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c-Myc Directly Regulates the Transcription of the NBS1 Gene Involved in DNA Double-strand Break Repair

2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês

10.1074/jbc.m212043200

ISSN

1083-351X

Autores

Yu-Chi Chiang, Shu‐Chun Teng, Yi‐Ning Su, Fon‐Jou Hsieh, Kou‐Juey Wu,

Tópico(s)

Genomics and Chromatin Dynamics

Resumo

The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. Here we show that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication. The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. Here we show that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication. The c-myc proto-oncogene codes for a nuclear phosphoprotein ubiquitously expressed in somatic cells (1Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1030) Google Scholar, 2Eisenman R.N. Genes Dev. 2001; 15: 2023-2030Crossref PubMed Scopus (321) Google Scholar). Alterations of the c-myc locus, caused by chromosomal translocation, amplification, retroviral insertion, or retroviral transduction, deregulate c-Myc expression and contribute to tumorigenesis in different species (1Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1030) Google Scholar, 3Bouchard C. Staller P. Eilers M. Trends Cell Biol. 1998; 8: 202-206Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 4Nesbit C.E. Tersak J.M. Prochownik E.V. Oncogene. 1999; 18: 3004-3016Crossref PubMed Scopus (961) Google Scholar, 5Lutz W. Leon J. Eilers M. Biochim. Biophy. Acta. 2002; 1602: 61-71PubMed Google Scholar). The c-Myc protein contains a carboxyl-terminal basic, helix-loop-helix and leucine-zipper domain, which associates with another basic, helix-loop-helix and leucine-zipper protein (MAX) 1The abbreviations used are: MAX, a basic, helix-loop-helix and leucine-zipper protein; NBS, Nijmegen breakage syndrome; DSB, double-strand break; ChIP, chromatin immunoprecipitation; CMV, cyto-megalovirus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BrdUrd, bromodeoxyuridine; E2, estrogen; TC, tetracycline; RNAi, RNA interference; TM, 4-hydroxytamoxifen; CX, cycloheximide;1The abbreviations used are: MAX, a basic, helix-loop-helix and leucine-zipper protein; NBS, Nijmegen breakage syndrome; DSB, double-strand break; ChIP, chromatin immunoprecipitation; CMV, cyto-megalovirus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BrdUrd, bromodeoxyuridine; E2, estrogen; TC, tetracycline; RNAi, RNA interference; TM, 4-hydroxytamoxifen; CX, cycloheximide; as heterodimers, and an amino-terminal domain necessary for transcriptional transactivation (2Eisenman R.N. Genes Dev. 2001; 15: 2023-2030Crossref PubMed Scopus (321) Google Scholar, 6Kato G.J. Barrett J. Villa-Garcia M. Dang C.V. Mol. Cell. Biol. 1990; 10: 5914-5920Crossref PubMed Scopus (312) Google Scholar, 7Blackwood E.M. Eisenman R.N. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1472) Google Scholar). The heterodimeric complexes c-Myc·MAX are capable of binding DNA at a specific site (E-box) and activate transcription of downstream target genes by recruiting protein complexes that can regulate histone acetylation and modify chromatin structure (8Blackwell T.K. Huang J. Ma A. Kretzner L Alt F.W. Eisenman R.N. Mol. Cell. Biol. 1993; 13: 5216-5224Crossref PubMed Scopus (332) Google Scholar, 9Bouchard C. Dittrich O. Kiermaier A. Dohmann K. Menkel A. Eilers M. Luscher B. Genes Dev. 2001; 15: 2042-2047Crossref PubMed Scopus (274) Google Scholar, 10Frank S.R. Schroeder M. Fernandez P. Taubert S. Amati B. Genes Dev. 2001; 15: 2069-2082Crossref PubMed Scopus (420) Google Scholar). The precise function of the c-Myc protein, and in particular the mechanism by which it promotes cell proliferation in normal and neoplastic cells, is not known. Using a variety of approaches, including genome-wide gene expression profiling, various c-Myc target genes have been identified and have been shown to be involved in heterogeneous functions, including cell cycle control, DNA synthesis, iron metabolism, protein synthesis, apoptosis, cell adhesion, and telomere maintenance (11Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1379) Google Scholar). In Drosophila as well as in mammalian cells, c-Myc has been shown to promote cell growth (12Iritani B.M. Eisenman R.N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13180-13185Crossref PubMed Scopus (296) Google Scholar, 13Johnston L.A. Prober D.A. Edgar B.A. Eisenman R.N. Gallant P. Cell. 1999; 98: 779-790Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar), although it has been recently reported that c-Myc may control organ and body size by regulating cell cycle entrance and cell number rather than cell size (14Trumpp A. Refaeli Y. Oskarsson T. Gasser S. Murphy M. Martin G.R. Bishop J.M. Nature. 