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Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis

Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis 446–453 Nucleic Acids Research, 2000, Vol. 28, No. 2 © 2000 Oxford University Press Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis Peter J. Greasley, Claude Bonnard and Bruno Amati* Cellular Growth Control Unit and Biocomputing Unit, Swiss Institute for Experimental Cancer Research (ISREC), 155 ch. des Boveresses, CH-1066 Epalinges, Switzerland Received October 4, 1999; Revised and Accepted November 11, 1999 DDBJ/EMBL/GenBank accession no. AF142779 ABSTRACT results in their constitutive expression (2). In normal cells, expression of c-myc is strictly dependent on mitogenic signals The c-Myc oncoprotein and its dimerization partner and is suppressed by growth inhibitory and differentiation Max bind the DNA core consensus sequence inducing signals (2,3). c-myc expression is also regulated by CACGTG (E-box) and activate gene transcription. the products of other oncogenes or tumor suppressor genes, However, the low levels of induction have hindered the such as tyrosine kinase receptors (4) or the APC/ -catenin/ identification of novel Myc target genes by differential TCF pathway (5). This may explain why c-myc is sometimes screening techniques. Here, we describe a computer- overexpressed in tumors without being mutated. based pre-selection of candidate Myc/Max target The c-myc protein product (Myc) conveys strong mitogenic and apoptotic stimuli. Constitutive expression of Myc reduces genes, based on two restrictive criteria: an extended growth factor requirements, prevents growth arrest by a variety of E-box consensus sequence for Myc/Max binding and growth inhibitory signals, and can block cellular differentiation the occurrence of this sequence within a potential (reviewed in 2,6). Conversely, activation of a conditional genomic CpG island. Candidate genes selected by Myc–estrogen receptor chimera (Myc-ER) (7) in quiescent these criteria were evaluated experimentally for their cells induces entry into the cell cycle in the absence of response to Myc. Two Myc target genes are charac- mitogens (8,9). However, concomitant with their mitogenic terized here in detail. These encode nucleolin, an action, Myc or Myc-ER can induce apoptosis if survival abundant nucleolar protein, and BN51, a co-factor of factors are absent from the extracellular environment (10,11; RNA polymerase III. Myc activates transcription of reviewed in 2,3). Myc-induced apoptosis has been linked to ARF1 both genes via E-boxes located in their first introns, activation of the p19 –p53 pathway and is separable from as seen for several well-characterized Myc targets. mitogenesis (12). Myc is a transcription factor of the basic helix–loop–helix For both genes, mutation of the E-boxes abolishes leucine zipper (bHLH-Zip) family. Myc dimerizes with transcriptional activation by Myc as well as repression another bHLH-Zip protein, Max, to bind the specific DNA by Mad1. In addition, the BN51 promoter is selectively sequence CACGTG (the E-box) and activate transcription activated by Myc and not by USF, another E-box- (13,14; reviewed in 3,15). Transcription-competent Myc/Max binding factor. Both nucleolin and BN51 are implicated dimers are the active form of Myc in inducing cell cycle in the maturation of ribosomal RNAs, albeit in progression, apoptosis and malignant transformation (3,15). In different ways. We propose that Myc, via regulation addition to its function as a transactivator, Myc can also of these and probably many other transcriptional repress transcription of several genes that are clearly relevant targets, may be an important regulator of ribosome to its biological function (16–18). In fact, some reports have biogenesis. suggested that transcription repression, rather than activation, correlates with the biological activity of Myc (19,20; reviewed in 3,21,22). There is, however, no clear demonstration that INTRODUCTION gene repression is a direct action of Myc in vivo, rather than the indirect consequence of activating as yet unknown Myc target The myc family of mammalian proto-oncogenes includes three genes (see also Discussion). The molecular mechanisms evolutionarily conserved genes c-, N- and L-myc,which proposed to explain transcription repression by Myc (21,22) encode related proteins. myc genes are differentially expressed therefore remain speculative. during embryonic development (1) and proliferating post-natal tissues, with few exceptions, express c-myc (2). Oncogenic Max also forms heterodimers with the other bHLH-Zip activation of these genes, in particular c-myc, is observed in a proteins Mad1, Mxi-1 (or Mad2), Mad3, Mad4 and Mnt (or wide range of human and animal neoplasias and generally Rox) (23–27). These alternative dimers also bind the E-box *To whom correspondence should be addressed at present address: DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304, USA. Tel: +1 650 858 7528; Fax: +1 650 496 1200; Email: [email protected] Present address: Peter J. Greasley, Institute of Pharmacology and Toxicology, University of Lausanne, 27 rue du