A method for identifying allele-specific hydroxymethylation

ABSTRACT

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.

KEYWORDS:

• DNA methylation
• hydroxymethylation
• allele-specific methylation
• restriction enzyme
• imprinting

Acknowledgments

This work was supported by Japan Society for the Promotion of Science KAKENHI under Grant Numbers 24510266 and 15K06894 and Shibuya Science Culture and Sports Foundation. We thank Daisuke Fukushima in School of Electrical and Computer Engineering, Kanazawa University for technical assistance.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

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Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.