2001; 414: 768-773Crossref PubMed Scopus (374) Google Scholar). A better understanding of the precise role of c-Myc depends upon the identification of the genes that are directly targeted by its transcriptional regulation. Nijmegen breakage syndrome (NBS) is an autosomal recessive hereditary disorder characterized by microcephaly, a “bird-like” facial appearance, growth retardation, immunodeficiency, radiosensitivity, chromosomal instability, and predisposition to tumor formation (15Petrini J.H.J. Curr. Opin. Cell Biol. 2000; 12: 293-296Crossref PubMed Scopus (203) Google Scholar, 16Karran P. Curr. Opin. Gen. Dev. 2000; 10: 144-150Crossref PubMed Scopus (386) Google Scholar). The gene defective in NBS has been cloned, and the gene product, NBS1 (p95, nibrin) is a member of the DNA double-strand break (DSB) repair complex (hMre11 complex), including hMre11, hRad50, and NBS1 (15Petrini J.H.J. Curr. Opin. Cell Biol. 2000; 12: 293-296Crossref PubMed Scopus (203) Google Scholar). Increased radiation sensitivity and radioresistant DNA synthesis of NBS fibroblasts are similar to the cellular features of ataxia telangiectasis cells (17van der Zwet M. Overkamp W.J. Friedl A.A. Klein B. Verhaegh G.W. Jaspers N.G. Midro A.T. Eckardt-Schupp F. Lohman P.H. Zdzienicka M.Z. Mutat. Res. 1999; 434: 17-27Crossref PubMed Scopus (99) Google Scholar), which is demonstrated by the recent results showing that ataxia telangiectasis-mutated protein phosphorylates NBS1 (18Lim D.S. Kim S.T. Xu B. Maser R.S. Lin J. Petrini J.H.J. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (673) Google Scholar, 19Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neil T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar, 20Zhao S. Weng Y.C. Yuan S.S. Lin Y.T. Hsu H.C. Lin S.C. Gerbino E. Song M.H. Zdzienicka M.Z. Gatti R.A. Shay J.W. Ziv Y. Shiloh Y. Lee E.Y. Nature. 2000; 405: 473-477Crossref PubMed Scopus (435) Google Scholar), linking these two proteins in the same pathway. NBS1 is a putative tumor suppressor gene as shown by the existence of NBS patients and some mutations discovered in different tumors (15Petrini J.H.J. Curr. Opin. Cell Biol. 2000; 12: 293-296Crossref PubMed Scopus (203) Google Scholar, 21Varon R. Reis A. Henze G. Einsiedel H.G. Sperling K. Seeger K. Cancer Res. 2001; 61: 3570-3572PubMed Google Scholar). However, NBS1 is expressed in highly proliferating tissues developmentally (22Wilda M. Demuth I. Concannon P. Sperling K. Hameister H. Hum. Mol. Genet. 2000; 9: 1739-1744Crossref PubMed Scopus (30) Google Scholar) and is located at sites of DNA synthesis through interaction with E2F (23Maser R.S. Mirzoeva O.K. Wells J. Olivares H. Williams B.R. Zinkel R.A. Farnham P.J. Petrini J.H.J. Mol. Cell. Biol. 2001; 21: 6006-6016Crossref PubMed Scopus (183) Google Scholar). In addition, Mre11 complex is able to prevent DSB accumulation during chromosomal DNA synthesis to ensure cell cycle progression (24Costanzo V. Robertson K. Bibikova M. Kim E. Grieco D. Gottesman M. Carroll D. Gautier J. Mol. Cell. 2001; 8: 137-147Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Obviously, the roles of NBS1 are multiple, and some of them are still subject to intensive investigation. Due to the correlation between c-Myc activity and physiological DNA synthesis during cell cycle progression, we investigated whether the expression of NBS1 could be regulated by c-Myc. In this report, we demonstrated that NBS1 is a direct c-Myc target gene, implicating that the DNA DSB repair pathway is regulated by c-Myc. The role of c-Myc during cell growth and proliferation is thereby linked to the physiological function of DNA DSB repair occurred during DNA replication to preserve the integrity of the genome and facilitate cell growth and proliferation. Cell Lines and Plasmids—The lymphoblastoid cell lines, EREB.TCMyc and EREB.MycER™ cells were previously described (25Gu W. Cechova K. Tassi V. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2935-2939Crossref PubMed Scopus (133) Google Scholar, 26Wu K.J. Polack A. Dalla-Favera R. Science. 1999; 283: 676-679Crossref PubMed Scopus (277) Google Scholar, 27Kempkes B. Spitkovsky D. Jansen-Durr P. Ellwart J.W. Kremmer E. Delecluse H.J. Rottenberger C. Bornkamm G.W. Hammerschmidt W. EMBO J. 1995; 14: 88-96Crossref PubMed Scopus (214) Google Scholar, 28Wu K.J. Grandori C. Amacker M. Simon-Vermot N. Polack A. Lingner J. Dalla-Favera R. Nat. Genet. 1999; 21: 220-224Crossref PubMed Scopus (769) Google Scholar). The NIH3T3.Myc cell line was generated by transfecting pSV2Neo and pMT2TMyc into NIH3T3 cells and selecting under G418 (250 μg/ml). 