Aim: To identify the relationship between DNA hyper-methylation and histone modification at a hyperme-thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification.
Methods: We used chromatin immunoprecipitation (ChIP) assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and mutL homolog 1 (MLH1) genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation-specific PCR (MSP) to evaluate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC), trichostatin A (TSA) or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression.
Results: For the p16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza-dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected after TSA treatment, and increased moderately at the silenced loci after 5-Aza-dC treatment.
Conclusion: Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate with DNA methylation status of each gene in gastric cancer cells. Alteration of DNA methylation affects histone modification.