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

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

Trans effects of chromosome aneuploidies on DNA methylation patterns in human Down syndrome and mouse models

Background Trisomy 21 causes Down syndrome (DS), but the mechanisms by which the extra chromosome leads to deficient intellectual and immune function are not well understood. Results Here, we profile CpG methylation in DS and control cerebral and cerebellar cortex of adults and cerebrum of fetuses. We purify neuronal and non-neuronal nuclei and T lymphocytes and find biologically relevant genes with DS-specific methylation (DS-DM) in each of these cell types. Some genes show brain-specific DS-DM, while others show stronger DS-DM in T cells. Both 5-methyl-cytosine and 5-hydroxy-methyl-cytosine contribute to the DS-DM. Thirty percent of genes with DS-DM in adult brain cells also show DS-DM in fetal brains, indicating early onset of these epigenetic changes, and we find early maturation of methylation patterns in DS brain and lymphocytes. Some, but not all, of the DS-DM genes show differential expression. DS-DM preferentially affected CpGs in or near specific transcription factor binding sites (TFBSs), implicating a mechanism involving altered TFBS occupancy. Methyl-seq of brain DNA from mouse models with sub-chromosomal duplications mimicking DS reveals partial but significant overlaps with human DS-DM and shows that multiple chromosome 21 genes contribute to the downstream epigenetic effects. Conclusions These data point to novel biological mechanisms in DS and have general implications for trans effects of chromosomal duplications and aneuploidies on epigenetic patterning

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

Local mapping of ASM in the <i>CYP2A7</i> gene shows a discrete DMR that precisely overlaps a CTCF binding site.

<p>Bis-seq of heterozygous liver samples for multiple <i>CYP2A7</i> amplicons shows ASM localized to a roughly 400 bp region (chr19: 41,386,227–41,386,613) spanning a CGI and a CTCF binding site in exon 2. ASM was evaluated visually and by a T-test on the percent methylation of individual clones, comparing the sets of clones for the two alleles.</p

Non-imprinted ASM in the <i>STEAP3-C2orf76</i> region and imprinted ASM in the <i>RPN1-C3orf27</i> regions are both associated with ASE.

<p><b>A,</b> ASE of the <i>C2orf76</i> gene, associated with the <i>C2orf76-STEAP3</i> DMR, is shown in duplicate for four PBL samples (rs6542522). In each case the C allele is preferentially expressed in the cDNA when compared to the genomic DNA, consistent with the sequence dependent nature of ASM in this locus. Overall, 12 informative PBL samples were analyzed in duplicate for ASE and 7 PBL samples showed preferential expression of the C allele. Likewise, preferential expression of the C allele was identified in 7 out of the 22 informative liver samples (data not shown). <b>B,</b> ASE of the lncRNA corresponding to the AK097792 EST, overlapping the index SNP rs2811488 in the <i>RPN1-C3orf27</i> intergenic DMR are shown on the left and those overlapping two additional informative SNPs, i.e. rs35604103 and rs61112519, are shown on the right for four placenta samples. Overall, the assays were performed in duplicates for 13 informative placenta samples. Ten of them showed a definite or complete ASE. In each of 6 informative samples assayed for both ASE and ASM, the hypermethylated maternal allele was the relatively repressed one.</p

ASM downstream of the <i>RPN1</i> gene and in the <i>VTRNA2-1</i> gene is due to genomic imprinting with hypermethylation of the maternal allele.

<p>Trios consisting of matched parents (PBL) and offspring (placental chorionic villi from the fetal surface) were analyzed for ASM to determine the parental origin of the hypermethylated and hypomethylated alleles in the <i>C3orf27-RPN1</i> and <i>VTRNA2-1</i> index regions. <b>A, </b><i>Hpa</i>II-predigestion/PCR/RFLP assay for ASM at non-polymorphic <i>Hpa</i>II sites in an amplicon including SNP rs12487604 (located immediately downstream of <i>RPN1</i>) which creates a <i>Bts</i>I restriction site. Undigested genomic DNA and genomic DNA pre-digested with the methylation-sensitive restriction enzyme <i>Hpa</i>II were amplified by PCR and then digested with <i>Bts</i>I. In the samples pre-digested with <i>Hpa</i>II (+) one allele drops out, indicating hypomethylation. A homozygous case for each allele is shown, as well as four heterozygotes; two cases show T-allele hypermethylation and two show A-allele hypermethylation. <b>B,</b> Trios analyzed by this assay showing parental imprinting of the index region downstream of <i>RPN1</i>. A total of 13 informative trios (two shown) were analyzed and each showed hypermethylation of the maternally-derived allele in the placenta (p<0.00311). <b>C,</b> Examples of bis-seq of trios for the index amplicon including SNP rs2346019 in the <i>VTRNA2-1</i> locus. The hypermethylated allele was found to be maternally derived in each of 10 informative trios (p<0.001565).</p

Bis-seq showing strong ASM in the <i>ELK3</i>, <i>STEAP3-C2orf76</i>, <i>C3orf27-RPN1</i>, and <i>VTRNA2-1</i> index regions.

<p><b>A,</b> Gene map and bis-seq of the index amplicon containing SNP rs2302902 in the first intron of the <i>ELK3</i> gene. The strong asymmetry in CpG methylation, with the G allele consistently hypermethylated in the PBL samples, indicates haplotype-dependent non-imprinted ASM (additional data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-t001" target="_blank">Table 1</a>; haplotype map in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen.1003622.s005" target="_blank">Figure S5</a>). There is some tissue-specificity, with biallelic hypermethylation in the HMEC sample. The grey (lower) bars indicate the index amplicons for initial bis-seq, each tagged by the index SNP that showed recurrent ASM in the MSNP data. The green bars indicate CGIs and the black rectangles are exons. The X's indicate polymorphic CpG sites. <b>B,</b> Gene map and bis-seq of the index amplicon containing SNP rs1530562, between the <i>STEAP3</i> and <i>C2orf76</i> genes in chromosome band 2q14, showing strong ASM with the G allele consistently hypermethylated in multiple tissues consistent with haplotype-dependent non-imprinted ASM, but with biallelic hypermethylation in sperm DNA (additional data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-t001" target="_blank">Table 1</a>). <b>C,</b> Gene map and bis-seq of the index amplicon containing SNP rs2811488 located downstream of the last exon of the <i>RPN1</i> gene in chromosome band 3q21, showing strong ASM in placenta, with the G allele or A allele hypermethylated, depending on parent-of-origin, as proven in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-g002" target="_blank">Figure 2</a>. For this imprinted DMR the ASM is highly tissue-specific, being seen in placenta but not in PBL, liver, lung, brain, HMEC or sperm (additional data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-t001" target="_blank">Table 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen.1003622.s002" target="_blank">Figure S2</a>). <b>D,</b> Gene map and bilsufite sequencing of the index amplicon containing SNP rs2346019 downstream of the <i>VTRNA2-1</i> vault-family RNA, located in chromosome band 5q31. Strong ASM is observed in multiple tissues with the A allele or G allele methylated, consistent with imprinting, which is proven by the data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-g002" target="_blank">Figure 2</a>.</p