293TMyci and HeLaMyci cell lines were generated by transfecting pSUPERMyci and pSV2Hygro into 293T cells or pSUPERMyci and pSV2Neo into HeLa cells and selecting under hygromycin (100 μg/ml) or G418 (400 μg/ml). RatNBS cell lines were generated by transfecting the HeBOCMVNBS plasmid into Rat1a cells and selecting under G418 (400 μg/ml). The pHeBOCMVNBS plasmid was constructed by PCR-mediated generation of a 2.34-kb fragment of the full-length human NBS1 cDNA from the plasmid pBS-NBS1 obtained from Dr. P. Concannon (29Desai-Mehta A. Cerosaletti K.M. Concannon P. Mol. Cell. Biol. 2001; 21: 2184-2191Crossref PubMed Scopus (158) Google Scholar). The primers used to generate this fragment are: 5′-CCGGTTACGCGCTAGCACGTCGGCCC-3′ and 5′-TTGGCCTGAAGCGGCCGCTTACTAGGAA-3′. This fragment was subcloned into the NheI-NotI sites of HeBOCMV vector (25Gu W. Cechova K. Tassi V. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2935-2939Crossref PubMed Scopus (133) Google Scholar) to make the HeBOCMVNBS expression vector, and it was verified by sequencing. The pSUPERMyci plasmid was generated by inserting the oligonucleotide of 5′-GATCCCCTTCAGAATAGAGTATGAGCCTTCAAGAGAGGCTCATACTCTATTCTGAATTTTTGGAAA-3′ into the pSUPER plasmid provided by Dr. R. Agami (30Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar). Northern Blot Analysis—3 μg of poly(A) RNA was used in each lane of the Northern blots. In EREB.MycER™ Northern blots, 6 μg of poly(A) RNA was used. The human NBS1 probe was generated by digestion of the pBS-NBS1 plasmid. The mouse Nbs1 probe was generated by reverse transcription-PCR using mouse-specific Nbs1 primers: 5′-GTACGTTGTTGGGAGGAA-3′ and 5′-CTGGAGGCTGTTTCTTAG-3′. The PCR fragment was subcloned into a pGEM-T vector and verified by sequencing. c-myc exon 3 and exon 1 probes were obtained from the pMC41RC and pMC41ER plasmids, respectively (31Dalla-Favera R. Gelmann E.P. Martinotti S. Franchini G. Papas T.S. Gallo R.C. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6497-6501Crossref PubMed Scopus (273) Google Scholar). The exon-3 genomic c-myc probe detects both endogenous and exogenous (pTCMyc plasmid) c-myc RNA species; however, the exon-1 c-myc probe detects only the endogenous c-myc RNA, because exon-1 sequences are not present in the transfected pTCMyc plasmid. The SV40 poly(A) tail probe and human β-actin probe were previously described (28Wu K.J. Grandori C. Amacker M. Simon-Vermot N. Polack A. Lingner J. Dalla-Favera R. Nat. Genet. 1999; 21: 220-224Crossref PubMed Scopus (769) Google Scholar). Data shown here are representative of two or more experiments from independent cell cultures. Western Blot Analysis and Immunoprecipitation Procedure—60 μg (CB control, CBMyc.Max, and CBMax, Fig. 1B)or100 μg (293T control, 293TMyci clone, HeLa control, and HeLaMyci clones, Fig. 3, A and B)of cellular extracts was used and probed with a polyclonal anti-NBS1 antibody (C-8580, Santa Cruz Biotechnology), an anti-c-Myc antibody (SC-764, Santa Cruz Biotechnology), and a polyclonal anti-β-actin antibody (C-11, Santa Cruz Biotechnology) as a control for protein loading. Signals were revealed using an ECL chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). Immunoprecipitation procedure was performed by incubating 600 μg of whole cell extract prepared under cell lysis buffer (26Wu K.J. Polack A. Dalla-Favera R. Science. 1999; 283: 676-679Crossref PubMed Scopus (277) Google Scholar) from each RatNBS or RatCMV clone with the anti-NBS1 antibody (1.2 μg), and the immune complexes were incubated overnight with protein-G (Pierce, Rockford, IL). The immunoprecipitate was washed three times and mixed with 1× Laemmli dye, boiled for 10 min, loaded onto an 8% SDS gel, and processed using a standard procedure with an anti-NBS1 antibody. Data shown here are representative of two or more experiments from independent cell cultures.Fig. 3Regulation of NBS1 by endogenous c-Myc as demonstrated by RNAi experiments.A, Western blot analysis showed the decrease in NBS1 levels in the 293T clone expressing small interfering RNA (293TMyci clone) to repress endogenous c-myc versus the control 293T clone. B, Western blot analysis showed the decrease in NBS1 levels in three different HeLa clones expressing small interfering RNA (HeLaMyci clones) to repress endogenous c-myc versus the control HeLa clone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cloning of the Human NBS1 Promoter Region, Generation of NBS1-driven Reporter Constructs, Transfection, and Luciferase Assays—The genomic region flanking the NBS1 gene promoter region was cloned by PCR amplification of human genomic DNA, which contains 360 bp 5′ upstream of the NBS1 transcription start site, first exon (88 bp), and ∼1 kb of intron 1. HindIII and BamHI sites were created to facilitate cloning. To construct the reporter construct driven by NBS1 promoter, the fragment mentioned above (containing the E-box site) was subcloned into the HindIII-BglII sites of the pXP2 vector to generate the NBSLuc parental construct (28Wu K.J. Grandori C. Amacker M. Simon-Vermot N. Polack A. Lingner J. Dalla-Favera R. Nat. Genet. 