Allele-specific DNA methylation: beyond imprinting

Abstract

Allele-specific DNA methylation (ASM) and allele-specific gene expression (ASE) have long been studied in genomic imprinting and X chromosome inactivation. But these types of allelic asymmetries, along with allele-specific transcription factor binding (ASTF), have turned out to be far more pervasive-affecting many non-imprinted autosomal genes in normal human tissues. ASM, ASE and ASTF have now been mapped genome-wide by microarray-based methods and NextGen sequencing. Multiple studies agree that all three types of allelic asymmetries, as well as the related phenomena of expression and methylation quantitative trait loci, are mostly accounted for by cis-acting regulatory polymorphisms. The precise mechanisms by which this occurs are not yet understood, but there are some testable hypotheses and already a few direct clues. Future challenges include achieving higher resolution maps to locate the epicenters of cis-regulated ASM, using this information to test mechanistic models, and applying genome-wide maps of ASE/ASM/ASTF to pinpoint functional regulatory polymorphisms influencing disease susceptibility.

Figure 1.

Example of primary data and validations showing non-imprinted ASM. (A) Primary data from MSNP on Affymetrix 6.0 arrays. Pre-digestion of the genomic DNA (PBL sample) with the methylation-sensitive restriction enzyme HpaII prior to linker ligation and PCR for probe synthesis leads to a dropout of the B-allele, suggesting that this allele is relatively hypomethylated. (B) Validation by bisulfite conversion of genomic DNA followed by PCR, cloning and sequencing of multiple clones. Black circles are methylated CpG dinucleotides; white circles are unmethylated CpGs; the dash indicates a CpG SNP in one individual. The two alleles are distinguished in the group of clones by an SNP (rs2302902) that is not destroyed by the bisulfite conversion. Using pre-digestion/PCR assays, the hypermethylated allele was the A-allele at rs4762138 in each of 11 informative PBL samples, consistent with cis-regulation of DNA methylation. (C) Map of the ELK3 gene, containing the intragenic index SNP on the microarrays (rs4762138) and the nearby SNP (rs2302902) that was utilized for distinguishing the alleles in the bisulfite sequencing. The region with ASM subjected to sequencing (gray bar) is CG-rich but is not a CpG island; two CpG islands are near the gene promoter (green bars). Black rectangles are exons 1 and 2. The functional role of the allelic asymmetry at this position in the ELK3 gene is under investigation. Unpublished data are from K. Kerkel and B.T.

Figure 2.

Possible mechanisms of cis-regulated ASE and ASM. The cis-acting effects of sequence polymorphisms can be either short-range (A and B) or long-range (C). Details of each model are discussed in the text. Working out the relevance of each mechanism will depend on developing efficient methods for high-resolution mapping of allelic asymmetries and for determining long-range haplotypes in large collections of primary tissues. Black circles, methylated CpG dinucleotides; white circles, unmethylated CpGs; dark gray rectangle and arrow, first exon of the gene.

Figure 3.

Allele-specific mapping for maximizing information from GWAS. This figure is adapted from Tycko (42). It shows the general strategy for overlaying information from genome-wide maps of allelic asymmetries with data from GWAS. Mapping ASE, ASTF and ASM can help to pinpoint bona fide regulatory polymorphisms, which reveal their presence by conferring the allelic asymmetries in the relevant disease target tissue. Supra-threshold and sub-threshold GWAS peaks that co-localize with these regulatory polymorphisms warrant attention as true-positive signals.