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Background There is increasing evidence for inter-individual methylation differences at CpG dinucleotides in the human genome, but the regional extent and function of these differences have not yet been studied in detail. For identifying regions of common methylation differences, we used whole genome bisulfite sequencing data of monocytes from five donors and a novel bioinformatic strategy. Results We identified 157 differentially methylated regions (DMRs) with four or more CpGs, almost none of which has been described before. The DMRs fall into different chromatin states, where methylation is inversely correlated with active, but not repressive histone marks. However, methylation is not correlated with the expression of associated genes. High-resolution single nucleotide polymorphism (SNP) genotyping of the five donors revealed evidence for a role of cis -acting genetic variation in establishing methylation patterns. To validate this finding in a larger cohort, we performed genome-wide association studies (GWAS) using SNP genotypes and 450k array methylation data from blood samples of 1128 individuals. Only 30/157 (19%) DMRs include at least one 450k CpG, which shows that these arrays miss a large proportion of DNA methylation variation. In most cases, the GWAS peak overlapped the CpG position, and these regions are enriched for CREB group, NF-1, Sp100 and CTCF binding motifs. In two cases, there was tentative evidence for a trans -effect by KRAB zinc finger proteins. Conclusions Allele-specific DNA methylation occurs in discrete chromosomal regions and is driven by genetic variation in cis and trans , but in general has little effect on gene expression.

The ability of nanopore sequencing to simultaneously detect modified nucleotides while producing long reads makes it ideal for detecting and phasing allele-specific methylation. However, there is currently no complete software for detecting SNPs, phasing haplotypes, and mapping methylation to these from nanopore sequence data. Here, we present NanoMethPhase, a software tool to phase 5-methylcytosine from nanopore sequencing. We also present SNVoter, which can post-process nanopore SNV calls to improve accuracy in low coverage regions. Together, these tools can accurately detect allele-specific methylation genome-wide using nanopore sequence data with low coverage of about ten-fold redundancy.

Triploid breeding is a promising avenue for generating seedless varieties with enhanced vigor, yet the underlying molecular mechanisms, particularly the relative contributions of hybridity, ploidy level, and parent-of-origin effects (POE), remain largely elusive in perennial fruit crops. This study focuses on loquat ( Eriobotrya japonica ), a highly heterozygous woody perennial, to explore the molecular mechanism of triploid seedling vigor heterosis. RNA-seq across a series of reciprocal diploid (2x), triploid (3x) and tetraploid (4x) hybrids with clear genetic background revealed POE as the predominant driver of triploid heterosis at the transcriptomic level. Specifically, 784 POE-responsive differentially expressed genes (DEGs) were identified between paternal-excess [3x(p)] and maternal-excess [3x(m)] triploids, exceeding the effects of ploidy (218–652 DEGs) and hybridity (8–90 DEGs). For an in-depth investigation, reciprocal crosses between L2 and L4 were further selected for an integrated transcriptome, allele-specific expression (ASE), and allele-specific methylation (ASM) analysis. Our findings demonstrate that POE orchestrates multilayered regulation, including (i) coordinated upregulation of vigor-related pathways (i.e., photosynthesis, starch metabolism, plant circadian rhythm) in 3x(p); (ii) a dual ASE pattern with maternal bias in gene quantity but paternal enhancement in levels, including five paternally expressed imprinted genes (PEGs); (iii) non-classical epigenetic regulation where paternal gene body hypermethylation (mCG) paradoxically enhances transcription, especially in circadian clock genes. Finally, qRT-PCR-based diurnal expression across all crosses validated that POE-dependent reprogramming of key circadian oscillator genes ( EjCCA1 , EjLHY , EjGI , EjTOC1 ), suggesting optimized metabolic efficiency through circadian clock modulation might contribute to enhanced vigor in 3x(p) hybrids. This study provides fundamental insights into the dosage-sensitive gene networks and epigenetic regulation underlying POE-driven heterosis in woody perennials, advancing polyploid heterosis theory and offering novel targets for genetic improvement.