Bugnon, CH-1005 Lausanne, Switzerland Nucleic Acids Research, 2000, Vol. 28, No. 2 447 and actively repress transcription and can therefore antagonize tamoxifen (OHT) (Laboratoires Besins-Isovesco, Paris, France). both the transcription and transforming activities of Myc. Cells were harvested at various time points by scraping and Furthermore, the myc and mad family genes are generally stored frozen until required for nuclear run-on or RNA preparation. regulated in opposite modes in growth control and development Preparation of filters for nuclear run-on and reverse (25,28; reviewed in 3). In summary, Myc, Max and Mad northern analysis proteins form a network that regulates gene expression, proliferation, apoptosis and differentiation. Plasmid DNAs containing the genes of interest were isolated To further understand the function of Myc, Max and Mad, it from Escherichia coli using the Promega Wizard miniprep is necessary to identify the genes that are directly regulated by DNA extraction kit. Aliquots of 12 g of each DNA were these proteins, in particular, Myc-induced and/or Mad-repressed ethanol precipitated, resuspended in 200 lH O and denatured genes. Known Myc target genes include -prothymosin ( PT), by addition of 20 l2 NNaOHand heating at95 C for 10 min. ornithine decarboxylase (ODC), Cdc25A, cad, MrDb, ECA39, Denatured DNA was then transferred to a second tube EIF-4E, LDH-A, rcl, E2F-2, RCC1, the telomerase catalytic containing 600 l ice-cold 10 SSC(1.5M NaCl, 0.15M Na- subunit hTERT and others (reviewed in 3,29,30). Although citrate, pH 7.0). The solution was neutralized by the addition of ODC, Cdc25A, LDH-A, rcl, E2F-2, PT and hTERT share 44 l 2 M HEPES (free acid) and divided equally between two some of the potential of Myc to induce cell cycle progression, wells on a 48-well slot blotter, so as to produce two identical apoptosis, immortalization and/or transformation (31–35), replica blots. Each blot contained actin, tubulin, GAPDH and additional target genes must be identified to explain the whole 36B4 as internal standards, and ODC as a positive control for a range of biological effects controlled by Myc (6). A common Myc-regulated gene. The filters were washed twice in 6 SSC feature of these genes is the low magnitude of their response to and allowed to air dry. After drying the filters were baked Myc. Generally, induction by Myc results in only 2- to 5-fold undervacuum for2 hat 80 C. increases, making identification of novel targets difficult. Nuclear run-on Indeed, only a few of the known targets have been identified by differential screening techniques, the majority of Myc-regulated Cells from two 15 cm dishes were resuspended in 8 ml ice-cold genes being postulated by the presence of E-box sequences buffer A (10 mM Tris–HCl, pH 7.5, 5 mM MgCl ,10mM (30). In the present study, we describe a computerized NaCl, 0.5 mM DTT), incubated on ice for 20 min, centrifuged, screening strategy so as to facilitate initial identification of resuspended in 2 ml buffer A and lysed by rapid pipetting of potential Myc-regulated genes, and characterize two novel the suspension. Nuclei were recovered by centrifugation at Myc targets, nucleolin and BN51. 2000 r.p.m. for 5 min, washed once in 2 ml buffer B (20 mM Tris pH 7.5, 10 mM MgCl , 140 mM KCl, 20% glycerol, 0.5 mM DTT), resuspended in buffer B supplemented with MATERIALS AND METHODS 0.5 mMATP,0.5 mM CTP, 0.5mMGTP,5 MUTP and Computer-based analysis of sequences in the GenBank 200 Ci [ - P]UTP, and incubated at 30 C for 15 min. The databases reaction was stopped with 1 ml ice-cold buffer C (10 mM Tris–HCl, pH 7.5, 50 mM MgCl , 500 mM NaCl, 2 mM CaCl ). RNase-free The program (cpg)was writteninCandusedthe GCG 2 2 DNase (200 U) was added to the mixture, followed by incubation package library functions (Wisconsin Package v.8, Genetics at room temperature for 5 min. The reaction mix was then Computer Group, Madison, WI) for the input of parameters, passed twice through a QIAshredder™ unit (Qiagen) and RNA nucleotide query sequences and GCG formatted database was extracted using the RNeasy™ midi-prep system (Qiagen), access (EMBL). With each perfect match for VCACGTGB in according to the manufacturer’s instructions. Prehybridization any nucleotide sequence entry of the database, the program of filters was performed in TESS2D (20 mM TES, pH 7.4, computed three values from within windows of determined 2 Denhardt’s solution, 400 mM NaCl, 2.6 mM EDTA and length around the detected match. First, the average GC 0.07% SDS) supplemented with 0.4 mg/ml yeast RNA at 65 C content expressed as the global percentage of G and C in the for 4 h. The eluant (200 l) from the RNeasy™ preparation of window, secondly, the CpG/GpC ratio and, finally, observed/ nuclear run-on RNA was added to 1 ml of TESS2D and expected CpG, defined as (no. CpG/length)/[(no. C no. G)/ hybridization allowed to take place for 24 h at 65 C. The blots length]. If the 5 -or3 -end of the sequence did not permit were then washed three times for 30 min in 1 SSC, 0.1% SDS application of the predetermined window length, the computation at 65 C. Run-on signals were quantified with a phosphorimager. was made