title: Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.
abstract: The enhancer of zeste homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2's role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and Apc(Min/+) mice were infected with CR (10(8) CFU); BLT1(-/-)Apc(Min/+) mice, azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated mice and de-identified human adenocarcinoma samples were the models of colon cancer. Following infection with wild-type but not mutant CR, elevated EZH2 levels in the crypt at days 6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3's occupancy on WIF1 (Wnt inhibitory factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via small interfering RNA or EZH2-inhibitor deazaneplanocin A (Dznep) either alone or in combination with histone deacetylase inhibitor suberoylanilide hydroxamic acid, WIF1 promoter activity increased significantly while the overexpression of EZH2 attenuated WIF1 reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels, whereas H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, although β-catenin levels were lower in EZH2-knocked down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days 6 and 12 post infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected Apc(Min/+) mice paralleled changes recorded in BLT1(-/-)Apc(Min/+), AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/β-catenin-dependent crypt hyperplasia in response to CR infection.
authors.affiliation: Departments of Molecular and Integrative Physiology and Family Medicine Research Division, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: B C
authors.initials: BC
authors.lastname: Roy
authors.affiliation: Departments of Molecular and Integrative Physiology and Family Medicine Research Division, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: D
authors.initials: D
authors.lastname: Subramaniam
authors.affiliation: Departments of Molecular and Integrative Physiology and Family Medicine Research Division, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: I
authors.initials: I
authors.lastname: Ahmed
authors.affiliation: James Graham Brown Cancer Center and Department of Microbiology and Immunology, University of Louisville, Louisville, KY, USA.
authors.forename: V R
authors.initials: VR
authors.lastname: Jala
authors.affiliation: Departments of Molecular and Integrative Physiology and Family Medicine Research Division, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Hester
authors.affiliation: Departments of Molecular and Integrative Physiology and Family Medicine Research Division, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Greiner
authors.affiliation: James Graham Brown Cancer Center and Department of Microbiology and Immunology, University of Louisville, Louisville, KY, USA.
authors.forename: B
authors.initials: B
authors.lastname: Haribabu
authors.affiliation: Departments of Molecular and Integrative Physiology and Family Medicine Research Division, University of Kansas Medical Center, Kansas City, KS, USA.|University of Kansas Cancer Center, Kansas City, KS, USA.
authors.forename: S
authors.initials: S
authors.lastname: Anant
authors.affiliation: Departments of Molecular and Integrative Physiology and Family Medicine Research Division, University of Kansas Medical Center, Kansas City, KS, USA.|University of Kansas Cancer Center, Kansas City, KS, USA.
authors.forename: S
authors.initials: S
authors.lastname: Umar
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D044082:Citrobacter rodentium; D000071221:Enhancer of Zeste Homolog 2 Protein; D004756:Enterobacteriaceae Infections / Q000378:metabolism*; D044127:Epigenesis, Genetic; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D045325:HCT116 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000502:physiology; D063151:Polycomb Repressive Complex 2 / Q000502:physiology*; D011401:Promoter Regions, Genetic; D018102:Receptors, Leukotriene B4 / Q000502:physiology; D014158:Transcription, Genetic; D051176:beta Catenin / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C495270:CTNNB1 protein, human; D016326:Extracellular Matrix Proteins; D036341:Intercellular Signaling Peptides and Proteins; C503704:Ltb4r1 protein, mouse; D035683:MicroRNAs; D018102:Receptors, Leukotriene B4; C118544:Wif1 protein, mouse; D051176:beta Catenin; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2