Relationship Of Histone Modifications And The Silencing Of Tumor-related Genes

IntroduceGastric cancer is one of the most common malignancies and a major cause of cancer-related death worldwide.The epigenetic alterations are involved in the carcinogenesis of this disease.Methylation of the CpG islands of some tumor related genes,such as p16,MLH1,MGMT and E-cadherin,leading to their transcriptional silencing is a highly consistent feature of carcinogenesis of gastric cancer.For example, hypermethylation induces the silencing of p16,a cyclin-dependent kinase inhibitor gene,leads to disruption of cell cycle regulation,provides a growth advantage to affected cells,and is involved in gastric cancer.A mismatch repair gene,MLH1,is often silenced with aberrant CpG island hypermethylation in gastric cancer.The silencing of O6-alkylguanine-DNA alkyltransferase(MGMT),a DNA repair gene,is also related to DNA methylation in gastric cancer.Hypermethylation-induced the silencing of E-cadherin is also involved in gastric cancer.Histone modification is closely associated with DNA methylation status,and is also important for gene regulation.Acetylation of histone H3 lysine 9 and methylation of histone H3 lysine 4 are associated with active gene transcription,and methylation of histone H3 lysine 9 is associated with gene repression.Interaction between DNA methylation and various histone modifications is now being investigated.A study indicates that histone H3 lysine 9 methylation directly correlates with DNA methylation, but histone H3 lysine 9 acetylation inversely correlates with DNA methylation of some tumor related genes.In Neurospora and Arabidopsis,epigenetic evidence indicates that histone H3 lysine 9 methylation is a prerequisite for DNA methylation to occur.It appears that DNA and histone methylation likely have a mutually reinforcing relationship,and both are required for stable and long-term epigenetic silencing. DNA methyltransferases(DNMTs),especially DNMT1,play important roles in maintaining DNA methylation.5-aza-2'-deoxycytidine(5-Aza-dC),an inhibitor of DNMT1,forms irreversible covalent bonds with DNMT1 after its incorporation into DNA,and induces degradation of DNMT1,then results in hypomethylation.Histone deacetylation is mediated by HDACs(histone deacetylases).Specific inhibition of HDACs by their inhibitors,such as trichostatin A(TSA),leads to hyperacetylation of histones.We treated the gastric cancer cells with 5-Aza-dC and/or TSA to identify which could reactivate the silenced tumor-related genes and to understand the effects of DNA methylation and histone modification on tumor-related genes silencing in four gastric cancer cell lines.Material and MethodsFour cell lines derived from human gastric cancer,MKN-45,SGC-7901, BGC-823 and MGC-803,were cultured and given one of the following treatments.①5-Aza-dC(5μM).Medium containing 5-Aza-dC was changed every 24 h.②Trichostatin A(300 nM)was used for 24 h.③5-Aza-dC(5μM)was used for 48 h followed by Trichostatin A(300 nM)for an additional 24 h.We used chromatin immunoprecipitation(CHIP)assay to assess the status of histone acetylation and methylation in promoter regions of p16,MLH1,MGMT and E-cadherin genes in four gastric cancer cell lines.We used methylation-specific PCR (MSP)to evaluate the effect of 5-Aza-dC,TSA or their combination of treatment on each promoter 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.We used western blot to test the effect of 5-Aza-dC,TSA or their combination on the level of histone H3,methyl-histone H3 lysine 9, methyl-histone H3 lysine 4,acetyl-histone H3 lysine 9.ResultsMSP:5-Aza-dC and the combined 5-Aza-dC and TSA resulted in demethylation of silenced gene which was associated with DNA hypermethylation.In contrast,TSA alone did not affect the DNA methylation status.RT-PCR:Tumor related gene silencing is associated with DNA methylation. 5-Aza-dC alone reactivated expression of the silenced genes.TSA had no effect on gene expression.The combined treatment with 5-Aza-dC and TSA increased gene expression.ChIP:The results of ChIP studies were almost identical in different regions of each promoter,and the values for each gene were averaged to present the data.Histone H3 Lysine 9 methylation directly correlated with DNA methylation,but histone H3 Lysine 9 acetylation and histone H3 Lysine 4 methylation inversely correlated with DNA methylation of p16,MLH1,MGMT and E-cadherin gene.Gene silencing associated with DNA methylation was accompanied by histone H3 Lysine 9 hypermethylation, histone H3 Lysine 4 hypomethylation and histone H3 Lysine 9 hypoacetylation.Histone H3 Lysine 9 methylation:5-Aza-dC had effects on histone H3 Lysine 9 methylation at silenced loci,reducing histone H3 Lysine 9 methylation in the promoter with partial methylation or hypermethylation.TSA alone had no effect on histone H3 Lysine 9 methylation,irrespective of DNA methylation status.The combination of 5-Aza-dC and TSA had similar effects on histone H3 Lysine 9 methylation to that of 5-Aza-dC.Histone H3 Lysine 9 acetylation:5-Aza-dC increased histone H3 Lysine 9 acetylation at loci with DNA hypermethylation,but had no effect on loci with partial or no DNA methylation.Treatment with TSA alone had no effect on histone H3 Lysine 9 acetylation in the promoter showed partial DNA methylation,but slightly increased histone H3 Lysine 9 acetylation in the silenced promoter.However,the combination of 5-Aza-dC and TSA increased histone H3 Lysine 9 acetylation effectively at all loci irrespective of DNA methylation status.Histone H3 Lysine 4 methylation:TSA did not affect histone H3 Lysine 4 methylation.5-Aza-dC,or the combination of 5-Aza-dC and TSA,increased histone H3 Lysine 4 methylation at all silenced loci.Western blot:5-Aza-dC and TSA didn't affecd the level of histone H3,methyl-H3K9,methyl-H3K4,acetyl-H3K9.ConclusionHypermethylation of DNA in the promoter region is related to transcriptional silencing of tumor related genes.Histone H3 Lysine 9 methylation in different regions of the promoter studied correlates with DNA methylation status of tumor related genes in gastric cancer cells. Histone H3 Lysine 4 methylation and Histone H3 Lysine 9 acetylation in different regions of the promoter studied inversely correlates with DNA methylation status of tumor related genes in gastric cancer cells.Alteration of DNA methylation affects histone H3 Lysine 9 methylation and histone H3 Lysine 4 methylation.Histone H3 Lysine 9 methylation and histone H3 Lysine 4 methylation are a critical modification responsible for maintenance of DNA methylation -related gene silencing in gastric cancer cells.5-Aza-dC is able to cause a regional remodeling of chromatin,by diminishing histone H3 Lysine 9 methylation and augmenting histone H3 Lysine acetylation and histone H3 Lysine 4 methylation,independently of its effects on DNA methylation or gene expression.