c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair.

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 (1,2). 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 (1,(3)(4)(5). 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) 1 as heterodimers, and an amino-terminal domain necessary for transcriptional transactivation (2,6,7). The heterodimeric complexes c-Myc⅐MAX 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 (8 -10).
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 (11). In Drosophila as well as in mammalian cells, c-Myc has been shown to promote cell growth (12,13), 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 (14). 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 (15,16). 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 (15). Increased radiation sensitivity and radioresistant DNA synthesis of NBS fibroblasts are similar to the cellular features of ataxia telangiectasis cells (17), which is demonstrated by the recent results showing that ataxia telangiectasis-mutated protein phosphorylates NBS1 (18 -20), 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 (15,21). However, NBS1 is expressed in highly proliferating tissues developmentally (22) and is located at sites of DNA synthesis through interaction with E2F (23). In addition, Mre11 complex is able to prevent DSB accumulation during chromosomal DNA synthesis to ensure cell cycle progression (24). 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.

EXPERIMENTAL PROCEDURES
Cell Lines and Plasmids-The lymphoblastoid cell lines, EREB.TC-Myc and EREB.MycER TM cells were previously described (25)(26)(27)(28). The NIH3T3.Myc cell line was generated by transfecting pSV2Neo and pMT2TMyc into NIH3T3 cells and selecting under G418 (250 g/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 g/ml) or G418 (400 g/ml). RatNBS cell lines were generated by transfecting the HeBOCMVNBS plasmid into Rat1a cells and selecting under G418 (400 g/ml). The pHeBOCMVNBS plasmid was constructed by PCRmediated generation of a 2.34-kb fragment of the full-length human NBS1 cDNA from the plasmid pBS-NBS1 obtained from Dr. P. Concannon (29). The primers used to generate this fragment are: 5Ј-CCGGT-TACGCGCTAGCACGTCGGCCC-3Ј and 5Ј-TTGGCCTGAAGCGGC-CGCTTACTAGGAA-3Ј. This fragment was subcloned into the NheI-NotI sites of HeBOCMV vector (25) to make the HeBOCMVNBS expression vector, and it was verified by sequencing. The pSUPERMyci plasmid was generated by inserting the oligonucleotide of 5Ј-GATCCCCTTCAGAATAGAGTATGAGCCTTCAAGAGAGGCTCAT-ACTCTATTCTGAATTTTTGGAAA-3Ј into the pSUPER plasmid provided by Dr. R. Agami (30).
Northern Blot Analysis-3 g of poly(A) RNA was used in each lane of the Northern blots. In EREB.MycER TM Northern blots, 6 g 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: 5Ј-GTACGTTGTTGGGAGGAA-3Ј and 5Ј-CTGGAGGCTGTTTCTTAG-3Ј. 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 (31). 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 ␤-actin probe were previously described (28). Data shown here are representative of two or more experiments from independent cell cultures.
Western Blot Analysis and Immunoprecipitation Procedure-60 g (CB control, CBMyc.Max, and CBMax, Fig. 1B) or 100 g (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-␤-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 g of whole cell extract prepared under cell lysis buffer (26) from each RatNBS or RatCMV clone with the anti-NBS1 antibody (1.2 g), and the immune complexes were incubated overnight with protein-G (Pierce, Rockford, IL). The immunoprecipitate was washed three times and mixed with 1ϫ 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.
Cloning of the Human NBS1 Promoter Region, Generation of NBS1driven 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 5Ј upstream of the NBS1 transcription start site, first exon (88 bp), and ϳ1 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 (28). 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 g 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 ␤-galactosidase gene (0.2 g, pCMV␤gal) was also cotransfected 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 ␤-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.