on the available sequence. The searches presented here used the following criteria: a window size of 100 nt in both Reverse northern and northern blotting directions and a GC content 65% (in both directions from the hit). Total cellular RNA was isolated using the RNeasy™ mini prep Induction of Myc-ER cells kit (Qiagen). An aliquot of 1 g of RNA was labeled by random 32 32 priming using a mixture of [ - P]dATP and [ - P]dCTP. The We used Myc-ER clone 3, a Rat1 cell line constitutively labeled probes were isolated by spin filtration using Sephadex expressing a Myc–ER fusion protein (7,10). Cells were grown to ~40% confluence in phenol red-free DMEM medium G-50. Hybridization of slot-blotted DNAs was performed as (Gibco BRL no. 11880-028) supplemented with 10% charcoal- for nuclear run-on, apart from the use of denatured salmon sperm DNA as non-specific competitor. Northern blotting was stripped fetal calf serum to prevent activation of Myc–ER. Subconfluent cultures were serum-starved for 24 h prior to performed by standard methods using random primed DNA activation of Myc–ER by addition of 200 nM trans-4-hydroxy- probes. All signals were quantified with a phosphorimager. 448 Nucleic Acids Research, 2000, Vol. 28, No. 2 Plasmid constructions Such a strategy is dependent upon defining the nucleotide sequence preferences of Myc/Max dimers. We have previously To generate the reporter pNucL14 (Fig. 2A), the promoter, identified the extended consensus VCACGTGB (where V = A, exon 1, intron1and the first 8bpofexon2wereclonedin C or G and B = T, C or G), as the optimal E-box site for Myc/Max, pGL3basic (Promega). Digestion with NcoI and subsequent both in vitro and in vivo (37). This E-box sequence contains a treatment with S1 nuclease followed by religation destroyed central CpG dinucleotide. CpG pairs throughout mammalian the natural translation start codon located in exon 1. The genomes are generally methylated at position N7 of cytosine, product of this reaction was confirmed by sequencing. To except in small regions of the genome (~0.3–1.5 kb) called generate the mutants pNucL14mut1, pNucL14mut2 and CpG islands. In vitro Myc/Max dimers fail to recognize the E-box pNucL14mut1/2 we first subcloned the EcoRV–HindIII fragment if the central CpG dinucleotide is methylated (38). By inference, of pNucL14 into pBluescript to generate pBSnuc. This most functional E-boxes should be located within CpG islands. construct was used for PCR mutagenesis of the nucleolin gene In fact, a feature that characterizes several Myc target genes, by mutating the respective CACGTG sequences to TACGTG. including ODC, cad and LDH-A, is the location of the E-boxes Subcloning the various mutants back into pNucL14 using within CpG islands. Furthermore, CpG islands are characterized EcoRV and HindIII then produced each reporter construct. To by a distinct sequence composition (39; see below) and are generate pGL3-BN51 (Fig. 3A), a genomic DNA fragment generally located at the 5 -end of genes, frequently encompassing spanning the promoter, exon 1, intron 1 and part of exon 2 of the promoter, exon 1 and intron 1. We thus designed a screen BN51 was recovered from plasmid pBN51eco (36) with XbaI based on three main steps: (i) computer-based identification of and AflIII. This fragment was subcloned in pGL3-basic VCACGTGB motifs located within potential CpG islands (Promega) cleaved with NheIand NcoI. The AflIII + NcoI ligation among mammalian genomic or cDNA sequences; (ii) large-scale allowed fusion of BN51 and luciferase on the natural BN51 experimental analysis of the computer-selected population to start codon. Mutagenesis of the E-box (pGL3-BN51-mut1, identify candidate Myc-regulated genes; (iii) detailed charac- Fig. 3A) was performed by PCR using native Pfu polymerase terization of individual genes. (Stratagene) in a modified buffer (30 mM Tricine pH 8.4, 2mM MgCl , 5 mM 2-mercaptoethanol, 0.01% Thesit). We Computer-based identification of putative Myc target amplified a 500 bp fragment of intron 1 encompassing the NarI sequences and AflIII sites and engineered new EcoRI and BamHI sites on We developed a program that analyses the base composition of each side of this fragment. The PCR product was cloned into DNA sequences surrounding E-boxes, permitting the selection pBluescript and sequenced (pBS-BN51). The mutant BN51 of potential CpG islands. The criteria adopted were a G+C fragment was recovered (NarI–AflIII) and subcloned in content >65% (compare with <50% in bulk genomic DNA) pGL3basic (XbaI–AflIII) in a three-way ligation together with and a ratio of CpG to GpC >0.6 (compare with <0.5 in bulk the XbaI–NarI fragment from pBN51eco. cDNAs expressing genomic DNA) (39–41). Our first search (June 1995, used as a humanMyc,Mad1orUSF were subclonedin the expression basis for this work) resulted in the selection of 977 E-boxes vector BJ3 (13). within potential CpG islands from a total of 42 971 E-box- Transient transfections and reporter gene assays containing sequences identified in the databases (Table 1). This represents 2.27% of the E-box-containing sequences and 293T cells were transiently transfected with a standard calcium 0.33% of