The methylation of CpG sites with 5mC mark is a dynamic epigenetic modification. However, the relationship between the methylation and the surrounding genomic sequence context remains poorly explored. Investigation of the allele methylation provides an opportunity to decipher the interplay between differences in the primary DNA sequence and epigenetic variation. Here, we performed high-coverage long-read whole-genome direct DNA sequencing of one individual using Oxford Nanopore technology. We also used Illumina whole-genome sequencing of the parental genomes in order to identify allele-specific methylation sites with a trio-binning approach. We have compared the results of the haplotype-specific methylation detection and revealed that trio binning outperformed other approaches that do not take into account parental information. Also, we analysed the cis-regulatory effects of the genomic variations for influence on CpG methylation. To this end, we have used available Deep Learning models trained on the primary DNA sequence to score the cis-regulatory potential of the genomic loci. We evaluated the functional role of the allele-specific epigenetic changes with respect to gene expression using long-read Nanopore RNA sequencing. Our analysis revealed that the frequency of SNVs near allele-specific methylation positions is approximately four times higher compared to the biallelic methylation positions. In addition, we identified that allele-specific methylation sites are more conserved and enriched at the chromatin states corresponding to bivalent promoters and enhancers. Together, these findings suggest that significant impact on methylation can be encoded in the DNA sequence context. In order to elucidate the effect of the SNVs around sites of allele-specific methylation, we applied the Deep Learning model for detection of the cis-regulatory modules and estimated the impact that a genomic variant brings with respect to changes to the regulatory activity of a DNA loci. We revealed higher cis-regulatory impact variants near differentially methylated sites that we further coupled with transcriptomic long-read sequencing results. Our investigation also highlights technical aspects of allele methylation analysis and the impact of sequencing coverage on the accuracy of genomic phasing. In particular, increasing coverage above 30X does not lead to a significant improvement in allele-specific methylation discovery, and only the addition of trio binning information significantly improves phasing. We investigated genomic variation in a single human individual and coupled computational discovery of cis-regulatory modules with allele-specific methylation (ASM) profiling. In this proof-of-concept analysis, we observed that SNPs located near methylated CpG sites on the same haplotype were enriched for sequence features suggestive of high-impact regulatory potential. This finding—derived from one deeply sequenced genome—illustrates how phased genetic and epigenetic data analyses can jointly put forward a hypotheses about the involvement of regulatory protein machinery in shaping allele-specific epigenetic states. Our investigation provides a methodological framework and candidate loci for future studies of genomic imprinting and cis-mediated epigenetic regulation in humans.

Imprinting is a critical part of normal embryonic development in mammals, controlled by defined parent-of-origin (PofO) differentially methylated regions (DMRs) known as imprinting control regions. Direct nanopore sequencing of DNA provides a means to detect allelic methylation and to overcome the drawbacks of methylation array and short-read technologies. Here, we used publicly available nanopore sequencing data for 12 standard B-lymphocyte cell lines to acquire the genome-wide mapping of imprinted intervals in humans. Using the sequencing data, we were able to phase 95% of the human methylome and detect 94% of the previously well-characterized, imprinted DMRs. In addition, we found 42 novel imprinted DMRs (16 germline and 26 somatic), which were confirmed using whole-genome bisulfite sequencing (WGBS) data. Analysis of WGBS data in mouse ( Mus musculus ), rhesus monkey ( Macaca mulatta ), and chimpanzee ( Pan troglodytes ) suggested that 17 of these imprinted DMRs are conserved. Some of the novel imprinted intervals are within or close to imprinted genes without a known DMR. We also detected subtle parental methylation bias, spanning several kilobases at seven known imprinted clusters. At these blocks, hypermethylation occurs at the gene body of expressed allele(s) with mutually exclusive H3K36me3 and H3K27me3 allelic histone marks. These results expand upon our current knowledge of imprinting and the potential of nanopore sequencing to identify imprinting regions using only parent-offspring trios, as opposed to the large multi-generational pedigrees that have previously been required.

Prostate-specific antigen remains a cornerstone biomarker for prostate cancer diagnosis and management. However, the molecular mechanisms regulating its expression, particularly through DNA methylation, are not fully understood. Here, we report a comprehensive analysis of allele-specific CpG and CCWGG methylation in the proximal PSA promoter across aggressive (PC3), indolent (LNCaP), benign (BPH1), and normal (HPrEpiC) prostate cell lines and provide insights into the unique methylation patterns associated with these states. Our findings reveal that PC3 cells, representing an aggressive PCa phenotype, exhibit complete biallelic methylation of the PSA promoter, leading to PSA gene silencing. Conversely, LNCaP cells display a fully unmethylated promoter with biallelic PSA expression. Interestingly, BPH1 cells display a monoallelic CG/CCWGG methylation pattern, yet fail to express PSA, suggesting imprinting defects or RNA decay mechanisms. Notably, acquisition of biallelic PSA promoter methylation status in PC3 was accompanied by upregulation of DNMT1, whereas unmethylated PSA promoter state in LNCaP was associated with downregulation of DNMT1. These findings highlight distinct methylation patterns in the PSA promoter that differentiate between aggressive, indolent, and benign prostate states. Translating this epigenetic insight into clinical diagnostics could enhance the precision of PSA-based diagnostics, addressing limitations such as false negatives in PSA testing for aggressive PCa. Further exploration of CCWGG methylation’s role in imprinting and monoallelic expression is warranted, particularly in patient-derived samples.

