Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

The miR-17-92 MicroRNA Cluster Is Regulated by Multiple Mechanisms in B-Cell Malignancies

2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2011.06.008

ISSN

1525-2191

Autores

Ming Ji, Enyu Rao, Himabindu Ramachandrareddy, Yulei Shen, Chunsun Jiang, Jianxiu Chen, Yiqiao Hu, Angie Rizzino, Wing C. Chan, Kai Fu, Timothy W. McKeithan,

Tópico(s)

Cancer-related molecular mechanisms research

Resumo

A cluster of six microRNAs (miRNAs), miR-17-92, is processed from the transcript of C13orf25, a gene amplified in some lymphomas and solid tumors. We find that levels of the miRNAs in the cluster do not vary entirely in parallel with each other or with the primary RNA in B-cell lines or normal cells, suggesting that processing or stability of the miRNAs is differentially regulated. Using luciferase reporter assays, we identified the region required for maximum promoter activity. Additional deletions and mutations indicated that the promoter is regulated by the collaborative activity of several transcription factors, most of which individually have only a moderate effect; mutation of a cluster of putative SP1-binding sites, however, reduces promoter activity by 70%. MYC is known to regulate C13orf25; surprisingly, mutation of a putative promoter MYC-binding site enhanced promoter activity. We found that the inhibitory MYC family member MXI1 bound to this region. The chromatin structure of a >22.5-kb region encompassing the gene contains peaks of activating histone marks, suggesting the presence of enhancers, and we confirmed that at least two regions have enhancer activity. Because the miR-17-92 cluster acts as an important oncogene in several cancers and targets genes important in regulating cell proliferation and survival, further studies of its transcriptional control are warranted. A cluster of six microRNAs (miRNAs), miR-17-92, is processed from the transcript of C13orf25, a gene amplified in some lymphomas and solid tumors. We find that levels of the miRNAs in the cluster do not vary entirely in parallel with each other or with the primary RNA in B-cell lines or normal cells, suggesting that processing or stability of the miRNAs is differentially regulated. Using luciferase reporter assays, we identified the region required for maximum promoter activity. Additional deletions and mutations indicated that the promoter is regulated by the collaborative activity of several transcription factors, most of which individually have only a moderate effect; mutation of a cluster of putative SP1-binding sites, however, reduces promoter activity by 70%. MYC is known to regulate C13orf25; surprisingly, mutation of a putative promoter MYC-binding site enhanced promoter activity. We found that the inhibitory MYC family member MXI1 bound to this region. The chromatin structure of a >22.5-kb region encompassing the gene contains peaks of activating histone marks, suggesting the presence of enhancers, and we confirmed that at least two regions have enhancer activity. Because the miR-17-92 cluster acts as an important oncogene in several cancers and targets genes important in regulating cell proliferation and survival, further studies of its transcriptional control are warranted. MicroRNAs (miRNAs) are single-stranded RNAs of ∼22 nucleotides that negatively regulate expression of their target genes posttranscriptionally.1Ambros V. The functions of animal microRNAs.Nature. 2004; 431: 350-355Crossref PubMed Scopus (9042) Google Scholar, 2Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29415) Google Scholar, 3He L. Hannon G.J. MicroRNAs: small RNAs with a big role in gene regulation.Nat Rev Genet. 