Abstract
Methyl-CpG binding domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.
Highlights
Aberrant hypermethylation of promoter CpG islands and the resulting transcriptional silencing is nowadays a widely accepted mechanism of inactivation of tumor suppressor genes in cancer that actively contributes to tumorigenesis [1,2,3,4]
The remaining members [methyl-CpG binding domain (MBD) 1, MBD2, MBD3 and MBD4] were identified from database searches done with the minimum portion able to recognize a single methylated CpG site, the MBD [10]
MBDs are differentially expressed in human cancer cell lines
Summary
Aberrant hypermethylation of promoter CpG islands and the resulting transcriptional silencing is nowadays a widely accepted mechanism of inactivation of tumor suppressor genes in cancer that actively contributes to tumorigenesis [1,2,3,4]. The criteria used to establish a positive result of MBD association was based on the following: first, for each promoter, conditions of amplification in the linear range were optimized by PCR amplifying serial dilutions of total DNA collected after sonication (input fraction); second, negative controls (no antibody) and input were included for each PCR experiment; and third, for each experiment (cell line and promoter), a minimum of three independent ChIP experiments were done. To identify statistical differences for the levels of MBD binding among several cell lines or among several genes, we used the Kruskal-Wallis nonparametric test.
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