Detection of allele-specific methylation through a generalized heterogeneous epigenome model

Motivations: High-throughput sequencing has made it possible to sequence DNA methylation of a whole genome at the single-base resolution. A sample, however, may contain a number of distinct methylation patterns. For instance, cells of different types and in different developmental stages may have different methylation patterns. Alleles may be differentially methylated, which may partially explain that the large portions of epigenomes from single cell types are partially methylated, and may have major effects on transcriptional output. Approaches relying on DNA sequence polymorphism to identify individual patterns from a mixture of heterogeneous epigenomes are insufficient as methylcytosines occur at a much higher density than SNPs

Genomic surveys by methylation-sensitive SNP analysis identify sequence-dependent allele-specific DNA methylation

Allele-specific DNA methylation (ASM) is a hallmark of imprinted genes, but ASM in the larger nonimprinted fraction of the genome is less well characterized. Using methylation-sensitive SNP analysis (MSNP), we surveyed the human genome at 50K and 250K resolution, identifying ASM as recurrent genotype call conversions from heterozygosity to homozygosity when genomic DNAs were predigested with the methylation-sensitive restriction enzyme HpaII. Using independent assays, we confirmed ASM at 16 SNP-tagged loci distributed across various chromosomes. At 12 of these loci (75%), the ASM tracked strongly with the sequence of adjacent SNPs. Further analysis showed allele-specific mRNA expression at two loci from this methylation-based screen—the vanin and CYP2A6-CYP2A7 gene clusters—both implicated in traits of medical importance. This recurrent phenomenon of sequence-dependent ASM has practical implications for mapping and interpreting associations of noncoding SNPs and haplotypes with human phenotypes

Extensive sequence-influenced DNA methylation polymorphism in the human genome

Background: Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters), but there are also interesting patterns of CpG methylation found outside of CpG islands. Results: We compared DNA methylation patterns on both alleles between many pairs (and larger groups) of related and unrelated individuals. Direct observation and simulation experiments revealed that around 10% of common single nucleotide polymorphisms (SNPs) reside in regions with differences in the propensity for local DNA methylation between the two alleles. We further showed that for the most common form of SNP, a polymorphism at a CpG dinucleotide, the presence of the CpG at the SNP positively affected local DNA methylation in cis. Conclusions: Taken together with the known effect of DNA methylation on mutation rate, our results suggest an interesting interdependence between genetics and epigenetics underlying diversity in the human genome

Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

<p>Abstract</p> <p>Background</p> <p>We previously developed a simple method termed <it>Hpa</it>II-<it>McrBC </it>PCR (HM-PCR) to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs) on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity.</p> <p>Findings</p> <p>We developed <it>Hpa</it>II-<it>McrBC </it>whole-genome-amplification PCR (HM-WGA-PCR) that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods.</p> <p>Conclusions</p> <p>HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.</p

Allele-skewed DNA modification in the brain:relevance to a schizophrenia GWAS

Numerous recent studies have suggested that phenotypic effects of DNA sequence variants can be mediated or modulated by their epigenetic marks, such as allele-skewed DNA modification (ASM). Using Affymetrix SNP microarrays, we performed a comprehensive search of ASM effects in human post-mortem brain and sperm samples (total n = 256) from individuals with major psychosis and control individuals. Depending on the phenotypic category of the brain samples, 1.4%-7.5% of interrogated SNPs exhibited ASM effects. Next, we investigated ASM in the context of genetic studies of schizophrenia and detected that brain ASM SNPs were significantly overrepresented among sub-threshold SNPs from a schizophrenia genome-wide association study (GWAS). Brain ASM SNPs showed a much stronger enrichment in a schizophrenia GWAS than in 17 large GWASs of non-psychiatric diseases and traits, arguing that ASM effects are at least partially tissue specific. Studies of germline and control brain ASM SNPs supported a causal association between ASM and schizophrenia. Finally, significantly higher proportions of ASM SNPs than of non-ASM SNPs were detected at loci exhibiting epigenetic signatures of enhancers and promoters, and they were overrepresented within transcription factor binding regions and DNase I hypersensitive sites. All of these findings collectively indicate that ASM SNPs should be prioritized in follow-up GWASs

CpG Deamination Creates Transcription Factor–Binding Sites with High Efficiency

The formation of new transcription factor–binding sites (TFBSs) has a major impact on the evolution of gene regulatory networks. Clearly, single nucleotide mutations arising within genomic DNA can lead to the creation of TFBSs. Are molecular processes inducing single nucleotide mutations contributing equally to the creation of TFBSs? In the human genome, a spontaneous deamination of methylated cytosine in the context of CpG dinucleotides results in the creation of thymine (C → T), and this mutation has the highest rate among all base substitutions. CpG deamination has been ascribed a role in silencing of transposons and induction of variation in regional methylation. We have previously shown that CpG deamination created thousands of p53-binding sites within genomic sequences of Alu transposons. Interestingly, we have defined a ∼30 bp region in Alu sequence, which, depending on a pattern of CpG deamination, can be converted to functional p53-, PAX-6-, and Myc-binding sites. Here, we have studied single nucleotide mutational events leading to creation of TFBSs in promoters of human genes and in genomic regions bound by such key transcription factors as Oct4, NANOG, and c-Myc. We document that CpG deamination events can create TFBSs with much higher efficiency than other types of mutational events. Our findings add a new role to CpG methylation: We propose that deamination of methylated CpGs constitutes one of the evolutionary forces acting on mutational trajectories of TFBSs formation contributing to variability in gene regulation

Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM

Human imprinted retrogenes exhibit non-canonical imprint chromatin signatures and reside in non-imprinted host genes

Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic ‘host’ gene. In the mouse, at least one transcript of the host gene is also subject to genomic imprinting. Human retrogene orthologues are imprinted and we reveal that human host genes are not imprinted. This coincides with genomic rearrangements that occurred during primate evolution, which increase the separation between the retrogene DMRs and the host genes. To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs. For the mouse retrogenes, the active mark H3K4me2 was associated with the unmethylated paternal allele, while the methylated maternal allele was enriched in repressive marks including H3K9me3 and H4K20me3. Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs. Finally, we show that the genes immediately flanking the host genes in mouse and human are biallelically expressed in a range of tissues, suggesting that these loci are distinct from large imprinted clusters

MethylomeDB: a database of DNA methylation profiles of the brain

MethylomeDB (http://epigenomics.columbia.edu/methylomedb/index.html) is a new database containing genome-wide brain DNA methylation profiles. DNA methylation is an important epigenetic mark in the mammalian brain. In human studies, aberrant DNA methylation alterations have been associated with various neurodevelopmental and neuropsychiatric disorders such as schizophrenia, and depression. In this database, we present methylation profiles of carefully selected non-psychiatric control, schizophrenia, and depression samples. We also include data on one mouse forebrain sample specimen to allow for cross-species comparisons. In addition to our DNA methylation data generated in-house, we have and will continue to include published DNA methylation data from other research groups with the focus on brain development and function. Users can view the methylation data at single-CpG resolution with the option of wiggle and microarray formats. They can also download methylation data for individual samples. MethylomeDB offers an important resource for research into brain function and behavior. It provides the first source of comprehensive brain methylome data, encompassing whole-genome DNA methylation profiles of human and mouse brain specimens that facilitate cross-species comparative epigenomic investigations, as well as investigations of schizophrenia and depression methylomes

Altered DNA Methylation in Leukocytes with Trisomy 21

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2′deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells