Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m309393200
ISSN1083-351X
AutoresKalpana Ghoshal, Sarmila Majumder, Jharna Datta, Tasneem Motiwala, Shoumei Bai, Sudarshana M. Sharma, Wendy L. Frankel, Samson T. Jacob,
Tópico(s)Cancer-related gene regulation
ResumoThe 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. 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. The transcriptional regulation of ribosomal RNA (rRNA) genes is a control point in the complex process of ribosome biogenesis. Diploid somatic cells harbor 300-400 copies of the rRNA genes that code for the most abundant cellular RNA. Only a fraction of these genes is transcribed, which depends on the growth stage of the cells and extracellular stimuli (for a review, see Refs. 1McStay B. Paule M. Schultz M.C. Willis I. Pikaard C.S. Gene Expr. 2002; 10: 263-269Crossref PubMed Scopus (5) Google Scholar and 2Leary D.J. Huang S. FEBS Lett. 2001; 509: 145-150Crossref PubMed Scopus (105) Google Scholar). In general, multiple copies of rRNA are found as repeated clusters, usually arranged in a head-to-tail fashion. The core promoter region spanning from -50 to +20 bp with respect to the initiation site is necessary and sufficient for the initiation of basal transcription in most species (for a review, see Refs. 3Jacob S.T. Biochem. J. 1995; 306: 617-626Crossref PubMed Scopus (100) Google Scholar, 4Hannan K.M. Hannan R.D. Rothblum L.I. Front. Biosci. 1998; 3: 376-398Crossref PubMed Google Scholar, 5Grummt I. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 109-154Crossref PubMed Scopus (205) Google Scholar, 6Paule M.R. White R.J. Nucleic Acids Res. 2000; 28: 1283-1298Crossref PubMed Google Scholar). Another key element is the upstream control element (UCE) 1The abbreviations used are: UCE, upstream control element; ETS1, external transcribed spacer region 1; pol I, II, and III, RNA polymerase I, II, and III, respectively; MBD, methyl-CpG-binding protein; DNMT, DNA methyltransferase; ChIP, chromatin immunoprecipitation; AdoMet, S-adenosyl-l-methionine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRES, internal ribosome entry site; TRITC, tetramethylrhodamine isothiocyanate; HCC, hepatocellular carcinoma. that extends 150-200 bp upstream of the transcription start site. Apart from core promoter and UCE, upstream enhancers and terminator also play a critical role in rRNA transcription. 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 (7Heix J. Grummt I. Curr. Opin. Genet. 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Methylation of DNA at the 5-position of cytosine of CpG base pairs, particularly in the promoter region is the predominant epigenetic modification of DNA in mammals and is known to suppress many RNA polymerase II (pol II) genes (10Bird A.P. Wolffe A.P. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar, 11Baylin S.B. Esteller M. Rountree M.R. Bachman K.E. Schuebel K. Herman J.G. Hum. Mol. Genet. 2001; 10: 687-692Crossref PubMed Google Scholar, 12Wade P.A. BioEssays. 2001; 23: 1131-1137Crossref PubMed Scopus (290) Google Scholar). DNA methylation is essential for development (13Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar, 14Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4739) Google Scholar). It regulates inactivation of X chromosome in females, genomic imprinting and suppresses spurious transcription from promoters of retroviruses and transposable elements integrated with the genome (15Robertson K.D. Wolffe A.P. Nat. Rev. Genet. 2000; 1: 11-19Crossref PubMed Scopus (883) Google Scholar). In addition, 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 (for a review, see Refs. 11Baylin S.B. Esteller M. Rountree M.R. Bachman K.E. Schuebel K. Herman J.G. Hum. Mol. Genet. 2001; 10: 687-692Crossref PubMed Google Scholar and 15Robertson K.D. Wolffe A.P. Nat. Rev. Genet. 2000; 1: 11-19Crossref PubMed Scopus (883) Google Scholar, 16Jones P.A. Baylin S.B. Nat. Rev. Genet. 2002; 3: 415-428Crossref PubMed Google Scholar, 17Plass C. Hum. Mol. Genet. 2002; 11: 2479-2488Crossref PubMed Scopus (118) Google Scholar). This modification is initiated by de novo DNA methyltransferases (DNMT3A and DNMT3B) and is propagated in successive cell divisions by the maintenance methyltransferase (DNMT1). DNMT1 transfers methyl group from S-adenosyl-l-methionine (AdoMet) to the newly replicated strand using hemimethylated strand as the template (for a review, see Refs. 18Bestor T.H. Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1611) Google Scholar and 19Jeltsch A. ChemBioChem. 2002; 3: 274-293Crossref PubMed Google Scholar). Aberrations in DNA methylation lead to a variety of diseases. For example, ICF (immunodeficiency, centromeric instability, and facial anomaly) syndrome is caused by mutations in the DNMT3B gene (20Hansen R.S. Wijmenga C. Luo P. Stanek A.M. Canfield T.K. Weemaes C.M. Gartler S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14412-14417Crossref PubMed Scopus (605) Google Scholar, 21Ehrlich M. Buchanan K.L. Tsien F. Jiang G. Sun B. Uicker W. Weemaes C.M. Smeets D. Sperling K. Belohradsky B.H. Tommerup N. Misek D.E. Rouillard J.M. Kuick R. Hanash S.M. Hum. Mol. Genet. 2001; 10: 2917-2931Crossref PubMed Scopus (104) Google Scholar, 22Xu G.L. Bestor T.H. Bourc'his D. Hsieh C.L. Tommerup N. Bugge M. Hulten M. Qu X. Russo J.J. Viegas-Pequignot E. Nature. 1999; 402: 187-191Crossref PubMed Scopus (537) Google Scholar). The drugs inhibiting DNMT, namely 5-deoxyazacytidine, 5-fluorocytidine, and zebularine, alone or in combination with histone deacetylase inhibitors are used clinically in certain types of cancer to activate methylated tumor suppressor or differentiation-inducing genes (23Zhu W.G. Otterson G.A. Curr. Med. Chem. Anti-cancer Agents. 2003; 3: 187-199Crossref PubMed Scopus (200) Google Scholar, 24Cheng J.C. Matsen C.B. Gonzales F.A. Ye W. Greer S. Marquez V.E. Jones P.A. Selker E.U. J. Natl. Cancer Inst. 2003; 95: 399-409Crossref PubMed Scopus (457) Google Scholar). DNA methylation can impede the transcriptional activity of a pol II gene (25Holliday R. Cancer Surv. 1996; 28: 103-115PubMed Google Scholar) directly by blocking the access of a transcription factor (e.g. AP-2, NF-κB, E2F, and c-MYC) to their cognate sites (19Jeltsch A. ChemBioChem. 2002; 3: 274-293Crossref PubMed Google Scholar). Most of the methylated promoters are, however, recognized by a group of proteins called methyl-CpG-binding proteins (MBDs) by virtue of their conserved methyl-CpG binding domain. Five such MBDs with highly conserved DNA binding domains have been identified (26Hendrich B. Bird A. Mol. Cell Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar). Among these proteins, MeCP2, MBD1, MBD2, and MBD4 can bind to methylated DNA. MBD4 is a uracil-DNA glycosylase involved in G-T mismatch repair (12Wade P.A. BioEssays. 2001; 23: 1131-1137Crossref PubMed Scopus (290) Google Scholar, 27Hendrich B. Hardeland U. Ng H.H. Jiricny J. Bird A. Nature. 1999; 401: 301-304Crossref PubMed Scopus (528) Google Scholar). MBDs repress transcription by recruiting a variety of proteins such as Sin3a, histone deacetylases, histone methyltransferases, and HP1α (heterochromatin protein 1α) as co-repressors (for reviews, see Refs. 10Bird A.P. Wolffe A.P. Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar, 14Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4739) Google Scholar, 28Hendrich B. Bird A. Curr. Top. Microbiol. Immunol. 2000; 249: 55-74PubMed Google Scholar, and 29Wade P.A. Oncogene. 2001; 20: 3166-3173Crossref PubMed Scopus (176) Google Scholar). Kaiso, a partner of β-catenin, lacking the signature methyl CpG binding domain can also bind to methyl-CpGs and repress methylated promoters (30Prokhortchouk A. Hendrich B. Jorgensen H. Ruzov A. Wilm M. Georgiev G. Bird A. Prokhortchouk E. Genes Dev. 2001; 15: 1613-1618Crossref PubMed Scopus (390) Google Scholar). A defect in the functions of these proteins also leads to various abnormalities. Rett syndrome, a prevalent X-linked neurological disorder among Caucasian females, is caused by dominant negative mutations in the MECP2 gene (31Nan X. Bird A. Brain Dev. 2001; 23 (suppl.): S32-S37Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Adult MBD1 knock-out mice, like MeCP2 null mice, also exhibit neurological abnormalities (32Zhao X. Ueba T. Christie B.R. Barkho B. McConnell M.J. Nakashima K. Lein E.S. Eadie B.D. Willhoite A.R. Muotri A.R. Summers R.G. Chun J. Lee K.F. Gage F.