the total number of sequences analyzed. Included in phosphate DNA precipitation procedure. Plasmid quantities the selected population were the E-boxes of known Myc target used in the transfections were 10 ng of nucleolin or BN51 genessuchasODC and cad. Not all E-box-containing CpG reporters driving expression of firefly luciferase (FL), 10 ng of islands will correspond to bona fide Myc-regulated genes. In aCMV-driven Renilla-luciferase (RL) construct (Promega) order to identify those that are indeed regulated by Myc in and 0.1 g of BJ3 derivatives (empty BJ3 vector or BJ3 cultured cells, the selected genes were requested from original expressing Myc, Mad1 or USF). Rat1 fibroblasts were trans- sources: 316 were obtained and analyzed experimentally as fected with Superfect™ (Qiagen AG) with 0.1 gofreporter, described below. 0.1 gofCMV-RLand 1 g of BJ3 derivatives. All transfections were performed in 12-well tissue culture plates. Transfected Experimental screens of candidate Myc target genes cells were incubated in DMEM (Gibco BRL catalog no. Upon Myc–ER activation, Myc target genes are induced, as are 31966-021) with 10% fetal calf serum and harvested 40 h post- their transcription rates and mRNA levels (8). These two transfection. Cells were lysed and luciferase activities were parameters were evaluated for our collection of computer- measured with the Dual Luciferase assay (Promega) using a identified clones by nuclear run-on and reverse northern Lumac Biocounter M2500. RL activity was used to normalize blotting, respectively (see Materials and Methods). The screen FL activity within each sample. consisted of 316 genes, as well as internal controls for genes previously shown to be induced (ODC, PT and cdc25a) and RESULTS not induced (36B4, tubulin, actin and GAPDH) by Myc. Plasmids were replica slot-blotted on duplicate nylon filters and probed Principle of the screen in parallel with radiolabeled nuclear run-on RNA or first strand Owing to their low level of induction, identification of novel cDNA probes, derived from parallel cultures of Rat1 Myc–ER Myc targets by differential screening strategies has been very cells,beforeand4hafter activationwith OHT.Each signal limited in its success. We therefore developed an alternative was normalized to that of the 36B4 gene under the same conditions. approach, aimed at identifying potential targets for which Table 2 gives the fold induction for various genes upon Myc–ER sequence information was already available in the databases. activation, as described below. 36B4 encodes acidic ribosomal Nucleic Acids Research, 2000, Vol. 28, No. 2 449 Table 1. Number of sequences selected and screened by the program cpg Species Sequences analyzed Sequences with E-boxes Sequences with E-boxes in CpG islands Primate 36 442 12 775 (35%) 473 (1.29%) Rodent 24 375 11 324 (46%) 142 (0.58%) Mammalian 6483 2200 (34%) 130 (2.01%) EST 224 784 12 327 (5.5%) 124 (0.06%) Vertebrate 7544 4345 (58%) 108 (1.43%) Total 299 628 42 971(14%) 977 (0.33%) The number of sequences fulfilling the requirement of an E-box located within a potential CpG island was determined on the June 1995 EMBL release. The fraction of each population selected is shown in parentheses. Table 2. Run-on and reverse northern analysis identifies a number of potential targets of Myc Gene Nuclear run-on Reverse northern 1 36B4 1.00 l.07 0.19 (n =8) 2 Tubulin n.d. l.14 0.21 (n =5) 3 Actin n.d. 1.14 0.28 (n =8) 4 ODC 2.77, 1.l1 2.30 1.11 (n =6) 5 -Prothymosin n.s. 1.37 0.83 (n =8) 6 cdc25a n.s. 1.06 0.31 (n =8) 7 Nucleolin 2.56, 2.01 2.98 1.86 (n =7) 8BN51 2.16 0.78 (n =4) 2.53 0.71 (n =7) 9 Cyclin D2 8.33, 1.91 2.09 0.46 (n =4) 10 Nucleolar protein p120 7.97, 2.41 3.27 1.54 (n =6) 11 wee I kinase 1.91, 1.88 3.20 1.29 (n =4) Figure 1. Run-on and reverse northern analysis of nucleolin and BN51 gene 12 a05 2.42, 0.95 1.94 0.87 (n =7) expression in Rat1 Myc–ER cells. Serum-starved Rat1 Myc–ER cells were treated with OHT for 4 h (+) or left untreated (–) prior to analysis of gene 13 HSC70 1.34,1.03 2.95 0.41 (n =4) expression by (A)run-onor (B) reverse northern analysis (see Materials and 14 c02 n.s. 1.94 0.54 (n =4) Methods). The genes analyzed are indicated at the top. 15 f11 n.s. 3.66 0.89 (n =4) 16 pl(mcm3) n.s. 1.96 0.37 (n =4) 17 c-met n.s. 2.34 0.47 (n =4) Six of the candidate genes showed increases in run-on 18 Topoisomerase I n.s. 2.02 0.98 (n =6) signals (Table 2, rows 7–13), although the results obtained with this technique were prone to variability. Figure 1A shows 19 Nucleolar antigen Ki67 n.s. 2.00 0.44 (n =4) an example of run-on analysis for nucleolin and BN51. The Rows 1–3 show the internal controls, rows 4–6 positive controls and rows 7–19 genes that had shown no response to Myc–ER in the nuclear genes identified as potential targets of Myc. Serum-starved Rat1 Myc–ER run-on also showed no changes by reverse northern. However, cells were treated with OHT for 4 h or left untreated prior to analysis of gene those genes that had shown an apparent increase in transcription expression by run-on or reverse northern blotting. All values were calculated relative to the expression of 36B4. n.d., not determined; n.s., no