Jump-seq: Genome-Wide Capture and Amplification of 5-Hydroxymethylcytosine Sites

摘要

5-Hydroxymethylcytosine (ShmC) arises from the oxidation of 5-methylcytosine (5mC) by Fe2+ and 2-oxoglutarate-dependent 10-11 translocation (TET) family proteins. Substantial levels of ShmC accumulate in many mammalian tissues, especially in neurons and embryonic stem cells, suggesting a potential active role for ShmC in epigenetic regulation beyond being simply an intermediate of active DNA demethylation. 5mC and ShmC undergo dynamic changes during embryogenesis, neurogenesis, hematopoietic development, and oncogenesis. While methods have been developed to map ShmC, more efficient approaches to detect 5hmC at base resolution are still highly desirable. Herein, we present a new method, Jump-seq, to capture and amplify ShmC in genomic DNA. The principle of this method is to label ShmC by the 6-N3-glucose moiety and connect a hairpin DNA oligonucleotide carrying an alkyne group to the azide-modified ShmC via Huisgen cycloaddition (click) chemistry. Primer extension starts from the hairpin motif to the modified ShmC site and then continues to "land" on genomic DNA. ShmC sites are inferred from genomic DNA sequences immediately spanning the 5-prime junction. This technology was validated, and its utility in ShmC identification was confirmed.