1999; 21: 220-224Crossref PubMed Scopus (769) Google Scholar). The ATG translation initiation codon of NBS1 was changed to GTG by site-directed mutagenesis to ensure translation of luciferase open reading frame and to make the NBSLuc1500 construct. This reporter construct was co-transfected with pMT2TMyc or its deletion mutants into 293T cells using the calcium phosphate transfection method, and their luciferase activities were assayed. The NBSLuc1500Mut construct was made by changing the sequence from CACGTG to CACCTG in the NBSLuc1500 construct by site-directed mutagenesis. 1 pmol of a c-myc wild type expression vector (pMT2TMyc) or a control vector (pMT2T) was co-transfected into 293T cells with 0.1 μg of reporter plasmids using the calcium phosphate transfection method. The total amount of transfected DNA and pMT2T sequences were kept constant in each experiment. A plasmid expressing the bacterial β-galactosidase gene (0.2 μg, pCMVβgal) was also co-transfected in each experiment to serve as an internal control for transfection efficiency. At 48 h after transfection, cells were harvested and transcriptional activity was assayed as a function of luciferase activity. The values are expressed as luciferase activity after normalization with β-galactosidase activity for efficiency of transfection. Each transfection was performed in triplicate, and standard deviation values are shown. Data shown here are representative of three or more experiments from independent transfections. Chromatin Immunoprecipitation Assay—A ChIP assay was performed following the detailed protocol as described previously (10Frank S.R. Schroeder M. Fernandez P. Taubert S. Amati B. Genes Dev. 2001; 15: 2069-2082Crossref PubMed Scopus (420) Google Scholar, 32Wu K.J. Mattioli M. Morse III, H.C. Dalla-Favera R. Oncogene. 2002; 21: 7872-7882Crossref PubMed Scopus (38) Google Scholar). Briefly, cells were cross-linked with 1% formaldehyde for 10 min and stopped by adding glycine to a final concentration of 0.125 m. Fixed cells were washed twice with TBS (20 mm Tris, pH 7.5, 150 mm NaCl) and harvested in 5 ml of SDS buffer (50 mm Tris, pH 8.0, 0.5% SDS, 100 mm NaCl, 5 mm EDTA, and protease inhibitors). Cells were pelleted by centrifugation and suspended in 2 ml of IP buffer (100 mm Tris, pH 8.6, 0.3% SDS, 1.7% Triton X-100, 5 mm EDTA). Cells were sonicated with a 0.25-inch diameter probe for 15 s twice using an MSE-Soniprep1500 sonicator (setting 18). For each immunoprecipitation, 1 ml of lysate was precleared by adding 50 μl of blocked protein A beads (50% protein A-Sepharose, Amersham Biosciences; 0.5 mg/ml bovine serum albumin, 0.2 mg/ml salmon sperm DNA) at 4 °C for 1 h. Samples were spun, and the supernatants were incubated at 4 °C for 3 h with no antibody, or a polyclonal antibody specific for either c-Rel (SC-71, Santa Cruz Biotechnology) or c-Myc (SC-764, Santa Cruz Biotechnology). Immune complexes were recovered by adding 50 μl of blocked protein A beads and incubated overnight at 4 °C. Beads were successively washed with 1) mixed micelle buffer (20 mm Tris, pH 8.1, 150 mm NaCl, 5 mm EDTA, 5% w/v sucrose, 1% Triton X-100, 0.2% SDS), 2) buffer 500 (50 mm Hepes, pH 7.5, 0.1% w/v deoxycholic acid, 1% Triton X-100, 500 mm NaCl, 1 mm EDTA), 3) LiCl detergent wash buffer (10 mm Tris, pH 8, 0.5% deoxycholic acid, 0.5% Nonidet P-40, 250 mm LiCl, 1 mm EDTA), and 4) TE buffer (10 mm Tris, 1 mm EDTA) and then eluted with 1% SDS and 0.1 m NaHCO3. 20 μl of 5 m NaCl was added to the elutes, and the mixture was incubated at 65 °C for 4 h to reverse the cross-linking. After digestion with proteinase K, the solution was phenol/chloroform-extracted and ethanol-precipitated. DNA fragments were resuspended in 50 μl of water. 5 μl of the resuspended DNA was used in a PCR reaction for 25 cycles. 28 cycles were used for immunoprecipitated chromatin from EREB.TCMyc cells. The primers used were: 5′-ATTGGCAAAGATCTATGTAGAG-3′ and 5′-TGCCCTACCAGATGGCAAAAT-3′ for intron 1 fragment (440 bp in size); 5′-GTTGCTTTGGAGTAAGCTTTTAAAATTCT-3′ and 5′-CTCTACATAGATCTTTGCCAAT-3′ for proximal promoter fragment (290 bp in size). Input was 2% of total input lysate. The data shown here are representative of two or more experiments from independent immunoprecipitations. MTT and BrdUrd Incorporation Assays—MTT assays were performed using the MTT kit (Roche Diagnostics GmbH, Mannheim, Germany). 