Abstract
The methylation status of the 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 with 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 the 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 an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with 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 complementary strands located in the promoter (-9, -102, -347 with respect to the +1 site) inhibited luciferase activity, whereas 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.
Highlights
The transcriptional regulation of ribosomal RNA genes is a control point in the complex process of ribosome biogenesis
We explored the methylation status of the CpG island that spans the ribosomal RNA (rRNA) promoter in human primary hepatocellular carcinomas and corresponding normal liver tissues and elucidated a potential molecular mechanism for the methylation-mediated alteration in rRNA promoter activity in human cells
Most of the CpG islands of housekeeping genes transcribed by RNA polymerase II are located in the promoter and exon 1 regions that are methylation-free in normal somatic cells
Summary
The transcriptional regulation of ribosomal RNA (rRNA) genes is a control point in the complex process of ribosome biogenesis. Whereas the transcription machineries of RNA polymerase II (pol II) and RNA polymerase III (pol III) are often compatible with genes from widely different species, RNA polymerase I (pol I) exhibits stringent [7] but not absolute [8] species specificity This could result from very little sequence similarity between rRNA promoters from different species despite the general conservation of functional transactivation domains of the transcription factors from mice to humans [6, 9]. Aberrations in DNA methylation cause activation of oncogenes, genomic instability, and silencing of a variety of tumor suppressor genes (e.g. P16, P15, P21, E-CAD, VHL, etc.), leading to uncontrolled cell proliferation We explored the methylation status of the CpG island that spans the rRNA promoter in human primary hepatocellular carcinomas and corresponding normal liver tissues and elucidated a potential molecular mechanism for the methylation-mediated alteration in rRNA promoter activity in human cells
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