hMLH1 promoter methylation and silencing in primary endometrial cancers are associated with specific alterations in MBDs occupancy and histone modifications

Abstract

Objective: To investigate the relationship between hMLH1 promoter methylation and changes in chromatin composition. To study how the occupancy of methyl CpG binding domain proteins (MBDs) and histone acetylation/methylation in hMLH1 promoter may participate in hMLH1 silencing.

Methods: 64 endometrial cancer samples were screened for hMLH1 mRNA expression. hMLH1 promoter methylation status was confirmed by methylation-specific PCR in cancers with high and low levels of hMLH1 expression. Chromatin immunoprecipitation was performed to compare the MBD occupancy and histone modifications between the methylated/silenced and unmethylated/active hMLH1 genes in multiple primary endometrial cancers.

Results: We demonstrated that MeCP2, MBD1 and MBD2, but not MBD3 and MBD4, specifically bind to methylated hMLH1 promoters. Hyperacetylated histones H3 and H4 were found to be associated with the unmethylated and transcriptionally active hMLH1 promoters. While H3 lysine-4 methylation was present in unmethylated hMLH1 promoters, H3 lysine-9 methylation was found exclusively in methylated promoters. Western blot analysis showed that similar global levels of MBDs and histones were present in the two cancer groups with high and low hMLH1 expression.

Conclusions: A distinct combination of MBDs and histone modification is associated with the silencing of the hMLH1 gene. The changes in hMLH1 chromatin composition are closely related to methylation status of hMLH1 promoters. These changes are not accounted by the global expression levels of MBDs and histones in endometrial cancers.

Fig. 1

hMLH1 levels in endometrial cancers. Sixty four endometrial cancer samples were screened for hMLH1 mRNA expression. The histological grade of the cancers was indicated for each sample. Real-time PCR was performed on hMLH1 and GAPDH as described in Materials and methods. Cancer samples with the highest (H) and lowest (L) hMLH1 mRNA levels were selected for further analysis. Note that samples marked by “*” failed to provide sufficient tissues and were excluded for further analysis.

Fig. 2

Confirmation of hMLH1 methylation. DNA methylation in the selected samples was examined by methylation-specific PCR. All the 7 cancers (H1–H7) expressing the highest levels of hMLH1 contain an unmethylated hMLH1 gene. Cancers with the lowest hMLH1 mRNA were found to contain completely methylated (L1–L7), partially methylated (marked by “*”) or unmethylated (marked by “**”) promoters.

Fig. 3

MBDs occupancy of the hMLH1 promoters. Chromatin immunoprecipitation experiments were performed on the cancer samples with unmethylated (H1–H7) or methylated (L1–L7) hMLH1 promoters. The left panels present typical ChIP results of three or more repeated experiments. The right panels show the results from densitometry analysis on average levels of MDB binding signals from multiple cancer samples. Significantly higher (P ≤ 0.05 as indicated by asterisk) average levels of occupancy by MeCP2, MBD1 and MBD2 were found in hMLH1-methylated cancers. Minimal binding of MDB3 and MBD4 was observed in both groups of cancer samples.

Fig. 4

ChIP studies on covalent histone modification in hMLH1 promoters. The left images are example results of three or more repeated experiments. The results of densitometry analysis on multiple patient samples are shown in the right panels. Statistical analysis indicates that similar levels of hMLH1 promoter DNA were recovered by antibodies against total histone H3 or H4 from the two cancer groups containing either unmethylated or methylated hMLH1 promoters. However, significantly higher (P ≤ 0.05 as indicted by asterisks) levels of acetylated histone H3 (Ac H3) and H4 (Ac H4), and K4 methylation in H3 (K4M H3), were found in unmethylated hMLH1 promoters. In contrast, decreased levels of K9 methylation (K9M H3) were found to be associated with the methylated hMLH1 promoters.

Fig. 5

Total cellular levels of MBD and histone proteins. Western blot analysis was performed on MBDs, total histone H3 and H4 and acetylated H3 and H4. The left panels are some example results representing at least three repeated Western blot experiments. The right panels show the result of densitometry analysis on multiple cancer samples. No statistically significant difference in average concentrations of MBDs and histones was found between cancer groups containing unmethylated and methylated MLH1 genes. For each blot, the β-actin protein levels were also measured for loading controls.