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6777-6782Crossref PubMed Scopus (313) Google Scholar), whereas MBD4 null mice are susceptible to cancer because of enhanced CpG to TpG mutation in their genome (33Millar C.B. Guy J. Sansom O.J. Selfridge J. MacDougall E. Hendrich B. Keightley P.D. Bishop S.M. Clarke A.R. Bird A. Science. 2002; 297: 403-405Crossref PubMed Scopus (261) Google Scholar). Most of the studies to date have focused on the up-regulation of rRNA promoter activity. Since methylation of DNA, particularly the promoter region, is known to silence many pol II genes (23Zhu W.G. Otterson G.A. Curr. Med. Chem. Anti-cancer Agents. 2003; 3: 187-199Crossref PubMed Scopus (200) Google Scholar, 24Cheng J.C. Matsen C.B. Gonzales F.A. Ye W. Greer S. Marquez V.E. Jones P.A. Selker E.U. J. Natl. Cancer Inst. 2003; 95: 399-409Crossref PubMed Scopus (457) Google Scholar, 25Holliday R. Cancer Surv. 1996; 28: 103-115PubMed Google Scholar, 26Hendrich B. Bird A. Mol. Cell Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1081) Google Scholar), it was of interest to investigate whether the methylation status of rRNA promoter modifies pol I-directed rRNA transcription. In the present study, 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. pIRES-Luc—The internal ribosome entry site (IRES; 16-518 bp) was amplified from pCITE vector (Novagen) using T3 (5′-AATTAACCCTCACTAAAGGG-3′) and T7 (5′-GTAATACGACTCACTATAGGGC-3′) oligonucleotides and digested with NcoI to generate the 503-bp IRES product. The Kozak sequence ((GCC)GCCRCCATGG, where R represents a purine) that directs translation of pol II-transcribed mRNAs was removed from pGL3-basic vector by digestion with NcoI and HindIII and was replaced by the amplified IRES fragment to generate the plasmid pIRES-Luc. The replacement of the Kozak sequence with IRES was essential to minimize pol II-directed spurious expression from human rRNA promoter-luciferase reporter constructs in transfected cells. Human rRNA-luciferase Vector (pHrD-IRES-Luc)—Human rRNA promoter spanning -410 to +314 bp (accession number K01105) with respect to the transcription initiation site was amplified from ∼2-kb fragments of HeLa genomic DNA digested with EcoRI. The following primer pairs with KpnI (forward) and BglII (reverse) restriction sites at the 5′ ends were used for PCR: forward, 5′-gtggtacccCGCGATCCTTTCTGGAGAGTCCC-3′; reverse, 5′-ggagatctGACGAGAACGCCTGACACGCAC-3′. The annealing temperature was 58 °C. The resultant 730-bp PCR product was treated with Pfu polymerase (Stratagene) to polish the ends following the manufacturer's protocol, digested with KpnI and BglII, and cloned into the same sites of pIRES-Luc to generate pHrD-IRES-Luc. Expression Vectors for MBD1, MBD2, MBD3, and MeCP2—These plasmids were constructed in mammalian expression vectors pcDNA 3.1(+/-) (Invitrogen) to obtain pcMBD1, pcMBD2, pcMBD3, pcMBD4, and pcMeCP2. Briefly, rat MeCP2 cDNA in pBlueScript-SK(-) (a generous gift from Adrian Bird) was digested with NotI-EcoRV enzymes, and the resultant ∼1.8-kb fragment was cloned into the same sites in pcDNA3.1(-) to generate pcMeCP2. Similarly, human MBD1 cDNA from pET6H (a generous gift from Adrian Bird) was excised with NcoI, and the sticky 5′-end of the ∼2-kb insert fragment was filled in with dNTPs catalyzed by Klenow and cloned in the EcoRV site of pcDNA3.1(-). Amplification with the vector- and insert-specific primers determined the correct orientation of MBD1. The mouse MBD2 cDNA in pBSK vector (a generous gift from Brian Hendrich) was digested with EcoRI and XhoI and ligated to the same sites in pcDNA3.1(-) to generate the mammalian expression vector. MBD3 cDNA was amplified from a mouse cDNA library and ligated to BamHI-HindIII sites of pcDNA3.1(-) to generate the plasmid pcMBD3. HepG2 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. For transfection assay, 2.5 × 105 cells were plated onto 60-mm dishes 24 h prior to transfection and then transfected using calcium phosphate co-precipitation method (34Ghosh A.K. Kermekchiev M. Jacob S.T. Gene (Amst.). 