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 p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C3W). 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. Although a strong correlation between promoter methylation and gene silencing has been extensively demonstrated (5,24,35), the molecular mechanisms of this methylation-modulated gene inactivation remains unclear. Two hypotheses have been proposed to explain transcriptional inactivation from promoter methylation. One of them is based on the finding that methyl-CpG-binding proteins (MBPs), such as MeCP2, specifically bind to symmetrically methylated DNA through a methyl-CpG-binding domain (11,41). MBPs then recruit transcriptional repressors such as Sin3, NuRD, and histone deacetylases (HDACs) through its transcriptional-repression domain (25,32,54). Since Sin3 and HDACs are known transcriptional repressors (2, 50), methylated DNA may repress gene expression indirectly through MeCP2 and other MBPs. In addition, deacetylation of histones results in a net increase in positively charged lysines and arginines at the N-terminal tail of the histones (18, 21), thus inducing a tighter noncovalent linkage between the positively charged histones and the negatively charged DNA (3, 47). Consequently, transcription factors have difficulty accessing their DNA-binding sites (4, 29, 47), with a reduction or silencing of gene transcription. This hypothesis, based on the interaction between DNA methylation and histone acetylation status, has been extensively supported by accumulated experimental evidence (7,16,37,40). For example, trichostatin A (TSA), an inhibitor of HDAC, induces a robust reexpression of silenced genes when used with minimal doses of the demethylating agent, 5-aza-2Ј-deoxycytidine (5-azaCdR), although TSA or 5-aza-CdR alone do not lead to gene reexpression (7). Our previous data also show a link between histone acetylation status and DNA methylation, such that 5-aza-CdR significantly enhances acetylation of histones H3 and H4 induced by a HDAC inhibitor, depsipeptide. Related to this, depsipeptide-induced apoptosis is dramatically increased in cells pretreated with 5-aza-CdR (56). In addition, p19 INK4D expression is greatly enhanced when human lung cancer cells are treated with depsipeptide and 5-aza-CdR together compared to treatment with each agent alone (55). These studies support the notion that methylation and histone acetylation work cooperatively to influence gene expression and other biological processes.A...

Multiple alternate p21 transcripts are regulated by p53 in human cells

“…The third possibility is that transcripts from one promoter may play a compensatory role in situations where the other promoter is silenced. There is some evidence of the main promoter being methylated in some instances of rhabdomyosarcoma tumors and lung cancer cells (Chen et al, 2000;Claus and Lubbert, 2003;Zhu et al, 2003). One way to find out the importance of the different transcripts is to selectively knockdown the alternate or classical transcripts and see if the cell can cope with the loss under different circumstances where p21 is required.…”

Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer

“…SP1 is involved in the control of cell growth and morphogenetic pathways (Black et al, 1999). Methylation outside of the consensus sequence for SP1 induced a significant decrease in SP1 binding, leading to a reduced target gene expression (Zhu et al, 2003). AP-2 regulates genes involved in various biological functions, including p21 WAF/CIP (Zeng et al, 1997), transforming growth factor-a (Wang et al, 1997), estrogen receptor (McPherson et al, 1997), and tyrosine kinase receptor gene c-KIT (Huang et al, 1998).…”

Epigenetic Up-Regulation of Leukemia Inhibitory Factor (LIF) Gene During the Progression to Breast Cancer

“…Methylation of CpG dinucleotides located within DNA recognition elements may interfere with the binding of transcription factors, which inhibits transcription (Siegfried et al, 1999). Methylated CpG dinucleotides interfere with SP1 binding and further the reduced binding of Sp1 may be involved in suppression of transcriptional activity of the genes such as AQP5, and CLDN4 (Clark et al, 1997;Honda et al, 2006;Kudo, 1998;Mancini et al, 1999;Motegi et al, 2005;Zhu et al, 2003). In agreement with these findings, we observed that the normal breast epithelial cells MCF-10A had a heavily methylated potential SP1-binding site, the ninth CG of the LIF promoter, while benign and carcinoma MCF10 variants did not.…”

“…The effect of methylation patterns of CpG sites on Sp1/Sp3 binding and activity in the p21 promoter was previously demonstrated (Clark et al, 1997;Zhu et al, 2003). We tested for the involvement of the transcription factor Sp1 in upregulation of LIF via binding to the potential consensus sequence containing the hypomethylated CpG pairs.…”

“…The Sp1 transcription factor has CG rich binding sites, indicating that Sp1 sites may be affected by DNA methylation both directly and indirectly. The methylation of adjacent CpG sites has been reported to affect Sp1/Sp3 binding and transcriptional activity in the p21 promoter (Clark et al, 1997;Zhu et al, 2003).…”