MTT and BrdUrd Incorporation Assays-MTT assays were performed using the MTT kit (Roche Diagnostics GmbH, Mannheim, Germany). 5 ϫ 10 3 Rat1a cells were seeded in 96-well microtiter plates and cultured for 24 and 72 h. MTT solution (30 l, 5 mg/ml) was added and incubated for 4 h. 100 l of Me 2 SO 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). 10 4 Rat1a cells were cultured in 96-well microtiter plates at 37°C for 8 h and then serumstarved 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 enzymelinked 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-Myc⅐MAX (CB-Myc.Max) or MAX⅐MAX (CBMax) complexes, representing the two regulatory situations of high and low c-Myc function, respectively (25). 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 (25). Northern blot analysis of these cell lines showed that the expression of NBS1 RNA was significantly increased (ϳ2.5-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 (ϳ3-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 (26). 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 (G 0 /G 1 ) associated with complete down-regulation of endogenous c-myc expression ( Fig.  2A, ϩTC/ϪE2 lanes) (27). 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 (26). 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 ϳ2.5-fold increase in the expression of NBS1 RNA ( Fig. 2A, ϪTC/ϪE2 lane). Twenty-four hours after E2 withdrawal, cells were examined for proliferation by flow cytometry analysis of DNA content and found arrested at G 0 /G 1 as previously described (26); 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 (ϳ3-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 upregulation of NBS1 gene expression was direct, we studied NBS1 expression in EREB cells (EREB.MycER TM ) expressing an inactive c-Myc fused to a mutant estrogen receptor domain (MYCER TM ) (28). This protein can be rapidly activated by 4-hydroxytamoxifen (TM), but not by estradiol; in turn, TM can activate c-MYCER TM , but not ER-EBNA-2, allowing for the selective activation of pre-existing c-MYCER TM in resting B cells (33,34). 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 TM in the absence of de novo protein synthesis. Fig. 4A showed that TM treatment led to a significant (ϳ6.5-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-Myc⅐MAX binding sites. The genomic organization of the 5Ј 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 ϳ1 kb of partial intron 1 sequence. An E-box site (CACGTG) is located within intron 1. We analyzed whether the c-Myc⅐MAX 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 5Ј-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) (28). 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 (ϳ2.5-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-medi-ated transactivation of NBS1, we tested the function of c-Myc mutants lacking the transactivation domain (pMT2TMyc(⌬7-91)) or heterodimerization domain (pMT2TMyc(⌬371-412)) (35). 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 (10,32). 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(ϩTC/ϩE2) 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(ϩTC/ϪE2) cells (data not shown). Positive control using the c-Myc-immunoprecipitated chromatin from CB-Myc.Max cells to amplify the promoter of a known c-Myc target gene, TERT (telomerase reverse transcriptase), generated an amplifiable fragment (data not shown) (32). 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 (ϳ70 to 100% increase) and DNA synthesis (ϳ25% increase) were ob-  500 nM). B, Northern blot analysis of EREB.MycER TM cells upon activation of MYCER TM by TM in the presence of cycloheximide (CX, 10 g/ ml) or with CX alone. Both blots were sequentially hybridized to an NBS1 probe, to a probe recognizing the MYC-ER TM RNA, to a c-myc exon-1 probe (endogenous c-myc RNA), and to a ␤-actin probe as a control for RNA loading. C, quantification analysis of the experiments shown in A and B by phosphorimaging analysis. served 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. DISCUSSION 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 (22), coinciding with the timing of myc expression. Nbs1 null mice are embryonic lethal (36). 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 (36). Primary or immortalized fibroblasts from NBS patients contain hypomorphic mutants of NBS1 (37). These hypomorphic NBS cells grow slower than NBS cells comple- mented with wild type NBS1 (38). In addition, mouse embryo fibroblast cells from Nbs1 m/m mice (similar to human mutants) have impaired cellular proliferation (39). The results of increased cellular proliferation in Rat1a cells overexpressing NBS1 (Fig. 6) are consistent with all of the above observations (22,36,38,39). 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 (1,11,40,41), 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 (41). 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 (42). 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 (24). 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.