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J. Biol. Chem., Vol. 279, Issue 8, 6783-6793, February 20, 2004

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Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression^*

Kalpana Ghoshal¶, Sarmila Majumder, Jharna Datta, Tasneem Motiwala, Shoumei Bai, Sudarshana M. Sharma, Wendy Frankel||, and Samson T. Jacob**

From the Department of Molecular and Cellular Biochemistry and ^||Department of Pathology, College of Medicine, Ohio State University, Columbus, Ohio 43210

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.

Received for publication, August 25, 2003 , and in revised form, October 22, 2003.

^* This work was supported, in part, by National Institutes of Health Grants ES 10874, CA 81024, and CA 86978. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

¶ To whom correspondence may be addressed. Tel.: 614-292-8865; Fax: 614-688-5600; E-mail: ghoshal.1{at}osu.edu. ** To whom correspondence may be addressed. Tel.: 614-688-5494; Fax: 614-688-5600; E-mail: jacob.42{at}osu.edu.

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