Abstract
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.
Highlights
NEK2 is a mammalian kinase that promotes centrosome separation during the cell cycle
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
Our data show that NEK2 expression is repressed by WT p53 through binding at the distal NEK2 promoter
Summary
NEK2 is a mammalian kinase that promotes centrosome separation during the cell cycle. Microarray profiling of transcripts from cells treated with DNA-demethylating agents, such as 5aza-dC ( known as decitabine), is often used to identify epigenetically regulated genes and has identified many derepressed genes. These global studies often report genes that are repressed by treatment; this repression is often regarded as nonspecific. We show that modulation of p53 levels affects both nucleosome positioning and the DNA methylation status of the distal promoter where the p53-binding site is located These results identify a novel p53-repressed target gene but provide evidence that identification of partially methylated regions can reveal important transcriptional regulatory elements in human cells
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