5 × 103 Rat1a cells were seeded in 96-well microtiter plates and cultured for 24 and 72 h. MTT solution (30 μl, 5 mg/ml) was added and incubated for 4 h. 100 μl of Me2SO was added to dissolve formazan crystals under vigorous shaking for 30 min to detect the absorption using an enzyme-linked immunosorbent assay reader. BrdUrd incorporation assays were performed using the BrdUrd incorporation assay kit (Roche Diagnostics GmbH, Mannheim, Germany). 104 Rat1a cells were cultured in 96-well microtiter plates at 37 °C for 8 h and then serum-starved for 3 days. Subsequently, BrdUrd was added and incubated with cells for 4 h to label newly synthesized cellular DNA. After the labeling period, the cells were fixed and the cellular DNA was denatured in one step by adding FixDenat. Anti-BrdUrd-POD (peroxidase) was added to detect the immune complexes by subsequent substrate reaction. The reaction product was quantified by measuring the absorbance from 370 (reading) to 492 nm (background) using an enzyme-linked immunosorbent assay reader. Activation of NBS1 Expression by c-Myc in Different Physiological Conditions and in Multiple Cell Types—To characterize NBS1 as a potential c-Myc target gene, we used a human Epstein-Barr virus-immortalized B cells (CB33) engineered by transfection to express high levels of either c-Myc·MAX (CBMyc.Max) or MAX·MAX (CBMax) complexes, representing the two regulatory situations of high and low c-Myc function, respectively (25Gu W. Cechova K. Tassi V. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2935-2939Crossref PubMed Scopus (133) Google Scholar). The phenotypes of these cells were previously characterized and shown to be consistent with their differential c-Myc levels, because CBMyc.Max cells display a short doubling time, have clonogenic properties in vitro and cause tumors in vivo, whereas CBMax cells proliferate slowly and lack any transformation-related phenotype (25Gu W. Cechova K. Tassi V. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2935-2939Crossref PubMed Scopus (133) Google Scholar). Northern blot analysis of these cell lines showed that the expression of NBS1 RNA was significantly increased (∼2.5-fold) in CBMyc.Max cells when compared with CB control or CBMax cells (Fig. 1A). To examine whether increased NBS1 expression was associated with increased protein levels, cell extracts from CB33 control, CBMyc.Max, and CBMax cells were analyzed by Western blot using an anti-NBS1 antibody (Fig. 1B). The results showed that CB33Myc.Max cells have increased levels (∼3-fold) of NBS1 protein. To further confirm the relationship between c-Myc and the NBS1 gene, we exploited a previously described B cell line (EREB.TCMyc) in which proliferation and c-Myc expression could be independently controlled (26Wu K.J. Polack A. Dalla-Favera R. Science. 1999; 283: 676-679Crossref PubMed Scopus (277) Google Scholar). Because in these cells the Epstein-Barr virus EBNA-2 gene (which induces B cell immortalization) is expressed as a chimeric fusion with the hormone binding domain of the estrogen receptor, estradiol (E2) removal leads to growth arrest (G0/G1) associated with complete down-regulation of endogenous c-myc expression (Fig. 2A, +TC/-E2 lanes) (27Kempkes B. Spitkovsky D. Jansen-Durr P. Ellwart J.W. Kremmer E. Delecluse H.J. Rottenberger C. Bornkamm G.W. Hammerschmidt W. EMBO J. 1995; 14: 88-96Crossref PubMed Scopus (214) Google Scholar). Because these cells have been transfected with a tetracycline (TC)-repressed c-myc vector, exogenous c-myc expression can then be induced by TC withdrawal. Induction of c-myc is not sufficient to cause cell cycle entrance, and the cells remain quiescent and viable for several days (26Wu K.J. Polack A. Dalla-Favera R. Science. 1999; 283: 676-679Crossref PubMed Scopus (277) Google Scholar). We analyzed NBS1 expression upon c-myc induction in the same cells by Northern blot analysis. The results showed that withdrawal of estrogen and tetracycline (causing induction of exogenous c-myc) causes a ∼2.5-fold increase in the expression of NBS1 RNA (Fig. 2A, -TC/-E2 lane). Twenty-four hours after E2 withdrawal, cells were examined for proliferation by flow cytometry analysis of DNA content and found arrested at G0/G1 as previously described (26Wu K.J. Polack A. Dalla-Favera R. Science. 