Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer

Abstract

9Biomolecular Engineering Research Institute (BERI), Japan O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic and mutagenic O6-alkylguanine produced by alkylating agents such as chemotherapeutic agents and mutagens. Recent studies have shown that in a subset of tumors, MGMT expression is inversely linked to hypermethylation of the CpG island in the promoter region; however, how the epigenetic silencing mechanism works, as it relates to hypermethylation, was still unclear. To understand the mechanism, we examined the detailed methylation status of the whole island with bisulfitesequencing in 19 MGMT non-expressed cancer cell lines. We found two highly methylated regions in the island. One was upstream of exon 1, including minimal promoter, and the other was downstream, including enhancer. Reporter gene assay showed that methylation of both the upstream and downstream regions suppressed luciferase activity drastically. Chromatin immunoprecipitation assay revealed that histone H3 lysine 9 was hypermethylated throughout the island in the MGMT negative line, whereas acetylation on H3 and H4 and methylation on H3 lysine 4 were at significantly high levels outside the minimal promoter in the MGMT-expressed line. Furthermore, MeCP2 preferentially bound to the CpG-methylated island in the MGMT negative line. Given these results, we propose a model for gene silencing of MGMT that is dependent on the epigenetic state in cancer.

PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation

“…To determine whether hypermethylation of CpG sites is associated with transcriptional silencing of the PRTFDC1 gene, we tested the promoter activity of this CpG island in OSCC cells (NA and HSC-2) using a 891-bp fragment (Fragment 1) covering the entire CpG island and 482-bp (Fragment 2) and 409-bp (Fragment 3) fragments covering uncommonly and commonly methylated regions, respectively, in cell lines without expression of PRTFDC1 (Figures 3a and b). We observed a remarkable increase in transcriptional activity of the reporter plasmid containing Fragments 1 and 3 compared with the mock reporter plasmid and reporter plasmid containing Fragment 2 in the plasmidcontaining cell lines (Figure 3b), suggesting that the CpG island around exon 1 of PRTFDC1, especially the region commonly methylated in PRTFDC1-nonexpressing cell lines, contains promoter activity, although few studies, including ours, have shown that promoter activity can be observed in fragments, especially CpG islands, not containing a 5 0 sequence around transcriptional start sites (Kolb 2003;Nakagawachi et al, 2003;Sonoda et al, 2004;Misawa et al, 2005).…”

Aberrant CpG island hypermethylation of multiple genes in colorectal neoplasia

“…A recent study performing a functional analysis of promoter activity has demonstrated that in vitro hypermethylation of promoter CpG island of MGMT was able to suppress gene transcription. 25 In conclusion, methylation analysis of a panel of genes provided gene-specific methylation patterns during colorectal carcinogenesis, and methylation profiles specific for colon adenoma and colorectal cancer. Our data indicate that aberrant CpG island hypermethylation occurs early and accumulates during multistep colorectal carcinogenesis, that a temporal order exist in the methylation of tumorrelated genes, and that CpG island hypermethylation is not a dichotomatous trait, challenging the concept of CIMP.…”

MGMT promoter methylation in malignant gliomas: ready for personalized medicine?

“…MethylCpG-binding proteins, such as methyl-CpG-binding protein 2 (MeCP2) and methyl-CpGbinding domain protein 2 (MBD2), bind to aberrantly methylated sequences, leading to alterations of chromatin structure and preventing binding of transcription factors, thereby silencing the gene (Figures 1 and 2a). 16 Some studies have provided insight into the relationship between gene expression and the patterns and localization of dense CpG methylation in the MGMT promoter. 16,17 Two regions that are prone to high levels of methylation have been identified, of which the region comprising the enhancer element seems to be more critical for the loss of MGMT gene expression upon methylation, on the basis of luciferase reporter assays interrogating different regions of the methylated promoter.…”

“…16 Some studies have provided insight into the relationship between gene expression and the patterns and localization of dense CpG methylation in the MGMT promoter. 16,17 Two regions that are prone to high levels of methylation have been identified, of which the region comprising the enhancer element seems to be more critical for the loss of MGMT gene expression upon methylation, on the basis of luciferase reporter assays interrogating different regions of the methylated promoter. 16,17 Hence, most methylation-specific tests are designed to interrogate this region ( Figure 2b).…”