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.

ACKNOWLEDGMENTS

We thank Y. Xiong, XH. Pei, T. Sakai, and KK Chan for their materials that are used in this study. We also thank Z. Hu, Y. Zhang, and J. Xiao for technical help. We also express appreciation for helpful discussions with Gustavo Leone.

This research was supported by grant P30 CA16058 from the National Cancer Institute, Bethesda, Md., and by a Translational Research Grant from the V Foundation.