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Papers In Press, published online ahead of print November 10, 2003
Molecular and Cellular Biochemistry, Ohio State University, College of Medicine, Columbus, OH. 43210
Corresponding Author: jacob.42{at}osu.edu
The methylation status of CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared to matching normal tissues, which was consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, we constructed pHrD-IRES-Luc (human rRNA promoter-luciferase reporter). In this plasmid, Kozak sequence of pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared to HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complimentary strands located in the promoter (-9, -102, -345 with respect to +1 site) inhibited luciferase activity while symmetrical methylation of a CpG in the transcribed region (+152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl CpG binding proteins, MBD1, MBD2, MBD3 and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylation-mediated inhibition of ribosomal RNA gene expression.
J. Biol. Chem, 10.1074/jbc.M309393200
Submitted on August 25, 2003
Revised on November 10, 2003
Accepted on November 10, 2003
Role of human Ribosomal RNA (rRNA) promoter methylation and of methyl CpG binding protein MBD2 in the suppression of rRNA gene expression
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