Local Depletion of DNA Methylation Identifies a Repressive p53 Regulatory Region in the NEK2 Promoter

Background: NEK2 is a mammalian kinase that promotes centrosome separation during the cell cycle. Results: Agents that demethylate the NEK2 promoter or induce DNA damage repress NEK2 expression in a p53-dependent manner. Conclusion: p53 represses NEK2 expression and protects its binding region from accumulating DNA methylation. Significance: Knowledge regarding novel mechanisms of NEK2 regulation may help inform clinical application of NEK2-based anticancer therapeutics.Genome-scale mapping suggests that the function of DNA methylation varies with genomic context beyond transcriptional repression. However, the use of DNA-demethylating agents (e.g. 5-aza-2-deoxycytidine (5aza-dC)) to study epigenetic regulation often focuses on gene activation and ignores repression elicited by 5aza-dC. Here, we show that repression of NEK2, which encodes the never in mitosis A (NIMA)-related kinase, by 5aza-dC is context-specific as NEK2 transcript levels were reduced in HCT116 colon cancer cells but not in isogenic p53(-/-) cells. Bisulfite sequencing showed that DNA methylation was restricted to the distal region of the NEK2 promoter. Demethylation by 5aza-dC was associated with increased accessibility to micrococcal nuclease, i.e. nucleosome depletion. Conversely, methyltransferase accessibility protocol for individual templates (MAPit) methylation footprinting showed that nucleosome occupancy and DNA methylation at the distal promoter were significantly increased in p53(-/-) cells, suggesting dynamic regulation of chromatin structure at this region by p53 in HCT116 cells. Stabilization of endogenous p53 by doxorubicin or ectopic expression of p53, but not a p53 DNA-binding mutant, decreased NEK2 expression. Chromatin immunoprecipitation demonstrated direct and specific association of p53 with the distal NEK2 promoter, which was enhanced by doxorubicin. Luciferase reporters confirmed that this region is required for p53-mediated repression of NEK2 promoter activity. Lastly, modulation of p53 abundance altered nucleosome occupancy and DNA methylation at its binding region. These results identify NEK2 as a novel p53-repressed gene, illustrate that its repression by 5aza-dC is specific and associated with nucleosome reorganization, and provide evidence that identification of partially methylated regions can reveal novel p53 target genes.