signal. levels also showed increases in mRNA levels (Table 2, rows 7–13, and Fig. 1B). Furthermore, a number of the genes that gave no signal in nuclear run-on were detectable by reverse northern. Of these, seven were induced upon Myc–ER activation phosphoprotein P0 and is not modulated by Myc, estradiol, (Table 2, rows 13–19). In summary, 316 clones were tested, tamoxifen, the Rb–E2F pathway or upon cell cycle entry (42–44; 123 gave a detectable signal with run-on and/or reverse Table 2, row 1). Actin and tubulin were also not induced northern, 110 were invariant upon Myc–ER activation and 13 (Table 2, rows 2 and 3). GAPDH was consistently induced by were induced in several experiments (Table 2, rows 7–19). We Myc–ER by a factor of 2.5 and was therefore no longer used as also attempted to see whether the sequences surrounding these a reference. Of the known Myc target genes that yielded a potential novel targets allowed, in conjunction with those of detectable signal in run-on and/or reverse northern, ODC was already established targets, the definition of a more precise induced ~2-fold, PT to a lesser extent and cdc25A not at all (Table 2, rows 4–6). consensus sequence surrounding the E-box. However, no 450 Nucleic Acids Research, 2000, Vol. 28, No. 2 additional similarity could be found beyond the VCACGTGB of our original search. At this stage, the 13 genes must still be considered as potential Myc target genes and should be studied in more detail to confirm their response to Myc. In particular: (i) direct and more quantitative analysis of mRNA levels should confirm induction of these genes by Myc–ER with, if possible, induction observed in the presence of the protein synthesis inhibitor cycloheximide, demonstrating that the gene is a direct tran- scriptional target of Myc; (ii) E-boxes in the regulatory regions of these genes should confer Myc-dependent transcriptional activation in transient transfection assays. Of the 13 genes identified, two, nucleolin and BN51, were studied in further detail and met these criteria. RNA was prepared from Rat1 Myc–ER cells before and after induction with OHT, and in the presence or absence of cycloheximide, and analyzed by northern blotting. Six hours following Myc–ER activation, nucleolin and BN51 mRNAs were induced ~2-fold (data not shown), consistent with the reverse northern data (Table 2). Although modest, these levels of induction by Myc–ER are similar to those observed for other Myc-induced genes in this system. The effect of cyclo- heximide on Myc–ER-induced transcription could not be tested, since expression of these genes was induced by cycloheximide alone, a phenomenon that has already been seen for other targets of Myc (45). Nucleolin and BN51 are induced by Myc in an E-box-dependent manner Nucleolin genes and/or cDNAs have been cloned from human (46,47), mouse (48), rat (49) and hamster (50), and related Figure 2. Myc activates the nucleolin promoter via E-boxes located in intron 1. proteins have been identified in budding and fission yeasts (A) Structure of the nucleolin reporter gene constructs. The mouse nucleolin promoter, exon 1, intron 1 and the first 8 nt of exon 2 were fused to the 5 -end (51,52). All the mammalian genes contain a first intron that of the luciferase gene in pGL3-basic to generate the reporter construct overlaps with a highly conserved CpG island (53). The mouse pNucL14. The ATG in exon 1 was deleted (see Materials and Methods). In gene contains five E-boxes spanning 150 bp of intron 1, within pNucL14-mut1 and pNucL14-mut2, two groups of E-boxes located in intron the CpG island (Fig. 2A). The first two and the fourth E-boxes 1 were mutated as shown (black dots). All E-boxes were mutated in are conserved in the human and hamster genes. We generated pNucL14-mut1/2. The reporters were transiently transfected into (B)Rat1 and (C) 293T cells, together with plasmids expressing Myc, Mad or USF, as a reporter construct containing the promoter, exon 1, intron 1 indicated. Luciferase activities are expressed relative to pNucL14 alone. The and the first 8 bp of exon 2 of mouse nucleolin driving expression data are averaged from three independent experiments. of luciferase (pNucL14, Fig. 2A). The nucleolin ATG in exon 1 was mutated. When transfected into either 293T or Rat1 cells, pNucL14 showed high basal transcriptional activity, comparable to a CMV–luciferase reporter (data not shown). E-boxes mutated. Mutation of CACGTG to TACGTG eliminates Co-transfection of a Myc expression construct (BJ3–myc3) all binding and responsiveness to Myc/Max complexes activated the reporter ~4-fold in Rat1 and 293T cells (Fig. 2B (13,39,54). pNucL14-mut1 had reduced basal activity and C). A Myc mutant deficient in DNA binding (MycR392/ compared with the wild-type construct and was neither activated 394/396A; 13) had no effect (data not shown). In contrast to by Myc nor repressed by Mad1 (Fig. 2B and C). pNucL14-mut2 Myc, a construct expressing Mad1 repressed pNucL14 activity had a basal level close to that of the native reporter and was (Fig. 2B and C). The reporter was also activated by USF, still induced by Myc and repressed by Mad1 (Fig. 2B and C). which