1994; 141: 271-275Crossref PubMed Scopus (4) Google Scholar, 35Majumder S. Ghoshal K. Gronostajski R.M. Jacob S.T. Gene Expr. 2001; 9: 203-215Crossref PubMed Scopus (26) Google Scholar). Unless mentioned otherwise, each transfection mixture contains a maximum of 8.8 μg of total plasmid DNA that includes the reporter plasmid (0.5-1.0 μg), pRLTK (Renilla luciferase reporter driven by HSV-tk promoter (Promega)) (50 ng), as an internal control and the eukaryotic expression vectors (4 μg) harboring the gene of interest in a total volume of 500 μl. Briefly, DNA was dissolved in 220 μl of 0.1× TE (1 mm Tris·HCl, pH 8.0, 0.1 mm EDTA, pH 8.0) and mixed with 250 μl of 2× Hepes-buffered saline (280 mm NaCl, 10 mm KCl, 1.5 mm Na2HPO4, 12 mm dextrose, and 50 mm HEPES). Next, 31 μl of 2 m CaCl2 was added slowly to the DNA mixture and incubated for 20 min at room temperature before adding to the cell culture. The cells were allowed to incubate with the transfection reagent in complete medium for 16 h at 37 °C, followed by replacement with fresh medium. After 24-48 h in the fresh medium, the cells were harvested in the lysis buffer (Promega), and luciferase activity was measured using the Dual Luciferase Assay kit (Promega) in a Luminometer (Lumat LB 9507; EG&G Berthold, Oak Ridge, TN). Genomic DNA isolated form hepatocellular carcinomas and matching liver tissues from the same individuals were treated with sodium bisulfite according to the protocol optimized in our laboratory (36Majumder S. Ghoshal K. Li Z. Jacob S.T. J. Biol. Chem. 1999; 274: 28584-28589Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 37Ghoshal K. Datta J. Majumder S. Bai S. Dong X. Parthun M. Jacob S.T. Mol. Cell Biol. 2002; 22: 8302-8319Crossref PubMed Scopus (142) Google Scholar). The rRNA promoter spanning -377 to +51 bp was amplified using two sets of nested primers from the bisulfite-treated DNA. The primers used were as follows: hrRNA BF1, 5′-AATTTTTTTGGAGAGTTTTCGTG-3′; hrRNA BR1, 5′-GAGTCGGAGAGCGTTTTTTGAG-3′. The annealing temperature used was 50 °C. The nested primers were hrRNA BF2 (5′-GAGTCGGAGAGCGTTTTTTGAG-3′) and hrRNA BR2 (5′-CATCCGAAAACCCAACCTCTCCAA-3′). The annealing temperature was 55 °C. To confirm complete conversion of unmethylated cytosines to uracils, the PCR products were digested with TaqI, the restriction sites of which were generated only after bisulfite conversion. Completely converted PCR products were then cloned into TA cloning vector (Invitrogen). Ten clones selected at random from each DNA were sequenced in automated DNA sequencer. The KpnI-BglII fragment of pHrD-IRES-Luc was methylated with M.SssI methylase (New England Biolabs) or with M.HhaI methylase (New England Biolabs) in the presence (methylated) or absence (mock-methylated) of 160 μm AdoMet in a manufacturer-supplied buffer at 37 °C for 4 h. An additional 10 units of enzyme and AdoMet were added to the same mixture, and the methylation reaction was continued for an additional 4 h. The completion of the methylation reaction was determined by digestion of the fragment with BstUI, HpaII, or HhaI for M.SssI, M.HpaII, and M.HhaI methylases, respectively. These enzymes cannot cleave DNA if their cognate restriction sites are methylated. The methylated promoter fragment was then ligated to the same sites of pIRES-Luc. The ligated plasmid was separated on an agarose gel and purified using a gel extraction kit (Qiagen). Before transfection, the concentrations of methylated and mock-methylated plasmids were measured in a Beckman spectrophotometer at 260 nm. Site-specific methylation was carried out following the methodology used for site-directed mutagenesis with some modifications. Single-stranded DNA was obtained by infection of XL1-blue-MRF′ bacteria harboring HrDNA plasmid with helper phage (R408; Stratagene). In a typical reaction, positive strand HrD-Luc plasmid (∼0.05 pmol) was annealed to 1.25 pmol of phosphorylated oligonucleotides (with a specific CpG either methylated denoted as "M" oligonucleotide or unmethylated control, depicted as "C" oligonucleotide) spanning different regions of the rRNA promoter or external transcribed spacer (external transcribed spacer region 1; ETS1). The annealing mixture (20 μl) contained 20 mm Tris·HCl (pH 7.5), 10 mm MgCl2, and 50 mm NaCl. The reaction was carried out in a thermocycler (PerkinElmer Life Sciences) programmed for a 5-min incubation at 96 °C followed by a slow descent (with a ramp of 3 min/degree Celsius) to 66 °C (annealing temperature of the oligonucleotide) and a 1-h incubation at 66 °C. Annealed oligonucleotide was then extended and ligated in a reaction mixture comprising 3 μl of 10× synthesis buffer (100 mm Tris·HCl, pH 7.5, 5 mm dNTPs, 10 mm ATP, and 20 mm dithiothreitol), 5 units of T4 DNA polymerase, and 1 unit of T4 DNA ligase in a total volume of 30 μl and was incubated at 37 °C for 90 min. The circularized plasmids were pooled, precipitated, and ligated overnight at 16 °C with a high concentration (10 units/μl) ligase (Invitrogen) in 10 μl of ligation mixture. These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C. Methylated (with AdoMet) or mock-methylated (without AdoMet), ligated plasmids were resolved by agarose gel electrophoresis and purified using gel extraction kit (Qiagen), and concentration was measured at 260 nm. Oligonucleotides used for site-specific methylation were C(-347) (5′-CGGCCAGGCCGCGACCTCTC-3′), M(-347) (5′-CGGCCAGGmCGCGACCTCTC-3′), C(-102) (5′-GCGCGACACGGACACCTGT-3′), M(-102) (5′-GCGCGACAmCGGACACCTGT-3′), C(-9) (5′-TCAGCAATAACCCGGCGGCC-3′), M(-9) (5′-TCAGCAATAACCmCGGCGGCC-3′), C(+152) (5′-CGGGAGTCGGGACGCTCGGA-3′), and M(+152) (5′-CGGGAGTCGGGACGCTmCGGA-3′). Methylation at the respective sites were confirmed by sequencing the bisulfite-converted plasmids with the primers 5′-TGTGTGGAGTTGGAGAGTG-3′ and 5′-TTGGGGTTGATTAGAGGG-3′ (for the positive strand) and 5′-CCCTCCTACAACCAAAAC-3′ and 5′-TGTGTGGTTGTGATGGTG-3′ (for the negative strand). Whole cell extract (200 μg) prepared from HepG2 cells overexpressing different MBDs were separated on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and blocked in 5% milk in Tris-buffered (pH 7.5) saline containing 0.1% Tween 20 (TBST). For detection of the overexpressed proteins, the membrane was subjected to immunoblot analysis with the respective antisera (37Ghoshal K. Datta J. Majumder S. Bai S. Dong X. Parthun M. Jacob S.T. Mol. Cell Biol. 2002; 22: 8302-8319Crossref PubMed Scopus (142) Google Scholar, 38Majumder S. Ghoshal K. Datta J. Bai S. Dong X. Quan N. Plass C. Jacob S.T. J. Biol. Chem. 2002; 277: 16048-16058Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) at appropriate dilution and the horseradish peroxidase-conjugated anti-rabbit IgG as the secondary antibody. The antigen-antibody complex was detected using the ECL™ kit (Amersham Biosciences), following the manufacturer's protocol. HepG2 cells at a density of 2.5 × 106 were plated on 150-mm tissue culture dish 24 h prior to the cross-linking. These cells were treated with formaldehyde (1%, final concentration) at 37 °C for 15 min to cross-link proteins to DNA. The reaction was stopped by the addition of 125 mm glycine. Soluble chromatin with an average size of 600-1000 bp was prepared following the protocol of Weinmann et al. (39Weinmann A.S. Bartley S.M. Zhang T. Zhang M.Q. Farnham P.J. Mol. Cell Biol. 2001; 21: 6820-6832Crossref PubMed Scopus (332) Google Scholar). For chromatin immunoprecipitation analysis, antisera raised against the methyl binding proteins MBD1, MBD3, and MeCP2 in our laboratory were used (37Ghoshal K. Datta J. Majumder S. Bai S. Dong X. Parthun M. Jacob S.T. Mol. Cell Biol. 2002; 22: 8302-8319Crossref PubMed Scopus (142) Google Scholar). Anti-MBD2 antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). The chromatin was