2004; 5: 522-531Crossref PubMed Scopus (5618) Google Scholar, 4Zamore P.D. Haley B. Ribo-gnome: the big world of small RNAs.Science. 2005; 309: 1519-1524Crossref PubMed Scopus (1129) Google Scholar Various miRNAs are overexpressed or underexpressed in different types of cancer in humans and may function as either tumor suppressors or oncogenes.5He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. A microRNA polycistron as a potential human oncogene.Nature. 2005; 435: 828-833Crossref PubMed Scopus (3137) Google Scholar, 6Kim H. Huang W. Jiang X. Pennicooke B. Park P.J. Johnson M.D. Integrative genome analysis reveals an oncomir/oncogene cluster regulating glioblastoma survivorship.Proc Natl Acad Sci U S A. 2010; 107: 2183-2188Crossref PubMed Scopus (193) Google Scholar, 7Johnson S.M. Grosshans H. Shingara J. Byrom M. Jarvis R. Cheng A. Labourier E. Reinert K.L. Brown D. Slack F.J. RAS is regulated by the let-7 microRNA family.Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3103) Google Scholar, 8O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. c-Myc-regulated microRNAs modulate E2F1 expression.Nature. 2005; 435: 839-843Crossref PubMed Scopus (2460) Google Scholar, 9Costinean S. Zanesi N. Pekarsky Y. Tili E. Volinia S. Heerema N. Croce C.M. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice.Proc Natl Acad Sci U S A. 2006; 103: 7024-7029Crossref PubMed Scopus (953) Google Scholar, 10Voorhoeve P.M. le Sage C. Schrier M. Gillis A.J. Stoop H. Nagel R. Liu Y.P. van Duijse J. Drost J. Griekspoor A. Zlotorynski E. Yabuta N. De Vita G. Nojima H. Looijenga L.H. Agami R. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.Cell. 2006; 124: 1169-1181Abstract Full Text Full Text PDF PubMed Scopus (1084) Google Scholar, 11He L. He X. Lim L.P. de Stanchina E. Xuan Z. Liang Y. Xue W. Zender L. Magnus J. Ridzon D. Jackson A.L. Linsley P.S. Chen C. Lowe S.W. Cleary M.A. Hannon G.J. A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2300) Google Scholar, 12Mestdagh P. Fredlund E. Pattyn F. Schulte J.H. Muth D. Vermeulen J. Kumps C. Schlierf S. De Preter K. Van Roy N. Noguera R. Laureys G. Schramm A. Eggert A. Westermann F. Speleman F. Vandesompele J. MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c-MYC-activated tumors.Oncogene. 2010; 29: 1394-1404Crossref PubMed Scopus (103) Google Scholar, 13Trang P. Medina P.P. Wiggins J.F. Ruffino L. Kelnar K. Omotola M. Homer R. Brown D. Bader A.G. Weidhaas J.B. Slack F.J. Regression of murine lung tumors by the let-7 microRNA.Oncogene. 2010; 29: 1580-1587Crossref PubMed Scopus (432) Google Scholar A cluster of six miRNAs (miR-17-92, comprising miR-17, miR-18a, miR-20a, miR-19a, miR-19b-1, and miR-92a-1) is processed from the transcript of C13orf25 (also known as MIR17HG or MIRHG1), a gene amplified in some lymphomas and solid tumors5He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. A microRNA polycistron as a potential human oncogene.Nature. 2005; 435: 828-833Crossref PubMed Scopus (3137) Google Scholar, 14Ota A. Tagawa H. Karnan S. Tsuzuki S. Karpas A. Kira S. Yoshida Y. Seto M. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma.Cancer Res. 2004; 64: 3087-3095Crossref PubMed Scopus (630) Google Scholar, 15Hayashita Y. Osada H. Tatematsu Y. Yamada H. Yanagisawa K. Tomida S. Yatabe Y. Kawahara K. Sekido Y. Takahashi T. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation.Cancer Res. 2005; 65: 9628-9632Crossref PubMed Scopus (1377) Google Scholar, 16Tagawa H. Seto M. A microRNA cluster as a target of genomic amplification in malignant lymphoma.Leukemia. 