Role of Nucleosomal Occupancy in the Epigenetic Silencing of the MLH1 CpG Island

Abstract

Epigenetic silencing of tumor suppressor genes is generally thought to involve DNA cytosine methylation, covalent modifications of histones, and chromatin compaction. Here, we show that silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells involves distinct changes in nucleosomal occupancy. Three nucleosomes, almost completely absent from the start sites in normal cells, are present on the methylated and silenced promoter, suggesting that epigenetic silencing may be accomplished by the stable placement of nucleosomes into previously vacant positions. Activation of the promoter by demethylation with 5-aza-2'-deoxycytidine involves nucleosome eviction. Epigenetic silencing of tumor suppressor genes may involve heritable changes in nucleosome occupancy enabled by cytosine methylation.

DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: Its possible involvement in the formation of epigenetic field defect

“…The other three genes, miR-137, miR-193a and miR-127, were expressed even in cell lines with complete methylation, and were unlikely to be silenced by promoter methylation in gastric cancers. Since methylation of putative promoter regions consistently represses transcription of their downstream genes, 29,30 the presence of the expression of the three genes in gastric cancer cell lines with complete methylation of their ''promoter'' CGI indicated that the three genes had additional or alternative promoters.…”

“…1a), whose methylation statuses are now known to be critical for induction of gene silencing. 29,30 qMSP was performed by real-time PCR using SYBR 1 Green I (BioWhittaker Molecular Applications, Rockland, ME) and an iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). Although the same primer set was used for qMSP, a specific annealing temperature in the presence of SYBR 1 Green I was redetermined using the fully methylated and unmethylated DNA.…”

Local Depletion of DNA Methylation Identifies a Repressive p53 Regulatory Region in the NEK2 Promoter

“…4B, middle). MLH1 is a target gene activated by p53, but it is unmethylated (25,35), and its expression is not significantly affected by 5aza-dC treatment of HCT116 cells (microarray signals: HCT116 and HCT116 ϩ 5aza-dC, 189 and 152 arbitrary units, respectively).…”

“…Both doxo and 5aza-dC have been shown to alter chromatin structure, including causing eviction or turnover of nucleosomes (35,38). To determine whether drug treatment causes changes in nucleosome occupancy at the NEK2 promoter, nuclei from HCT116 cells treated with or without 5aza-dC or doxo were digested with MNase and analyzed by qPCR using convergent primers spanning the p53-binding region (Fig.…”

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

“…10,[15][16][17] These studies implicated several factors in the propensity of given CpG sites to remethylate, including proximity to a transcription start site (TSS), occupancy of RNA Polymerase II (RNAP II), and the presence of certain histone modifications. 10,16 Given the widespread use of these agents and the promising clinical trials underway utilizing DAC or related compounds as a synergistic or sensitizing agents, [4][5][6] it is vital to understand both the extent and stability of DACinduced demethylation given its immediate potential to improve cancer treatment.…”