DNA methylation is an essential epigenetic modification that plays a crucial role in regulating gene expression and maintaining genomic stability. With the advancement in sequencing technology, methylation studies have provided valuable insights into the diagnosis of rare diseases through the various identification of episignatures, epivariation, epioutliers, and allele-specific methylation. However, current methylation studies are not without limitations. This mini-review explores the current understanding of DNA methylation in rare diseases, highlighting the key mechanisms and diagnostic potential, and emphasizing the need for advanced methodologies and integrative approaches to enhance the understanding of disease progression and design more personable treatment for patients, given the nature of rare diseases.

Identifying genetic variants that are associated with methylation variation—an analysis commonly referred to as methylation quantitative trait locus (mQTL) mapping—is important for understanding the epigenetic mechanisms underlying genotype-trait associations. Here, we develop a statistical method, IMAGE, for mQTL mapping in sequencing-based methylation studies. IMAGE properly accounts for the count nature of bisulfite sequencing data and incorporates allele-specific methylation patterns from heterozygous individuals to enable more powerful mQTL discovery. We compare IMAGE with existing approaches through extensive simulation. We also apply IMAGE to analyze two bisulfite sequencing studies, in which IMAGE identifies more mQTL than existing approaches.

We describe a new procedure for the analysis of the methlyation status of imprinted genes based on methylation‐specific PCR followed by denaturing high performance liquid chromatography (MSP/DHPLC). The method offers a rapid and very reliable alternative to conventional methods used for such purposes such as Southern blots and methylation specific PCR (allele‐specific MSP). The efficient resolution of the differentially methylated alleles is demonstrated for two human imprinted genes, namely the SNRPN gene and the LIT1 gene (KCNQ1OT1). Abnormal imprinting of the two genes is associated with the Angelman/Prader‐Willi syndromes and the Beckwith‐Wiedemann syndrome, respectively. The MSP/DHPLC method is based on PCR amplification of gene segments which show parent‐of‐origin specific methylation. Genomic DNA is subjected to an in vitro bisulfite treatment prior to PCR amplifications using primers specific for modified DNA. Both alleles are theoretically amplified with equal efficiency and are represented by identically sized PCR products; they differ, however, at a number of positions within the amplified DNA segment. The DHPLC analysis allows a very efficient resolution of the two populations of PCR products. The high sensitivity and quantitative properties of the MSP/DHPLC method are illustrated based on its ability to reveal a low cell mosaicism in an infant with a maternal uniparental disomy 15 (i.e., Prader‐Willi syndrome patient). The minor cell line (approximately 8% in blood) was not detectable with conventional molecular analysis. While the detection of low cell mosaicisms of structurally abnormal chromosomes usually relies on cytogenetic studies, the MSP/DHPLC method described here not only offers an alternative at the molecular level, but may also reveal mosaicisms concerning structurally intact chromosomes. Hum Mutat 17:423–430, 2001. © 2001 Wiley‐Liss, Inc.

Background Intersexual genomic conflict sometimes leads to unequal expression of paternal and maternal alleles in offspring, resulting in parent-of-origin effects. In honey bees reciprocal crosses can show strong parent-of-origin effects, supporting theoretical predictions that genomic imprinting occurs in this species. Mechanisms behind imprinting in honey bees are unclear but differential DNA methylation in eggs and sperm suggests that DNA methylation could be involved. Nonetheless, because DNA methylation is multifunctional, it is difficult to separate imprinting from other roles of methylation. Here we use a novel approach to investigate parent-of-origin DNA methylation in honey bees. In the subspecies Apis mellifera capensis, reproduction of females occurs either sexually by fertilization of eggs with sperm, or via thelytokous parthenogenesis, producing female embryos derived from two maternal genomes. Results We compared genome-wide methylation patterns of sexually-produced, diploid embryos laid by a queen, with parthenogenetically-produced diploid embryos laid by her daughters. Thelytokous embryos inheriting two maternal genomes had fewer hypermethylated genes compared to fertilized embryos, supporting the prediction that fertilized embryos have increased methylation due to inheritance of a paternal genome. However, bisulfite PCR and sequencing of a differentially methylated gene, Stan (GB18207) showed strong allele-specific methylation that was maintained in both fertilized and thelytokous embryos. For this gene, methylation was associated with haplotype, not parent of origin. Conclusions The results of our study are consistent with predictions from the kin theory of genomic imprinting. However, our demonstration of allele-specific methylation based on sequence shows that genome-wide differential methylation studies can potentially confound imprinting and allele-specific methylation. It further suggests that methylation patterns are heritable or that specific sequence motifs are targets for methylation in some genes.