A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA.

Recently, 5-hydroxymethyluracil (5hmU) was identified in mammalian genomic DNA as an oxidative product of thymine by the ten-eleven translocation (TET) proteins. While the biological role of this modification remains unclear, identifying its genomic location will assist in elucidating function. Here we present a rapid and robust method to selectively tag and enrich genomic regions containing 5hmU. This method involves the selective glucosylation of 5hmU residues by the base J glucosyltransferase from trypanosomes creating glucosylhydroxymethyluracil (base J). The base J can then be efficiently and selectively pulled down by antibodies against base J or by J-binding protein 1. DNA that is enriched is suitable for analysis by quantitative PCR or sequencing. We utilized this tagging reaction to provide proof of concept for the enrichment of 5hmU containing DNA from a pool that contains modified and unmodified DNA. Furthermore, we demonstrate that the base J pull-down assay identifies 5hmU at specific regions of the trypanosome genome involved in transcriptional repression. The method described here will allow for a greater understanding of the functional role and dynamics of 5hmU in biology.