1999; 283: 676-679Crossref PubMed Scopus (277) Google Scholar); no change in cell cycle activity was detectable after induction of c-myc by TC withdrawal. This result also indicates that c-Myc induces NBS1 expression in the absence of cell proliferation. The levels of mouse Nbs1 expression also increased (∼3-fold) in NIH3T3 cells overexpressing c-Myc when compared with control NIH3T3 cells (Fig. 2B), indicating the activation of mouse Nbs1 by c-Myc in a fibroblast background. Taken together, these results indicate that increased c-myc expression is associated with increased NBS1 RNA and protein levels in different physiological conditions and in multiple cell types. Regulation of NBS1 by Endogenous c-Myc—To test whether NBS1 is regulated by endogenous c-Myc, we performed RNA interference (RNAi) experiments on 293T and HeLa cells to knockdown endogenous c-Myc expression and examine the subsequent decrease in NBS1 expression. Fig. 3A shows that the endogenous c-Myc levels were repressed in the 293TMyci clone compared with the control 293T clone. The NBS1 protein level also decreased in the 293TMyci clone. In addition, Fig. 3B showed that the endogenous c-Myc expression was repressed in three different HeLaMyci clones compared with the control HeLa clone. The downstream NBS1 expression also significantly decreased in these HeLaMyci clones. The results of RNAi experiments on two different cell lines demonstrated the regulation of NBS1 by endogenous c-Myc. Activation of NBS1 by c-Myc in the Absence of de Novo Protein Synthesis—To investigate whether c-Myc-mediated up-regulation of NBS1 gene expression was direct, we studied NBS1 expression in EREB cells (EREB.MycER™) expressing an inactive c-Myc fused to a mutant estrogen receptor domain (MYCER™) (28Wu K.J. Grandori C. Amacker M. Simon-Vermot N. Polack A. Lingner J. Dalla-Favera R. Nat. Genet. 1999; 21: 220-224Crossref PubMed Scopus (769) Google Scholar). This protein can be rapidly activated by 4-hydroxytamoxifen (TM), but not by estradiol; in turn, TM can activate c-MYCER™, but not ER-EBNA-2, allowing for the selective activation of pre-existing c-MYCER™ in resting B cells (33Sasson S. Pathol. Biol. (Paris). 1991; 39: 59-69PubMed Google Scholar, 34Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (701) Google Scholar). By this approach and by simultaneous treatment with the protein synthesis inhibitor cycloheximide (CX), we examined whether NBS1 gene expression could be regulated upon activation of pre-existing c-MYCER™ in the absence of de novo protein synthesis. Fig. 4A showed that TM treatment led to a significant (∼6.5- to 8-fold) and rapid (detectable after 1 h) increase in NBS1 mRNA levels. This induction was only slightly abolished by co-treatment with CX (CX plus TM) (Fig. 4, B and C), indicating that induction of the NBS1 gene expression involves a mechanism that is mostly independent of de novo protein synthesis. Control experiments using EREB control cells treated with TM did not cause the induction of NBS1 expression (data not shown). The rapid kinetics of c-Myc-induced up-regulation and its independence of cellular proliferation and new protein synthesis are consistent with a direct effect of c-Myc activation on NBS1 gene expression. Activation of NBS1 by c-Myc Requires an E-box Site Located in the Intron 1 Region—To ascertain whether c-Myc up-regulates NBS1 gene expression at the transcriptional level, we first investigated whether the NBS1 promoter region contained c-Myc·MAX binding sites. The genomic organization of the 5′ flanking region of human NBS1 gene, including exon 1, is schematically reported in Fig. 5A. This region includes 360 bp of upstream sequence from the NBS1 transcription start site, exon 1 (88 bp), and ∼1 kb of partial intron 1 sequence. An E-box site (CACGTG) is located within intron 1. We analyzed whether the c-Myc·MAX binding site could mediate transcriptional activation of the NBS1 gene by c-Myc, by studying whether c-Myc could activate the transcription of a reporter gene linked to NBS1 promoter sequences. To this end, we constructed a reporter vector (NBSLuc1500, Fig. 5A) containing segments of the 5′-region, exon 1, and part of the intron 1 sequence of the NBS1 gene linked to the promoterless coding domain of the luciferase gene (Luc) (28Wu K.J. Grandori C. Amacker M. Simon-Vermot N. Polack A. Lingner J. Dalla-Favera R. Nat. Genet. 