DNA hydroxymethylation increases the susceptibility of reactivation of methylated P16 alleles in cancer cells

ABSTRACT

It is well established that 5-methylcytosine (5mC) in genomic DNA of mammalian cells can be oxidized into 5-hydroxymethylcytosine (5hmC) and other derivates by DNA dioxygenase TETs. While conversion of 5mC to 5hmC plays an important role in active DNA demethylation through further oxidation steps, a certain proportion of 5hmCs remain in the genome. Although 5hmCs contribute to the flexibility of chromatin and protect bivalent promoters from hypermethylation, the direct effect of 5hmCs on gene transcription is unknown. In this present study, we have engineered a zinc-finger protein-based P16-specific DNA dioxygenase (P16-TET) to induce P16 hydroxymethylation and demethylation in cancer cells. Our results demonstrate, for the first time, that although the hydroxymethylated P16 alleles retain transcriptionally inactive, hydroxymethylation could increase the susceptibility of reactivation of methylated P16 alleles.

KEYWORDS:

• P16 gene
• CpG islands
• methylation
• hydroxymethylation
• epigenetic editing

Acknowledgments

We thank Mr. Jordan M. Grainger (Predoctoral student, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, USA), Dr. Huidong Shi (Cancer Center, Georgia Medical College, Augusta, USA), and Mr. James Hyun (Pharmacology Department, University of Illinois, Chicago, USA) for English language editing.

Author contributions

PL and YG: elucidated the biological function of P16 hydroxymethylation; SQ: discovered P16 hydroxymethylation; XH: demonstrated the strand-bias distribution of 5hmCs in the P16 alleles; CC, Z-mL, and BZ: constructed the P16-specific oxygenase; LG performed the animal experiments; BZ performed immunostaining and cell sorting assays and codesigned the study; ZL and JZ: carried out other experiments; DD: designed the study, analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Ethics approval and consent to participate

The Peking University Cancer Hospital and Institutional Review Boards approved this study. Ethical approval for the animal experiments was obtained.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China under Grant number 81672770; Beijing Municipal Commission of Health and Family Planning under Grant number PXM2018_026279_000005, Beijing Municipal Administration of Hospital Clinical Medical Development of Special Funding Support under Grant number XM201303.