al, 2019). PCC was rare neuroendocrine tumors that arise from chromaffin cells, the prognosis of malignant PCC was poor, and the average 5-year survival rate was 40% (Hamidi, 2019).…”

“…The PLAGL1 gene was located in chromosome 6q24-q25 and identified as a tumor suppressor gene. PLAGL1 provides instructions for producing zinc finger proteins and acts as a transcription factor involved in inducing cell apoptosis through regulating Wnt/β-catenin signaling pathway (Ahn et al, 2021;Zhu et al, 2009). This study is aimed at investigating the demethylation effects of EGCG in the PLAGL1 gene and exploring whether EGCG inhibits cell proliferation through inducing the PLAGL1 gene demethylation via regulating Wnt/β-catenin signaling pathway in PCC.…”