2005; 19: 2013-2016Crossref PubMed Scopus (185) Google Scholar, 17Rinaldi A. Poretti G. Kwee I. Zucca E. Catapano C.V. Tibiletti M.G. Bertoni F. Concomitant MYC and microRNA cluster miR-17-92 (C13orf25) amplification in human mantle cell lymphoma.Leuk Lymphoma. 2007; 48: 410-412Crossref PubMed Scopus (86) Google Scholar and overexpressed in a large fraction of lymphomas.5He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. A microRNA polycistron as a potential human oncogene.Nature. 2005; 435: 828-833Crossref PubMed Scopus (3137) Google Scholar Overexpression of miR-17-92 accelerates lymphomagenesis in mouse models.5He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. A microRNA polycistron as a potential human oncogene.Nature. 2005; 435: 828-833Crossref PubMed Scopus (3137) Google Scholar, 8O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. c-Myc-regulated microRNAs modulate E2F1 expression.Nature. 2005; 435: 839-843Crossref PubMed Scopus (2460) Google Scholar The miRNAs in the cluster can target transcripts of genes, such as E2F1, PTEN, and BIM, which are important in cell proliferation and apoptosis.18Koralov S.B. Muljo S.A. Galler G.R. Krek A. Chakraborty T. Kanellopoulou C. Jensen K. Cobb B.S. Merkenschlager M. Rajewsky N. Rajewsky K. Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage.Cell. 2008; 132: 860-874Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 19Ventura A. Young A.G. Winslow M.M. Lintault L. Meissner A. Erkeland S.J. Newman J. Bronson R.T. Crowley D. Stone J.R. Jaenisch R. Sharp P.A. Jacks T. Targeted deletion reveals essential and overlapping functions of the miR-17∼92 family of miRNA clusters.Cell. 2008; 132: 875-886Abstract Full Text Full Text PDF PubMed Scopus (1335) Google Scholar, 20Xiao C. Srinivasan L. Calado D.P. Patterson H.C. Zhang B. Wang J. Henderson J.M. Kutok J.L. Rajewsky K. Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes.Nat Immunol. 2008; 9: 405-414Crossref PubMed Scopus (1061) Google Scholar, 21Sylvestre Y. De Guire V. Querido E. Mukhopadhyay U.K. Bourdeau V. Major F. Ferbeyre G. Chartrand P. An E2F/miR-20a autoregulatory feedback loop.J Biol Chem. 2007; 282: 2135-2143Crossref PubMed Scopus (507) Google Scholar, 22Pickering M.T. Stadler B.M. Kowalik T.F. miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression.Oncogene. 2009; 28: 140-145Crossref PubMed Scopus (141) Google Scholar The transcriptional regulation of the miR-17-92 cluster, however, is poorly understood. C13orf25 is activated by MYC and E2F transcription factors, and MYC was first shown to bind to a region containing a conserved CATGTG sequence in the first intron of the gene locus.8O'Donnell K.A. Wentzel E.A. Zeller K.I. Dang C.V. Mendell J.T. c-Myc-regulated microRNAs modulate E2F1 expression.Nature. 2005; 435: 839-843Crossref PubMed Scopus (2460) Google Scholar By chromatin immunoprecipitation (ChIP) in HeLa cells, endogenous E2F1, E2F2, and E2F3 were found to directly bind the promoter of the gene and regulate its transcription.21Sylvestre Y. De Guire V. Querido E. Mukhopadhyay U.K. Bourdeau V. Major F. Ferbeyre G. Chartrand P. An E2F/miR-20a autoregulatory feedback loop.J Biol Chem. 2007; 282: 2135-2143Crossref PubMed Scopus (507) Google Scholar The primary transcript initiates from a consensus initiator sequence downstream of a nonconsensus TATA box.23Woods K. Thomson J.M. Hammond S.M. Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors.J Biol Chem. 2007; 282: 2130-2134Crossref PubMed Scopus (416) Google Scholar This TATA box is flanked by a TP53 binding site that mediates repression under hypoxic conditions.24Yan H.L. Xue G. Mei Q. Wang Y.Z. Ding F.X. Liu M.F. Lu M.H. Tang Y. Yu H.Y. Sun S.H. Repression of the miR-17-92 cluster by p53 has an important function in hypoxia-induced apoptosis.EMBO J. 2009; 28: 2719-2732Crossref PubMed Scopus (261) Google Scholar A recent study showed a direct link between the action of IL-6 and the expression of C13orf25 mediated by STAT3.25Brock M. Trenkmann M. Gay R.E. Michel B.A. Gay S. Fischler M. Ulrich S. Speich R. Huber L.C. Interleukin-6 modulates the expression of the bone morphogenic protein receptor type II through a novel STAT3-microRNA cluster 17/92 pathway.Circ Res. 2009; 104: 1184-1191Crossref PubMed Scopus (319) Google Scholar To explore transcriptional regulation of the mir17-92 cluster, we have begun to analyze the C13orf25 promoter and the first intron of the gene in several cell culture model systems. Because it is now clear that epigenetic mechanisms regulate miRNA expression,26Huang Y.W. Liu J.C. Deatherage D.E. Luo J. Mutch D.G. Goodfellow P.J. Miller D.S. Huang T.H. Epigenetic repression of microRNA-129-2 leads to overexpression of SOX4 oncogene in endometrial cancer.Cancer Res. 2009; 69: 9038-9046Crossref PubMed Scopus (250) Google Scholar, 27Roman-Gomez J. Agirre X. Jimenez-Velasco A. Arqueros V. Vilas-Zornoza A. Rodriguez-Otero P. Martin-Subero I. Garate L. Cordeu L. San Jose-Eneriz E. Martin V. Castillejo J.A. Bandres E. Calasanz M.J. Siebert R. Heiniger A. Torres A. Prosper F. Epigenetic regulation of microRNAs in acute lymphoblastic leukemia.J Clin Oncol. 2009; 27: 1316-1322Crossref PubMed Scopus (120) Google Scholar, 28Tsai K.W. Kao H.W. Chen H.C. Chen S.J. Lin W.C. Epigenetic control of the expression of a primate-specific microRNA cluster in human cancer cells.Epigenetics. 2009; 4: 587-592Crossref PubMed Scopus (105) Google Scholar, 29Saetrom P. Snove Jr, O. Rossi J.J. Epigenetics and microRNAs.Pediatr Res. 2007; 61: 17R-23RCrossref PubMed Scopus (81) Google Scholar, 30Saito Y. Jones P.A. Epigenetic activation of tumor suppressor microRNAs in human cancer cells.Cell Cycle. 2006; 5: 2220-2222Crossref PubMed Scopus (247) Google Scholar we also investigated histone posttranslational modifications (PTMs) in C13orf25 in normal B cells and in B-cell lines. Further studies of the transcriptional control of C13orf25 are warranted because the miR-17-92 cluster targets genes important in regulating cell proliferation and survival and acts as an important oncogene in several cancers, including lymphoma. Mantle cell lymphoma–derived cell lines (JeKo-1 and JVM2), Burkitt's lymphoma cell lines (CL-01, Daudi, Raji, and P3HR-1), a diffuse large B-cell lymphoma (DLBCL) cell line of germinal center B-cell type [SU-DHL16 (hereafter, DHL16)], a mediastinal large B-cell lymphoma cell line (Karpas 1106), a Hodgkin's lymphoma cell line (L428), and a myeloma cell line (U266) were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). A DLBCL line of germinal center B-cell type (DHL6) and two DLBCL lines of activated B-cell type (OCI-Ly3 and OCI-Ly10) were maintained in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 20% human serum, 50 μmol/L 2-mercaptoethanol, and 1% penicillin/streptomycin. HEK293T and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HEK293T, NIH3T3, JeKo-1, JVM2, and U266 were obtained from American Type Culture Collection (Manassas, VA) and Karpas 1106 from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). P3HR-1 and CL-01 were gifts from Dr. Luwen Zhang (University of Nebraska, Lincoln, NE) and Dr. Paolo Casali (University of California, Irvine, CA), respectively. All the other lines were a gift from Dr. Louis Staudt (National Cancer