also recognizes and transactivates from E-box elements, pNucL14-mut1/2 behaved very similarly to pNucL14mut1. albeit to a lesser extent. The pNucL14 reporter was also induced by USF. In Rat1 cells, A deletion mutant of the reporter was made in which the this response was largely dependent upon the first three E-boxes, region encompassing the five E-boxes was deleted (pNucL8). although a residual response of pNucL14-mut1 and mut1/2 to This reporter had a decreased basal transcriptional activity USF was observed (Fig. 2B). In 293T cells, the response to (40% of that of the native reporter) and was unresponsive to USF was largely independent of all E-boxes (Fig. 2C). Altogether, co-transfection of Myc or Mad expression plasmids (data not our data show that E-boxes 1, 2 and/or 3 of intron 1 are responsible shown). To further address which of the E-boxes were responsible for activation of the nucleolin promoter by Myc, its repression for mediating the effects of Myc and Mad we constructed three by Mad and are limiting for the basal activity of the promoter. series of point mutations. pNucL14-mut1 had the first three CACGTG motifs mutated to TACGTG, pNucL14-mut2 had In contrast, E-boxes 4 and 5 play a minimal role, if any. Given the last two E-boxes mutated and pNucL14-mut1/2 had all five that E-box 3 is not conserved in all species (see above), we Nucleic Acids Research, 2000, Vol. 28, No. 2 451 was significantly induced by USF. Thus, BN51 appears to be a Myc-specific target, as has been previously shown for cad and PT. DISCUSSION We have shown here that the application of a computer-based analysis of DNA databases using restrictive sequence- dependent criteria permits the selection of potential Myc target genes. Furthermore, several criteria establish that the genes for BN51 and nucleolin are bona fide Myc targets: (i) BN51 and nucleolin gene transcription (as assessed by run-on) and mRNA levels (as assessed by reverse northern) are induced upon activation of Myc–ER in Rat1 cells; (ii) Myc activates transcription from the BN51 and nucleolin promoters via one and three (probably two) E-boxes, respectively; (iii) in either case, the E-boxes are located in the first intron and within a CpG island, as defined by the initial conditions of our screen. Consistent with our identification of BN51 as a Myc target, BN51 transcription is induced after serum stimulation of quiescent fibroblasts, but this induction is abolished by cycloheximide (36). Thus, BN51 is a ‘delayed early’ serum response gene that must be induced by the product of an ‘immediate early’ gene, such as c-myc (reviewed in 2). Nucleolin expression is enhanced in tumors and in proliferating cells, is low in serum-deprived cells, increases upon cell cycle re-entry (55–58) and is induced by interleukin-2 in T cells (59). In human neuroblastoma cells, nucleolin and N-myc expression decrease concomitantly during differentiation (60). Nucleolin is an abundant nucleolar and multifunctional protein involved in rRNA processing and ribosome assembly Figure 3. Myc activates the BN51 promoter via a single E-box located in (61–65 and references therein). The human BN51 gene was intron 1. (A) Structure of the BN51–reporter gene constructs. The human cloned by virtue of its ability to suppress G arrest in a temperature- BN51 promoter, exon 1, intron 1 and the first 6 nt of exon 2 were fused to the sensitive hamster cell line (tsBN51) (66). BN51 encodes a 5 -end of the luciferase gene in pGL3-basic (pGL3-BN51). The natural BN51 subunit of RNA polymerase (pol) III homologous to the ATG in exon 2 was fused in-frame to luciferase. The E-box located in intron 1 was mutated to TACGTG (pGL3-BN51-mut). During construction of the RPC53 subunit of Saccharomyces cerevisiae RNA pol C (the reporters, we resequenced the exon 1–intron 1 region of the BN51 gene, pol III counterpart in yeast; 67–69). RNA pol III activity is revealing several errors in the published sequence (GenBank accession no. temperature sensitive in extracts of tsBN51 cells, suggesting L15301) (73). These included the omission of 81 nt at the 5 -end of intron 1 that loss of BN51/pol III function is directly responsible for G and several nucleotide substitutions. In the published sequence there were two 1 arrest (68). Consistent with this notion, tsRPC53 mutants of E-boxes, one in intron 1 and one spanning the exon 1–intron 1 boundary. Resequencing demonstrated that the latter E-box does not exist (GenBank S.cerevisiae also arrest in G at the restrictive temperature (67). accession no. for the corrected sequence AF142779). The reporters were transiently It will be interesting to address whether Myc is rate limiting for transfected in (B) Rat1 and (C) 293T cells, together with plasmids expressing RNA pol III transcription in vivo. RNA pol III is involved in Myc, Mad or USF, as indicated. Luciferase activities are expressed relative to transcription of the 5S rRNA and tRNAs. While RNA pol I pGL3-BN51 alone. The data are averaged from three independent experiments. transcribes the 45S precursor of the 28S, 18S and 5.8S rRNAs, the maturation of these rRNAs is defective in tsBN51 cells at the restrictive