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Molecular and Cellular Biology, June 2003, p. 4056-4065, Vol. 23, No. 12
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.12.4056-4065.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Methylation of Adjacent CpG Sites Affects Sp1/Sp3 Binding and Activity in the p21Cip1 Promoter

Wei-Guo Zhu,1 Kanur Srinivasan,1 Zunyan Dai,2,3 Wenrui Duan,1 Lawrence J. Druhan,1 Haiming Ding,1 Lisa Yee,4 Miguel A. Villalona-Calero,1 Christoph Plass,3 and Gregory A. Otterson1*

Division of Hematology/Oncology, Department of Internal Medicine,1 Department of Pathology,2 Division of Human Cancer Genetics, Department of Molecular Virology, Immunology, and Medical Genetics,3 Department of Surgery, The Ohio State University-Comprehensive Cancer Center, Columbus, Ohio 432104

Received 10 January 2003/ Returned for modification 18 February 2003/ Accepted 21 March 2003

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 p21Cip1 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 p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. By analyzing luciferase activity of transfected p21Cip1 promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21Cip1 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 p21Cip1 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 p21Cip1 expression in response to depsipeptide treatment.

* Corresponding author. Mailing address: Division of Hematology and Oncology, Department of Internal Medicine, The Ohio State University, Room B415, Starling Loving Hall, 320 West 10th Ave., Columbus, OH 43210-1240. Phone: (614) 293-6786. Fax: (614) 293-7529. E-mail: otterson-1{at}medctr.osu.edu.

Molecular and Cellular Biology, June 2003, p. 4056-4065, Vol. 23, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.12.4056-4065.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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