Institute, Bethesda, MD). Primary cells isolated for miRNA studies, including naive B cells (IgD+) and centroblasts (CD77+), were isolated from tonsils using magnetic microbeads (Miltenyi Biotec Inc., Auburn, CA), as described.31Ramachandrareddy H. Bouska A. Shen Y. Ji M. Rizzino A. Chan W.C. McKeithan T.W. BCL6 promoter interacts with far upstream sequences with greatly enhanced activating histone modifications in germinal center B cells.Proc Natl Acad Sci U S A. 2010; 107: 11930-11935Crossref PubMed Scopus (29) Google Scholar miRNA expression profiling was conducted using the TaqMan human miRNA array set v2.0 (Applied Biosystems, Foster City, CA) with 300 ng of total RNA from cell lines or normal cells. All the RNA samples were reverse transcribed using Megaplex RT primers (Invitrogen) for 40 cycles at 16°C for 2 minutes, 42°C for 1 minute, and 50°C for 1 second; were held at 85°C for 5 minutes to inactivate the reverse transcriptase; and were preamplified for 12 cycles using Megaplex PreAmp primers (Invitrogen). The diluted preamplified DNA was loaded onto the 384-well plate per the manufacturer's instructions, and real-time PCR was run using a 7900HT fast real-time PCR system (Applied Biosystems) in 384-well plates at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The CT is defined as the fractional cycle number at which the fluorescence exceeds the fixed threshold of 0.1 using RQ Manager 2.1 software (Applied Biosystems). Data from each 384-well plate were normalized with the average expression of U6 small nuclear RNA, the least variable control RNA analyzed on the plate. RNA was isolated using the RNeasy mini kit (Qiagen Inc., Valencia, CA), and miR-17-92 pri-miRNA and RPL13A mRNA, for normalization, were quantified by SYBR Green PCR (New England BioLabs, Inc., Ipswich, MA) using the DNA Engine Opticon 2 real-time PCR detection system (MJ Research, Waltham, MA) according to the manufacturer's instructions; the primers used are listed in Table 1. To determine the expression levels of mature miRNAs, TaqMan miRNA assays (Applied Biosystems) were performed using an Applied Biosystems 7900HT fast real-time PCR system and were normalized to U6 expression.Table 1Oligonucleotides Used in This StudyPrimer nameSequencePurposemiR-17∼92-pri-F5′-CAGTAAAGGTAAGGAGAGCTCAATCTG-3′RT-PCRmiR-17∼92-pri-R5′-CATACAACCACTAAGCTAAAGAATAATCTGA-3′RT-PCRRPL13A-F5′-ACCGTCTCAAGGTGTTTGACG-3′RT-PCR (normalization)RPL13A-R5′-GTACTTCCAGCCAACCTCGTG-3′RT-PCR (normalization)ODC1-F5′-TGGGCGCTCTGAGGTGAGGG-3′ChIP of ODC1 (positive control)ODC1-R5′-ATCCAGCCGCGGGAGAACCC-3′ChIP of ODC1 (positive control)C13orf25-1-F5′-AAACGTTCTGAATGTTCTGGATTGT-3′ChIP of site 1C13orf25-1-R5′-CACAGCCTTCTCAAGTCAGCTAAA-3′ChIP of site 1C13orf25-2-F5′-AAGGAAGTATGAGCGAAACCCT-3′ChIP of site 2C13orf25-2-R5′-CAGGTTTCTTAGCAGGAGTTATTCA-3′ChIP of site 2C13orf25-3-F5′-GACTTGTACAATCACGAACCAGTG-3′ChIP of site 3C13orf25-3-R5′-TCTGGGTTCTCCAGTAGAAATAGC-3′ChIP of site 3C13orf25-4-F5′-TTTATGCTAATGAGGGAGTGG-3′ChIP of site 4C13orf25-4-R5′-GCTTTACTACGACCGGAGG-3′ChIP of site 4C13orf25-5-F5′-GACGGCGAACACAATGGC-3′ChIP of site 5C13orf25-5-R5′-GCGTACAAAGTTTGGGGAACC-3′ChIP of site 5C13orf25-6-F5′-AAGAATAGTCTGTGGGCTGCT-3′ChIP of site 6C13orf25-6-R5′-GCTATTTTCCAACCTCCTAACAGC-3′ChIP of site 6Cloning and deletions⁎In the “Cloning and deletions” section, added nucleotides are in lowercase and restriction sites are underlined. LucSeqR25′-CCAGGAACCAGGGCGTATC-3′Preparing promoter deletions and mutations P482-F5′-atttggtaccGCTCCCGGCTCCCGCTCTC-3′Preparing P482 deletion P435-F5′-atttggtacCGGGGCCTGCGGTGATTGG-3′Preparing P435 deletion P512-F5′-ataggtaccgagGGGGAAGGCTG-3′Preparing 3′ promoter deletions P339-R5′-gaaactcgagCCCGCGAGGAGAGCTTCG-3′Preparing P339 deletion 301-R5′-caaactcgaGCGGCGCTGCGGTAGTCGTC-3′Preparing P301 deletion 217-R5′-caaactcgagGGCACCTCGAAGGACCATG-3′Preparing P217 deletion IntronA-F5′-gaaaggatccCGGACGGCGAACACAATG-3′Cloning “intron A” from BAC IntronA-R5′-cattgtcgaCCTCCTTTGTGTCCCCCAG-3′Cloning “intron A” from BAC IntronB-F5′-caaaggatccCTGGGGGACACAAAGGAG-3′Cloning “intron B” from BAC IntronB-R5′-catagtcgacCTTTTACTCTCTAAGAAACCAATCC-3′Cloning “intron B” from BACMutations by overlap PCR†In the “Mutations by overlap PCR” section, altered nucleotides are in italics and new restriction sites are underlined. P512-F25′-gtttttcgtacgaattcCCGGGGAAGGCTGC-3′Preparing promoter mutations NFYmut-F5′-GGCCTGCGGTGGCTAGCGGGCGGGCG-3′Preparing promoter NFY mutation NFYmut-R5′-CGCCCGCCCGCTAGCCACCGCAGGCC-3′Preparing promoter NFY mutation SP1mut1-F5′-CGAGCCGCCGTTAACCGGGCCTGCGGTG-3′Preparing promoter SP1 mutation 1 SP1mut1-R5′-CGAGCCGCCGTTAACCGGGCCTGCGGTG-3′Preparing promoter SP1 mutation 1 SP1mut2-F5′-GGTGATTGGCGAGAATTCGGGGAGGTCGG-3′Preparing promoter SP1 mutation 2 SP1mut2-R5′-CCGACCTCCCCGAATTCTCGCCAATCACC-3′Preparing promoter SP1 mutation 2 ETSmut-F5′-GCGGGGAGGTCCGCGGTACTTTGTTTTTTATG-3′Preparing promoter ETS mutation ETSmut-R5′-CATAAAAAACAAAGTACCGCGGACCTCCCCGC-3′Preparing promoter ETS mutation OCTmut-F5′-CTTTGTTTTTTATCGATTTGAGGGAGTGGG p-3′Reparing promoter OCT mutation OCTmut-R5′-CCCACTCCCTCAAATCGATAAAAAACAAAG-3′Preparing promoter OCT mutation Ebox1mut-F5′-CCTTCATTCACCCGATCGGTCCTTCGAG-3′Preparing promoter E-box mutation Ebox1mut-R5′-CTCGAAGGACCGATCGGGTGAATGAAGG-3′Preparing promoter E-box mutation E2Fmut1-F5′-CTGCGCCTTGGGCCCACTTCGCGCCCTCGGGCG-3′Preparing promoter E2F mutation 1 E2Fmut1-R5′-CGCCCGAGGGCGCGAAGTGGGCCCAAGGCGCAG-3′Preparing promoter E2F mutation 1 E2Fmut2-F5′-CGCCACTTCGGTACCTCGGGCGAG-3′Preparing promoter E2F mutation 2 E2Fmut2-R5′-CTCGCCCGAGGTACCGAAGTGGCG-3′Preparing promoter E2F mutation 2 E2Fmut1,2-F5′-CCTTGGGCCCACTTCGGTACCTCGGGCGAGGCC-3′Preparing combined E2F mutations 1 & 2 E2Fmut1,2-R5′-CGCCCGAGGTACCGAAGTGGGCCCAAGGCGCAG-3′Preparing combined E2F mutations 1 & 2 Ebox2mut-F5′-GCGGCGGCGGCGATCGGGGCAGGCCG-3′Preparing intron E-box mutation 2 Ebox2mut-R5′-CGGCCTGCCCCGATCGCCGCCGCCGC-3′Preparing intron E-box mutation 2 Ebox3mut-F5′-CCTTGTGCGACGATCGCTGCCGGCCC-3′Preparing intron E-box mutation 3 Ebox3mut-R5′-GGGCCGGCAGCGATCGTCGCACAAGG-3′Preparing intron E-box mutation 3 Ebox4mut-F5′-CCAGTCATACGATCGGACCTAACTGC-3′Preparing intron E-box mutation 4 Ebox4mut-R5′-GCAGTTAGGTCCGATCGTATGACTGG-3′Preparing intron E-box mutation 4 In the “Cloning and deletions” section, added nucleotides are in lowercase and restriction sites are underlined.† In the “Mutations by overlap PCR” section, altered nucleotides are in italics and new restriction sites are underlined. Open table in a new tab The firefly luciferase reporter vectors pGL3 basic, pGL3 promoter, and Renilla luciferase vectors pRL-SV40 are from Promega (Madison, WI). From BAC RP11-97P7 (BACPAC Resources Center, Children's Hospital Oakland Research Institute, Oakland, CA), a fragment containing the human C13orf25 promoter (−659 to 323 bp) was PCR amplified with the addition of an XhoI site at the 3′ end and was cloned into the pBLCAT7 plasmid. Four fragments of the promoter region were cut out from the human C13orf25-pBLCAT7 plasmid by digestion with XhoI and either PvuII (P982; names correspond to the insert size in bp), HpaI (P606), SmaI (P512), or ScaI (P395) and were ligated into the Ecl136II and XhoI sites of pGL3 basic vector. Additional 5′ deletions (P482 and P435) and 3′ deletions (P339, P301, and P217) of P512 were