A method for identifying allele-specific hydroxymethylation.

We previously identified sequence-dependent allele-specific methylation (sd-ASM) in adult human peripheral blood leukocytes, in which ASM occurs in cis depending on adjacent polymorphic sequences. A number of groups have identified sd-ASM sites in the human and mouse genomes, illustrating the prevalence of sd-ASM in mammalian genomes. In addition, sd-ASM can lead to sequence-dependent allele-specific expression of neighbouring genes. Imprinted genes also often exhibit parent-of-origin-dependent allele-specific methylation (pd-ASM), which causes parent-of-origin-dependent allele-specific expression. However, whether most of the already known sd-ASM and pd-ASM sites are methylated or hydroxymethylated remains unclear due to technical restrictions. Accordingly, a novel method that enables examination of allelic methylation and hydroxymethylation status and also overcomes the drawbacks of conventional methods is needed. Such a method could also be used to elucidate the mechanisms underlying polymorphism-associated inter-individual differences in disease susceptibility and the mechanism of genomic imprinting. Here, we developed a simple method to determine allelic hydroxymethylation status and identified novel sequence- and parent-of-origin-dependent allele-specific hydroxymethylation sites. Correlation analyses of TF binding sequences and methylation or hydroxymethylation between three mouse strains revealed the involvement of Pax5 in strain-specific methylation and hydroxymethylation in exon 7 of Pdgfrb.