title: Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter.
abstract: DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21(Cip1) expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C-->W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21(Cip1) expression because the promoter of p21(Cip1) in these cells is hypermethylated. By analyzing luciferase activity of transfected p21(Cip1) promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21(Cip1) expression. We hypothesize that hypermethylation may interfere with Sp1/Sp3 binding. By using an electrophoretic mobility shift assay, we show that, although methylation within the consensus Sp1-binding site did not reduce Sp1/Sp3 binding, methylation outside of the consensus Sp1 element induced a significant decrease in Sp1/Sp3 binding. Depsipeptide induced p21(Cip1) expression was reconstituted when cells were pretreated with 5-aza-2'-deoxycytidine. Our data suggest, for the first time, that hypermethylation around the consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, therefore leading to a reduced p21(Cip1) expression in response to depsipeptide treatment.
authors.affiliation: Department of Internal Medicine. Department of Pathology, The Ohio State University-Comprehensive Cancer Center, Columbus, Ohio 43210, USA.
authors.forename: Wei-Guo
authors.initials: WG
authors.lastname: Zhu
authors.forename: Kanur
authors.initials: K
authors.lastname: Srinivasan
authors.forename: Zunyan
authors.initials: Z
authors.lastname: Dai
authors.forename: Wenrui
authors.initials: W
authors.lastname: Duan
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Druhan
authors.forename: Haiming
authors.initials: H
authors.lastname: Ding
authors.forename: Lisa
authors.initials: L
authors.lastname: Yee
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Villalona-Calero
authors.forename: Christoph
authors.initials: C
authors.lastname: Plass
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Otterson
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003062:Codon; D018899:CpG Islands; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D019175:DNA Methylation; D047630:Depsipeptides; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000166:cytology; D008745:Methylation; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010456:Peptides, Cyclic / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012978:Sodium Oxybate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: C493956:CDKN1A protein, human; D003062:Codon; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D047630:Depsipeptides; D010455:Peptides; D010456:Peptides, Cyclic; D005047:Etoposide; D012978:Sodium Oxybate; C087123:romidepsin; D008156:Luciferases