generated by PCR. Downstream regions A, B, and AB were PCR amplified from BAC DNA and then inserted into BamHI-SalI sites located downstream of the luciferase gene in the pGL3 C13orf25-P301 promoter or pGL3 promoter reporter vector. All the mutations were generated by the overlap PCR method from P512. Primers used in preparing these mutations and other constructs are listed in Table 1. The sequences of all the constructs prepared using PCR were confirmed by complete sequencing. HEK293T or NIH3T3 cells were plated at 5 × 104 cells per well in a 24-well plate 24 hours before transfection. The plasmids were cotransfected with pRL-SV40 using TurboFect transfection reagent (Fermentas, Hanover, MD) according to the manufacturer's instructions. DHL16 cells were transiently transfected using an Amaxa Nucleofector (Lonza, Basel, Switzerland) with cell line solution V combined with program L-29. Luciferase assays were performed 24 hours after transfection using the Dual-Luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each reaction. Triplicate transfected wells were analyzed for each group. ChIP assay was performed in P3HR-1 and DHL16 cells according to the protocol of the ChampionChIP one-day kit (SABiosciences Corp., Frederick, MD). The antibodies used in this study were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA): MYC (N-262, sc-764), MNT (M-132, sc-769), and MXI1 (G-16, sc-1042). Real-time PCR amplifications were performed using DyNAmo Capillary SYBR Green PCR master mix (New England BioLabs, Inc.) and the DNA Engine Opticon 2 real-time PCR detection system according to the manufacturer's instruction. Primer sequences are listed in Table 1. ChIP of modified histones and RNA polymerase II (polII), amplification of ChIP DNA, and hybridization to a custom chip were performed as described previously.31Ramachandrareddy H. Bouska A. Shen Y. Ji M. Rizzino A. Chan W.C. McKeithan T.W. BCL6 promoter interacts with far upstream sequences with greatly enhanced activating histone modifications in germinal center B cells.Proc Natl Acad Sci U S A. 2010; 107: 11930-11935Crossref PubMed Scopus (29) Google Scholar For analysis of histone modifications, we performed native ChIP on 20 × 106 cells without formaldehyde cross-linking. Nuclei were digested by micrococcal nuclease enzyme followed by mild sonication to obtain mononucleosomes. For analysis of polII binding, we performed ChIP after sonication after formaldehyde cross-linking to obtain DNA fragments ranging from 200 to 400 bp. Chromatin was then dialyzed into radioimmunoprecipitation assay buffer at 4°C. We conjugated 4 μg of H3-K9, K14-diacetyl–specific, and lysine-27 trimethylation–specific antibodies from Upstate Biotechnology (Millipore, Billerica, MA) (06–559 and 07–449) and lysine-4 monomethylation–specific, histone 3 lysine-4 trimethylation (H3K4me3)–specific, and polII-specific antibodies from Abcam Inc. (Cambridge, MA) (ab8895, ab8580, and ab5408) to Dynabeads protein A (Dynal Biotech, Oslo, Norway). We performed ChIP by overnight incubation of dialyzed chromatin with the antibody-loaded beads. We thoroughly washed the beads containing antibody and chromatin complexes, and the DNA was finally eluted from the beads. The eluted DNA was tested for enrichment by quantitative PCR. Input and enriched ChIP DNA were amplified by ligation-mediated PCR. Ligation-mediated PCR–amplified input DNA and ChIP-enriched DNA were labeled with Cy3 and Cy5, respectively, and were hybridized to a custom 385K array (Roche NimbleGen, Inc., Madison, WI) by the NimbleGen service laboratory in Reykjavik, Iceland. Coordinates for individual reads from