Maternal 5mCpG Imprints at the PARD6G-AS1 and GCSAML Differentially Methylated Regions Are Decoupled From Parent-of-Origin Expression Effects in Multiple Human Tissues

Abstract

A hallmark of imprinted genes in mammals is the occurrence of parent-of-origin-dependent asymmetry of DNA cytosine methylation (5mC) of alleles at CpG islands (CGIs) in their promoter regions. This 5mCpG asymmetry between the parental alleles creates allele-specific imprinted differentially methylated regions (iDMRs). iDMRs are often coupled to the transcriptional repression of the methylated allele and the activation of the unmethylated allele in a tissue-specific, developmental-stage-specific and/or isoform-specific fashion. iDMRs function as regulatory platforms, built through the recruitment of chemical modifications to histones to achieve differential, parent-of-origin-dependent chromatin segmentation states. Here, we used a comparative computational data mining approach to identify 125 novel constitutive candidate iDMRs that integrate the maximal number of allele-specific methylation region records overlapping CGIs in human methylomes. Twenty-nine candidate iDMRs display gametic 5mCpG asymmetry, and another 96 are candidate secondary iDMRs. We established the maternal origin of the 5mCpG imprints of one gametic (PARD6G-AS1) and one secondary (GCSAML) iDMRs. We also found a constitutively hemimethylated, nonimprinted domain at the PWWP2AP1 promoter CGI with oocyte-derived methylation asymmetry. Given that the 5mCpG level at the iDMRs is not a sufficient criterion to predict active or silent locus states and that iDMRs can regulate genes from a distance of more than 1 Mb, we used RNA-Seq experiments from the Genotype-Tissue Expression project and public archives to assess the transcriptional expression profiles of SNPs across 4.6 Mb spans around the novel maternal iDMRs. We showed that PARD6G-AS1 and GCSAML are expressed biallelically in multiple tissues. We found evidence of tissue-specific monoallelic expression of ZNF124 and OR2L13, located 363 kb upstream and 419 kb downstream, respectively, of the GCSAML iDMR. We hypothesize that the GCSAML iDMR regulates the tissue-specific, monoallelic expression of ZNF124 but not of OR2L13. We annotated the non-coding epigenomic marks in the two maternal iDMRs using data from the Roadmap Epigenomics project and showed that the PARD6G-AS1 and GCSAML iDMRs achieve contrasting activation and repression chromatin segmentations. Lastly, we found that the maternal 5mCpG imprints are perturbed in several hematopoietic cancers. We conclude that the maternal 5mCpG imprints at PARD6G-AS1 and GCSAML iDMRs are decoupled from parent-of-origin transcriptional expression effects in multiple tissues.

Whole-Genome Methylation Analysis of Phenotype Discordant Monozygotic Twins Reveals Novel Epigenetic Perturbation Contributing to the Pathogenesis of Adolescent Idiopathic Scoliosis

“…Besides, NDN is a well-known maternally imprinted gene and is expressed exclusively from the paternal allele, and its methylation are persistent markers of gene regulation (Lau et al, 2004). Previous studies had proved that imprinted DMRs (iDMRs) could be perturbed in kinds of diseases (de Sa Machado Araujo et al, 2018). In our study, we hypothesized that higher methylation could decrease the expression of NDN, which predisposed the patient to AIS.…”

“…Significant methylation level difference between the twins may influence the function of DMR-related genes and contribute to the etiology of this disease. Interestingly, these DMRs overlapped with 25 allelespecific methylated regions, suggesting that they were related to abnormal genomic imprinting ( Figure S3) (de Sa Machado Araujo et al, 2018). We enriched the genes associated with these DMRs and then subjected these genes to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis.…”

Parent-of-origin-specific allelic expression in the human placenta is limited to established imprinted loci and it is stably maintained across pregnancy

“…Interestingly, for KLHDC10 showing clearly maternally biased expression, mDMR has been reported within the gene body [14]. The discrepancy between the reported parent-of-origin allelic methylation vs. transcription is supported by the emerging evidence that in a number of genomic regions, constitutive parental DNA methylation imprints are actually decoupled from the parent of origin expression effects [13, 46]. Several studies have shown that candidate loci associated with placenta-specific maternal methylation are associated with actual parental allelic transcriptional bias at only half the loci [6, 13, 14].…”

A Differentially Methylated CpG Site in theIL4Gene Associated with Anti-FVIII Inhibitor Antibody Development in Hemophilia A

“…The methylation level at the individual CpG sites was estimated using methylation-sensitive restriction enzyme PCR (MSRE-PCR) triplex specific assays and the products analyzed by Quantitative Fluorescent PCR. We have outlined the concept of the MSRE-PCR triplex assay for the identification of differentially methylated regions in the context of genomic imprinting (Alves da Silva et al, 2016; de Sa Machado Araujo et al, 2018). Briefly, for the intended individual CpG sites herein, each CpG sites was interrogated by a specific assay composed primarily of a primer pair encompassing a region in the context of the CpG site of interest, containing at least one recognition site for methylation-sensitive restriction enzymes.…”

“…The electropherograms were produced with the GeneScan® Analysis and Genotyper® software version 3.7 packages and GeneMapper® ID version 3.2 (Applied Biosystems ® from Thermo Fisher Scientific, Waltham, MA, USA). The level of methylation at the individual CpG sites was estimated as the normalized ratio of restriction enzyme-resistant amplimer after digestion as compared with the undigested product and corrected by the proportion of resistant positive control amplimer, using the formula previously reported (Alves da Silva et al, 2016; de Sa Machado Araujo et al, 2018). Target amplimers ranged 150 bp to 440 bp in length Control amplimers (resistant to enzymatic digestion) were ten bp larger than the target amplimers (Supplemental Table S3).…”