first precleared with preimmune sera coupled to protein A and protein G beads, followed by overnight incubation with preimmune or immune sera. The immune complex was then captured by protein G (for anti-MBD2) and protein A (for all other antibodies) beads, washed extensively as described (37Ghoshal K. Datta J. Majumder S. Bai S. Dong X. Parthun M. Jacob S.T. Mol. Cell Biol. 2002; 22: 8302-8319Crossref PubMed Scopus (142) Google Scholar, 39Weinmann A.S. Bartley S.M. Zhang T. Zhang M.Q. Farnham P.J. Mol. Cell Biol. 2001; 21: 6820-6832Crossref PubMed Scopus (332) Google Scholar). Immunoprecipitated DNA-protein complex was eluted, decrosslinked, treated with RNase A and proteinase K, and purified as described (37Ghoshal K. Datta J. Majumder S. Bai S. Dong X. Parthun M. Jacob S.T. Mol. Cell Biol. 2002; 22: 8302-8319Crossref PubMed Scopus (142) Google Scholar). The immunoprecipitated as well as the input DNA was digested with HpaII or MspI, and the digests along with an equal amount of the undigested immunoprecipitated DNA were amplified using the radiolabeled primers specific for the human rRNA promoter region: HrDChIP-F (5′-CTGCGATGGTGGCGTTTTTG-3′) and HrDCh-IP-R (5′-ACAGCGTGTCAGCAATAACC-3′). The primers used for the GAPDH promoter (accession number AY340484) are GAPDH-F (5′-GTGCCCAGTTGAACCAG-3′) and GAPDH-R (5′-AACAGGAGGAGCAGAGAGCGAAGC-3′). The annealing temperatures for rRNA and GAPDH primers were 58 and 60 °C, respectively. The sizes of the PCR products for rRNA and GAPDH are 152 and 223 bp, respectively. The amplicons were run on polyacrylamide gels (6% acrylamide), and the dried gels were subjected to autoradiography and PhosphorImager analysis. 32P-labeled PCR products were quantified using ImageQuant software (Amersham Biosciences), and results were depicted as the ratio of methylated and unmethylated DNA precipitated with the antibodies to the input methylated and unmethylated DNA, respectively. HepG2 cells were grown overnight on Labtek chamber slides (2 × 104 cells/chamber). Cells were fixed with 1:1 methanol and acetone mixture for 15 min at 4 °C, washed with PBS, and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. Next the cells were incubated with 1% bovine serum albumin for 1 h to block nonspecific binding. Blocked chambers were subsequently washed and incubated overnight at 4 °C with a mixture of anti-nucleolin monoclonal antibody (anti-C23) and antibodies raised against recombinant MBD1, MBD3, and MeCP2 in our laboratory or MBD2 (Upstate Biotechnology). MBD1, MBD3, and MeCP2 antibodies were used at a dilution of 1:500 in PBS, whereas MBD2 antibody was used at a dilution of 1:20. Fluorescein isothiocyanate-conjugated anti-rabbit (for MBD1, MBD3, and MeCP2) and anti-sheep (for MBD2) were used for green channel detection, whereas monoclonal anti-C23 antibody was detected by TRITC-conjugated monoclonal anti-mouse antibody (Sigma) for detection in the red channel. Nuclei were stained using 4′,6-diamidino-2-phenylindole in the mounting fluid. Ribosomal RNA Promoter Is Hypomethylated in Human Hepatocellular Carcinomas—The sequence analysis of rRNA transcriptional initiation region of the human rRNA gene showed that it is highly enriched in CpG and that the promoter harbors a CpG island encompassing 19 CpGs in the upstream control element (UCE) and six CpGs in the core promoter regions. On the other hand, the mouse and rat rRNA promoters contain only one and five CpGs, respectively (Fig. 1A). 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. Although ribosomal RNA genes are housekeeping genes, they are highly reiterated, and only a fraction of these genes is transcribed by pol I. Therefore, we sought to investigate the methylation status of each CpG within different cis elements (spanning from -200 to -9 bp) in the rRNA promoter in human livers and their potential alterations in hepatocellular carcinomas (HCCs). To determine the methylation status of individual CpGs in the human rRNA promoter region, we performed bisulfite genomic sequ
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