title: Local depletion of DNA methylation identifies a repressive p53 regulatory region in the NEK2 promoter.
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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Florida Health Cancer Center, University of Florida College of Medicine, Gainesville, Florida 32610.
authors.forename: Nancy H
authors.initials: NH
authors.lastname: Nabilsi
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Ryder
authors.forename: Ashley C
authors.initials: AC
authors.lastname: Peraza-Penton
authors.forename: Rosha
authors.initials: R
authors.lastname: Poudyal
authors.forename: David S
authors.initials: DS
authors.lastname: Loose
authors.forename: Michael P
authors.initials: MP
authors.lastname: Kladde
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D000072379:NIMA-Related Kinases; D009707:Nucleosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D009707:Nucleosomes; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D000077209:Decitabine; D004247:DNA; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D001374:Azacitidine
keywords: Chromatin Regulation; Chromatin Structure; DNA Methylation; Epigenetics; MAPit; NEK2; Nucleosome; p53