Promoter Methylation Inhibits APC Gene Expression by Causing Changes in Chromatin Conformation and Interfering with the Binding of Transcription Factor CCAAT-Binding Factor

Abstract

As an important regulator in Wnt-signaling pathway, the APC gene is involved in apoptosis and cell cycle arrest. The loss of APC function is observed in most familial adenomatous polyposis-associated and sporadic colorectal cancer. APC gene is frequently inactivated by DNA mutations. However, hypermethylation in APC gene promoter was also observed in different cancers. In this study, by analyzing the methylation status of APC promoter in 22 colorectal cancer cell lines with different APC expression levels, we identified Regions A and B in the promoter, where the methylation of CpG sites was invariably correlated with the loss of gene expression. By nuclease accessibility assay, we also observed a correlation between the closed chromatin conformation in APC promoter and loss of gene expression. When the nonexpressing cell lines were treated with a DNA methyltransferase inhibitor, 5-Aza-2-Deoxycytidine, the APC expression in these cells was induced, CpG sites were demethylated, and closed chromatin conformation was opened. However, when these cell lines were treated with a histone deacetylase inhibitor, Trichostatin A, no significant changes in APC expression, methylation status, and chromatin conformation were observed. Using transient transfection assay, a CCAAT box located in Region B was identified, which was involved in up-regulation of APC expression. Methylation of CpG sites around the CCAAT box resulted in a significant inhibition in the gene expression. The specific binding of a transcription factor CCAAT-binding factor (CBF) to the CCAAT box was determined by electrophoretic mobility shift analysis. The binding was inhibited after CpG sites close to the CCAAT box were methylated, indicating that DNA methylation can silence gene expression through interfering with the binding of transcription factors to the promoter. The biological function of CBF in APC gene regulation was further indicated by the decrease of luciferase activities in cells cotransfected with a plasmid carrying APC promoter/luciferase gene and a plasmid expressing dominant negative CBF mutant. In summary, methylation of CpG sites around CCAAT box in APC promoter inhibits the gene expression by changing the chromatin conformation and interfering with the binding of transcription factor CBF to CCAAT box.

Involvement of adenomatous polyposis coli (APC) gene in testicular yolk sac tumor of infants

“…Such a high frequency of methylation seems to be concordant with the result previously reported [10]. Promoter methylation of the APC gene has been detected in various human cancers [15] and demonstrated to be one of the mechanisms of gene inactivation [15,16]. Among 7 YSTs showing APC promoter methylation, at least 3 YSTs also harbored LOH at 5q21.…”

Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions

“…Cystatin M contains a large CpG island that flanks the transcription start site and spans the proximal promoter and exon 1 regions. CpG islands represent well-characterized sites for DNA methylation, impairing transcription activation related to regional or specific methylation events (Deng et al, 2004;Douglas et al, 2004;Prendergast and Ziff, 1991). In a previous microarray-based analysis of differential gene expression in MCF-7 breast cancer cells, we identified CST6 as a methylation-sensitive gene, and demonstrated an inverse relationship between CST6 mRNA expression and methylation of the proximal promoter region of its CpG island (Rivenbark et al, 2006b).…”

Correlation between the methylation of APC gene promoter and colon cancer

“…Deng et al (20) revealed that the abnormal APC gene methylation was correlated well with the deactivation of APC protein functions. In the present study, APC gene was found with high methylation in the tumor tissues of colon cancer patients.…”