1999; 21: 220-224Crossref PubMed Scopus (769) Google Scholar). NBSLuc1500 was then co-transfected with either the control pMT2T or the pMT2TMyc expression vector into 293T cells. Fig. 5B showed that c-Myc was able to activate the expression of the NBSLuc1500 construct at levels (∼2.5-fold) comparable to those observed for other c-Myc target genes in this type of assay. To determine whether the intronic E-box site was responsible for c-Myc-mediated activation, its sequence was changed from CACGTG to CACCTG using site-directed mutagenesis, and the resulting reporter construct (NBSLuc1500Mut) was tested for c-Myc responsiveness in transient co-transfection assay. The results (Fig. 5B) showed that this mutant construct could not be activated by c-Myc, thus indicating that the E-box within intron 1 of the NBS1 gene mediates transcriptional activation by c-Myc. To further characterize the mechanism involved in c-Myc-mediated transactivation of NBS1, we tested the function of c-Myc mutants lacking the transactivation domain (pMT2TMyc(Δ7–91)) or heterodimerization domain (pMT2TMyc(Δ371–412)) (35Stone J. de Lange T. Ramsay G. Jakobovits E. Bishop J.M. Varmus H. Lee W. Mol. Cell. Biol. 1987; 7: 1697-1709Crossref PubMed Scopus (331) Google Scholar). These two mutants were unable to induce the expression of the NBSLuc1500 reporter gene (Fig. 5C), indicating that c-Myc-mediated activation of NBS1 transcription required both the transactivation domain and heterodimerization with MAX, as expected for physiological c-Myc function. To determine whether c-Myc binds to the intronic E box in vivo, a chromatin immunoprecipitation (ChIP) assay using CB33Myc.Max cells was performed (10Frank S.R. Schroeder M. Fernandez P. Taubert S. Amati B. Genes Dev. 2001; 15: 2069-2082Crossref PubMed Scopus (420) Google Scholar, 32Wu K.J. Mattioli M. Morse III, H.C. Dalla-Favera R. Oncogene. 2002; 21: 7872-7882Crossref PubMed Scopus (38) Google Scholar). Fig. 5D (panel a) showed that c-Myc, but not c-Rel, binds to the intronic E-box as demonstrated by the c-Myc immunoprecipitated chromatin containing the intronic fragment amplifiable to generate a 440-bp fragment. The control experiment carried out to amplify an NBS1 proximal promoter fragment (290 bp), which did not contain an E-box, failed to generate an amplifiable fragment (Fig. 5D, panel b). The 440-bp fragment was also amplifiable from the c-Mycimmunoprecipitated EREB.TCMyc(+TC/+E2) chromatin, demonstrating the binding of endogenous c-Myc to the intronic E-box site (Fig. 5D, panel c). In addition, the 440-bp fragment was not amplifiable in c-Myc-immunoprecipitated chromatin prepared from CBMax cells (Fig. 5D, panel d) or quiescent EREB.TCMyc(+TC/-E2) cells (data not shown). Positive control using the c-Myc-immunoprecipitated chromatin from CBMyc.Max cells to amplify the promoter of a known c-Myc target gene, TERT (telomerase reverse transcriptase), generated an amplifiable fragment (data not shown) (32Wu K.J. Mattioli M. Morse III, H.C. Dalla-Favera R. Oncogene. 2002; 21: 7872-7882Crossref PubMed Scopus (38) Google Scholar). Taken together, these results indicate that c-Myc can induce direct activation of the NBS1 transcription by binding to the E-box site within the NBS1 intron 1 region. Induction of Cell Proliferation in Rat1a Cells by Overexpression of NBS1—To explore the role of NBS1 induction in c-Mycinduced cell growth and proliferation, we tested whether NBS1-overexpressing Rat1a cells exhibit accelerated growth and proliferation phenotypes. An NBS1 expression vector was stably transfected into Rat1a cells, and the transfected Rat1a clones (RatNBS) were tested for their ability to increase cell proliferation and DNA synthesis rate by MTT and BrdUrd incorporation assays. Fig. 6A showed that two different Rat1a clones overexpressed NBS1 protein levels (RatNBS) compared with two control clones (RatCMV) by immunoprecipitation and Western blot analysis. Increased cell proliferation rate (∼70 to 100% increase) and DNA synthesis (∼25% increase) were observed in the RatNBS clones compared with the control RatCMV clones using MTT and BrdUrd incorporation assays (Fig. 6, B and C). These results indicate that overexpression of NBS1 in Rat1a cells increased cell proliferation and DNA synthesis rate, consistent with the role of NBS1 in c-Myc-mediated cell growth and proliferation. These results present the first demonstration of a DNA DSB repair gene, NBS1, as a direct c-Myc target gene. Our results demonstrated that NBS1 is activated by c-Myc in different c-Myc-overexpressing cell lines. RNAi experiments showed the regulation of NBS1 by endogenous c-Myc. EREB.MycER experiments, transient transfection, and chromatin immunoprecipitation assays showed that the activation of NBS1 by c-Myc is direct. Nbs1 is expressed in highly proliferating tissues developmentally (22Wilda M. Demuth I. Concannon P. Sperling K. Hameister H. Hum. Mol. Genet. 