Abstract
We have previously demonstrated that the expression of human ribosomal RNA genes (rDNA) in normal and cancer cells is differentially regulated by methylation of the promoter CpG islands. Furthermore, we showed that the methyl CpG-binding protein MBD2 plays a selective role in the methylation-mediated block in rDNA expression. Here, we analyzed the role of three functional mammalian DNA methyltransferases (DNMTs) in regulating the rDNA promoters activity. Immunofluorescence analysis and biochemical fractionation showed that all three DNMTs (DNMT1, DNMT3A, and DNMT3B) are associated with the inactive rDNA in the nucleolus. Although DNMTs associate with both methylated and unmethylated rDNA promoters, DNMT1 preferentially associates with the methylated genes. The rDNA primary transcript level was significantly elevated in DNMT1-/- or DNMT3B-/- human colon carcinoma (HCT116) cells. Southern blot analysis demonstrated a moderate level of rDNA promoter hypomethylation in DNMT1-/- cells and a dramatic loss of rDNA promoter methylation in double knockout cells. Transient overexpression of DNMT1 or DNMT3B suppressed the luciferase expression from both methylated and unmethylated pHrD-IRES-Luc, a reporter plasmid where the rDNA promoter drives luciferase expression. DNMT1-mediated suppression of the unmethylated promoter involves de novo methylation of the promoter, whereas histone deacetylase 2 cooperates with DNMT1 to inhibit the methylated rDNA promoter. Unlike DNMT1, both the wild type and catalytically inactive DNMT3B mutant can suppress rDNA promoter irrespective of its methylation status. DNMT3B-mediated suppression of the rDNA promoter also involves histone deacetylation. Treatment of HCT116 cells with Decitabine (a DNMT inhibitor) or trichostatin A (a histone deacetylase inhibitor) up-regulated endogenous rDNA expression. These inhibitors synergistically activated methylated pHrD-IRES-Luc, whereas they exhibited additive effects on the unmethylated promoter. These results demonstrate localization of DNMTs with the inactive rDNA in the nucleolus, the specific role of DNMT1 and DNMT3B in rDNA expression and the differential regulation of rDNA expression from the methylated and unmethylated rDNA promoters.
Highlights
CpG-binding protein MBD2 plays a selective role in the II).4 Recent studies from a few laboratories including our own methylation-mediated block in rDNA expression
Western blot analysis confirmed the absence of the respective proteins in null cells, whereas the third functional enzyme DNMT3A was detectable in all three cell lines (Fig. 1A)
We addressed whether any alteration in methylation status of the rDNA promoter occurred in DNMT1 or DNMT3B null cells
Summary
The factors involved in the epigenetic regulation of pol II-directed genes have been well studied, such an approach has not been fully used in deciphering their role in pol I-directed ribosomal gene expression. The existence of CGI in the human rDNA promoter compared with only a few CpGs in the rodents [9], in the mouse promoter [16], suggests distinct mechanism of transcriptional regulation in the two systems. Three distinct DNMTs, namely DNMT1, -3A, and -3B encoded by different genes direct DNA methylation in mammalian cells [19, 20]. Recent studies have associated DNMT1 with methylation of unmethylated human CGIs in cancer cells [21]. Large NH2-terminal domains of DNMTs can mediate transcriptional repression of genes independent of their methyltransferase activity (24 –27). We focused on the role of DNA methyltransferases in regulating rDNA promoter activity in both the methylated as well as the unmethylated state, and the involvement of the transcriptional repressor domains of these enzymes in suppressing rDNA promoter
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