Nucleosome Positions and Differential Methylation Status of Various Regions within MLH1 CpG Island

Objective: To determine the relationship between nucleosome positions and formation of differential methylation of the reported region A, B, C, and D within the MLH1 CpG island. Methods: Methylation of the MLH1 promoter was analyzed by combined of bisulfite restriction assay. Chromatin of RKO and MGC803 cells were extracted and digested by MNase. Mono-nucleosomal DNA fragment was isolated and used as templates for detection of nucleosomal distribution by a battery of quantitative PCRs covering the full MLH1 promoter region. Results: The MLH1 was methylated in RKO and unmethylated in MGC803. At the region B, where methylation of CpG sites did not correlated with transcription of this gene well, qPCR product of the M-3 (-599nt similar to -475nt) fragment was amplified in both RKO and MGC803 cells. However, at the region C and D within the core promoter, where methylation of CpG sites correlated with loss of MLH1 transcription well, the M-7 (-257nt similar to -153nt)and M-8 (-189nt similar to -71nt) fragments were amplified remarkably only in RKO cells. Conclusion: Nucleosome may be the basic unit for both CpG methylation and methylation-related regulation of gene transcription. Methylation status of CpG sites within the same nucleosome may be homogeneous; between different nucleosomes, homogeneous or heterogeneous.