2000; 9: 1739-1744Crossref PubMed Scopus (30) Google Scholar), coinciding with the timing of myc expression. Nbs1 null mice are embryonic lethal (36Zhu J. Petersen S. Tessarollo L. Nussenzweig A. Curr. Biol. 2001; 11: 105-109Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Mutant blastocysts from these Nbs1 null mice showed greatly diminished expansion of the inner cell mass in culture, suggesting that Nbs1 mediates essential functions during cell growth and proliferation (36Zhu J. Petersen S. Tessarollo L. Nussenzweig A. Curr. Biol. 2001; 11: 105-109Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Primary or immortalized fibroblasts from NBS patients contain hypomorphic mutants of NBS1 (37Maser R.S. Zinkel R. Petrini J.H.J. Nat. Genet. 2001; 27: 417-421Crossref PubMed Scopus (182) Google Scholar). These hypomorphic NBS cells grow slower than NBS cells complemented with wild type NBS1 (38Ranganathan V. Heine W.F. Ciccone D.N. Rudolph K.L. Wu X. Chang S. Hai H. Ahearn I.M. Livingston D.M. Resnick I. Rosen F. Seemanova E. Jarolim P. DePinho R.A. Weaver D.T. Curr. Biol. 2001; 11: 962-968Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In addition, mouse embryo fibroblast cells from Nbs1m/m mice (similar to human mutants) have impaired cellular proliferation (39Kang J. Bronson R.T. Xu Y. EMBO J. 2002; 21: 1447-1455Crossref PubMed Scopus (140) Google Scholar). The results of increased cellular proliferation in Rat1a cells overexpressing NBS1 (Fig. 6) are consistent with all of the above observations (22Wilda M. Demuth I. Concannon P. Sperling K. Hameister H. Hum. Mol. Genet. 2000; 9: 1739-1744Crossref PubMed Scopus (30) Google Scholar, 36Zhu J. Petersen S. Tessarollo L. Nussenzweig A. Curr. Biol. 2001; 11: 105-109Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 38Ranganathan V. Heine W.F. Ciccone D.N. Rudolph K.L. Wu X. Chang S. Hai H. Ahearn I.M. Livingston D.M. Resnick I. Rosen F. Seemanova E. Jarolim P. DePinho R.A. Weaver D.T. Curr. Biol. 2001; 11: 962-968Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 39Kang J. Bronson R.T. Xu Y. EMBO J. 2002; 21: 1447-1455Crossref PubMed Scopus (140) Google Scholar). All these results support the role of increased NBS1 expression in promoting c-Myc-mediated cell growth and proliferation. Although many c-Myc target genes were identified (1Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1030) Google Scholar, 11Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1379) Google Scholar, 40Watson J.D. Oster S.K. Shago M. Khosravi F. Penn L.Z. J. Biol. Chem. 2002; 277: 36921-36930Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 41Menssen A. Hermeking H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6274-6279Crossref PubMed Scopus (314) Google Scholar), NBS1 represents one of the relatively limited numbers of genes that are direct as opposed to secondary targets for c-Myc transcriptional activation. NBS1 represents the first c-Myc target gene linked to DNA DSB repair, recombination, and replication. Recent experiments using the serial assessment of gene expression approach also identified APEX/Ref-1, BRCA1, and MSH2 as putative c-Myc target genes related to DNA repair (41Menssen A. Hermeking H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6274-6279Crossref PubMed Scopus (314) Google Scholar). However, these putative target genes were not yet extensively characterized. In Saccharomyces cerevisiae, DNA repair genes are required during DNA replication to preserve the integrity of the genome (42Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Given the fact that spontaneous DNA DSBs are generated frequently during DNA replication, activation of NBS1 by c-Myc may be required physiologically to preserve the integrity of the genome and facilitate DNA synthesis/replication during cell cycle progression in highly proliferating tissues. Prevention of DSB accumulation by Mre11 complex as demonstrated in Xenopus experiments is consistent with this model (24Costanzo V. Robertson K. Bibikova M. Kim E. Grieco D. Gottesman M. Carroll D. Gautier J. Mol. Cell. 2001; 8: 137-147Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Taken together, NBS1 is the first c-Myc target gene linked to DNA DSB repair, thereby further elucidating the molecular mechanism of cell growth promotion induced by c-Myc. We thank Dr. R. Dalla-Favera for critical comments on the manuscript and Drs. P. Concannon and R. Agami for the generous gift of full-length human NBS1 cDNA and pSUPER plasmid. We thank C. T. Lee, C. P. Lin, S. F. Tseng, and Y. Y. Chen for their excellent technical assistance. K. J. W. thanks Dr. S. Y. Shieh of the Academia Sinica for the initial experiments performed at her laboratory.

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