genome-wide ChIP-Seq experiments in K562 and HeLa-S3 cells and DNase sensitivity analysis in K562 were downloaded (http://genome.ucsc.edu, last accessed July 27, 2010) and sorted; for each experimental replicate, the average fragment size was estimated using an Excel spreadsheet (Microsoft Corp., Redmond, WA) by determining the coordinate displacement that resulted in maximal overlap of forward and reverse reads in a continuous block of ≥200,000 reads. Coordinates of forward and reverse reads in the C13orf25 region were adjusted based on these values, and the numbers of reads over all replicates and both orientations were combined and plotted in 20-bp increments using a 100-bp window. Evolutionary conservation in the C13orf25 promoter and close matches conserved consensus binding sites for transcription factors were determined using the MatInspector program (Genomatix Software GmbH, Munich, Germany). miRNA expression profiling was initially conducted using the TaqMan human miRNA array. Because the miR-17-92 cluster is expressed in proliferating cells, we normalized the values to those of normal centroblasts, which proliferate rapidly. We observed decreased miR-17-92 expression in normal naive B cells compared with that in centroblasts for each of the six miRNAs in the cluster but to varying extents, ranging from 1.28-fold in miR-92a to 7.8-fold in miR-18a (Figure 1A). Various tumor cell lines showed higher expression of the miRNA cluster than in normal centroblasts, with a ratio varying from 1.8 to 12.7, averaging over the six miRNAs (Figure 1A). In general, there are strong correlations between the levels of the different miRNAs in the cluster in the 11 cell lines analyzed, representing different stages of B-cell differentiation. The miRNA whose level correlated least well with the others was miR-18a (Figure 1A and inset). Processing of this miRNA is known to be regulated by an RNA-binding protein32Michlewski G. Guil S. Semple C.A. Caceres J.F. Posttranscriptional regulation of miRNAs harboring conserved terminal loops.Mol Cell. 2008; 32: 383-393Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar; variation in efficiency of miR-18a processing among different cell lines is likely to be responsible for the relatively low correlation between miR-18a levels and those of the other members of the cluster. Of the 11 cell lines, the greatest amounts of miR-17-92 expression were seen in JeKo-1 (a mantle cell lymphoma line) and in DHL16 (a DLBCL line of the germinal center B-cell subtype). Because JeKo-1 cells are known to have amplification of the C13orf25 locus, we wished to determine whether gene amplification was also responsible for the high-level miR-17-92 expression in DHL16 cells. This was done using two approaches. First, we performed quantitative PCR of genomic DNA with primers in C13orf25 and the control locus RPL13 in DHL16 and seven other lines. Four of the lines (DHL6, OCI-Ly1, OCI-Ly3, and JVM2) had virtually identical ratios of C13orf25 to RPL13, and it was assumed that they contain two copies of C13orf25. Relative to the average normalized quantity of C13orf25 DNA in these four lines, DHL16 showed a mean ± SD ratio of 1.33 ± 0.60 and JeKo-1 a ratio of 5.8 ± 1.6 (see Supplemental Figure S1A at http://ajp.amjpathol.org). These data suggest that DHL16 has either two or three copies of C13orf25, whereas JeKo-1 has approximately 12. As a second approach to test for possible C13orf25 amplification in DHL16, we used the ChIP with hybridization to a custom oligonucleotide chip (ChIP-chip) data (described later herein) to estimate the relative copy numbers of the

Referência(s)