temperature (69). Recent data suggest that Myc function is critical not only for cell surmise that E-boxes 1 and 2 are critical for regulation by Myc cycle progression, but also for cell growth, i.e. the accumulation of and Mad1. cell mass in both Drosophila (70) and mammalian cells The human BN51 gene contains a single E-box within intron 1 (discussedin6). Deletionofbothc-myc alleles by homologous (Fig. 3A legend). We constructed a reporter gene (pGL3-BN51) recombination in a diploid clone of Rat1 fibroblasts impaired, containing the BN51 promoter, exon 1, intron 1 and the first but surprisingly did not abrogate, cell proliferation (71). These 6 bp of exon 2, allowing the use of the natural BN51 start –/– Rat1 myc cells grew and proliferated very slowly, with codon to initiate translation of luciferase. This reporter showed prolonged G and G phases and decreased rates of total RNA 1 2 a significant level of basal activity (~20% of CMV–luciferase), and protein accumulation (as well as protein degradation), wasinduced by Myc ~3-fold, and repressedbyMad1inboth whilst maintaining a normal cell size. This is the phenotype Rat1 and 293T cells (Fig. 3B and C). Mutation of the E-box expected if the rates of cell proliferation and cell growth are (pGL3-BN51mut) abolished the response to both Myc and concomitantly reduced. We speculate that two of the main –/– Mad (Fig. 3B and C). Neither pGL3-BN51wt nor pGL3-BN51mut defects in Rat1 myc cells are decreases in RNA pol III 452 Nucleic Acids Research, 2000, Vol. 28, No. 2 activity and in the production of ribosomes, leading to the discussions that took place in the course of this work. Our observed generalized defects in cellular protein synthesis. thanks also go to Dr Phil Shaw and Prof. Susanna Cotecchia for helpful comments on the manuscript. We would also like to It was recently reported that most known Myc-induced thank Prof. Susanna Cotecchia for her consideration during the genes, with the exception of cad, were not down-regulated in –/– preparation of this manuscript. P.J.G. was supported by Rat1 myc cells (72). It should be recalled, however, that those fellowships from EMBO and the European Community. B.A. cells were derived from immortalized Rat1 cells and were selected was the recipient of a START fellowship and of a research in culture to grow without myc. Thus, Myc-dependent genes that grant from the Swiss National Science Foundation. are essential for cell proliferation might have been re-expressed in –/– Rat1 myc cells, owing to genetic or epigenetic alterations that might have occurred either before of after disruption of c-myc.It REFERENCES will be important to address whether BN51, nucleolin or other 1. Downs,K.M., Martin,G.R. and Bishop,J.M. 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Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis

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446–453 Nucleic Acids Research, 2000, Vol. 28, No. 2 © 2000 Oxford University Press Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis Peter J. Greasley, Claude Bonnard and Bruno Amati* Cellular Growth Control Unit and Biocomputing Unit, Swiss Institute for Experimental Cancer Research (ISREC), 155 ch. des Boveresses, CH-1066 Epalinges, Switzerland Received October 4, 1999; Revised and Accepted November 11, 1999 DDBJ/EMBL/GenBank accession no. AF142779 ABSTRACT results in their constitutive expression (2). In normal cells, expression of c-myc is strictly dependent on mitogenic signals The c-Myc oncoprotein and its dimerization partner and is suppressed by growth inhibitory and differentiation Max bind the DNA core consensus sequence inducing signals (2,3). c-myc expression is also regulated by CACGTG (E-box) and activate gene transcription. the products of other oncogenes or tumor suppressor genes, However, the low levels of induction have hindered the such as tyrosine kinase receptors (4) or the APC/ -catenin/ identification of novel Myc target genes by differential TCF pathway (5). This may explain why c-myc is sometimes screening techniques. Here, we describe a computer- overexpressed in tumors without being mutated. based pre-selection of candidate Myc/Max target The c-myc protein product (Myc) conveys strong mitogenic and apoptotic stimuli. Constitutive expression of Myc reduces genes, based on two restrictive criteria: an extended growth factor requirements, prevents growth arrest by a variety of E-box consensus sequence for Myc/Max binding and growth inhibitory signals, and can block cellular differentiation the occurrence of this sequence within a potential (reviewed in 2,6). Conversely, activation of a conditional genomic CpG island. Candidate genes selected by Myc–estrogen receptor chimera (Myc-ER) (7) in quiescent these criteria were evaluated experimentally for their cells induces entry into the cell cycle in the absence of response to Myc. Two Myc target genes are charac- mitogens (8,9). However, concomitant with their mitogenic terized here in detail. These encode nucleolin, an action, Myc or Myc-ER can induce apoptosis if survival abundant nucleolar protein, and BN51, a co-factor of factors are absent from the extracellular environment (10,11; RNA polymerase III. Myc activates transcription of reviewed in 2,3). Myc-induced apoptosis has been linked to ARF1 both genes via E-boxes located in their first introns, activation of the p19 –p53 pathway and is separable from as seen for several well-characterized Myc targets. mitogenesis (12). Myc is a transcription factor of the basic helix–loop–helix For both genes, mutation of the E-boxes abolishes leucine zipper (bHLH-Zip) family. Myc dimerizes with transcriptional activation by Myc as well as repression another bHLH-Zip protein, Max, to bind the specific DNA by Mad1. In addition, the BN51 promoter is selectively sequence CACGTG (the E-box) and activate transcription activated by Myc and not by USF, another E-box- (13,14; reviewed in 3,15). Transcription-competent Myc/Max binding factor. Both nucleolin and BN51 are implicated dimers are the active form of Myc in inducing cell cycle in the maturation of ribosomal RNAs, albeit in progression, apoptosis and malignant transformation (3,15). In different ways. We propose that Myc, via regulation addition to its function as a transactivator, Myc can also of these and probably many other transcriptional repress transcription of several genes that are clearly relevant targets, may be an important regulator of ribosome to its biological function (16–18). In fact, some reports have biogenesis. suggested that transcription repression, rather than activation, correlates with the biological activity of Myc (19,20; reviewed in 3,21,22). There is, however, no clear demonstration that INTRODUCTION gene repression is a direct action of Myc in vivo, rather than the indirect consequence of activating as yet unknown Myc target The myc family of mammalian proto-oncogenes includes three genes (see also Discussion). The molecular mechanisms evolutionarily conserved genes c-, N- and L-myc,which proposed to explain transcription repression by Myc (21,22) encode related proteins. myc genes are differentially expressed therefore remain speculative. during embryonic development (1) and proliferating post-natal tissues, with few exceptions, express c-myc (2). Oncogenic Max also forms heterodimers with the other bHLH-Zip activation of these genes, in particular c-myc, is observed in a proteins Mad1, Mxi-1 (or Mad2), Mad3, Mad4 and Mnt (or wide range of human and animal neoplasias and generally Rox) (23–27). These alternative dimers also bind the E-box *To whom correspondence should be addressed at present address: DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304, USA. Tel: +1 650 858 7528; Fax: +1 650 496 1200; Email: [email protected] Present address: Peter J. Greasley, Institute of Pharmacology and Toxicology, University of Lausanne, 27 rue du Bugnon, CH-1005 Lausanne, Switzerland Nucleic Acids Research, 2000, Vol. 28, No. 2 447 and actively repress transcription and can therefore antagonize tamoxifen (OHT) (Laboratoires Besins-Isovesco, Paris, France). both the transcription and transforming activities of Myc. Cells were harvested at various time points by scraping and Furthermore, the myc and mad family genes are generally stored frozen until required for nuclear run-on or RNA preparation. regulated in opposite modes in growth control and development Preparation of filters for nuclear run-on and reverse (25,28; reviewed in 3). In summary, Myc, Max and Mad northern analysis proteins form a network that regulates gene expression, proliferation, apoptosis and differentiation. Plasmid DNAs containing the genes of interest were isolated To further understand the function of Myc, Max and Mad, it from Escherichia coli using the Promega Wizard miniprep is necessary to identify the genes that are directly regulated by DNA extraction kit. Aliquots of 12 g of each DNA were these proteins, in particular, Myc-induced and/or Mad-repressed ethanol precipitated, resuspended in 200 lH O and denatured genes. Known Myc target genes include -prothymosin ( PT), by addition of 20 l2 NNaOHand heating at95 C for 10 min. ornithine decarboxylase (ODC), Cdc25A, cad, MrDb, ECA39, Denatured DNA was then transferred to a second tube EIF-4E, LDH-A, rcl, E2F-2, RCC1, the telomerase catalytic containing 600 l ice-cold 10 SSC(1.5M NaCl, 0.15M Na- subunit hTERT and others (reviewed in 3,29,30). Although citrate, pH 7.0). The solution was neutralized by the addition of ODC, Cdc25A, LDH-A, rcl, E2F-2, PT and hTERT share 44 l 2 M HEPES (free acid) and divided equally between two some of the potential of Myc to induce cell cycle progression, wells on a 48-well slot blotter, so as to produce two identical apoptosis, immortalization and/or transformation (31–35), replica blots. Each blot contained actin, tubulin, GAPDH and additional target genes must be identified to explain the whole 36B4 as internal standards, and ODC as a positive control for a range of biological effects controlled by Myc (6). A common Myc-regulated gene. The filters were washed twice in 6 SSC feature of these genes is the low magnitude of their response to and allowed to air dry. After drying the filters were baked Myc. Generally, induction by Myc results in only 2- to 5-fold undervacuum for2 hat 80 C. increases, making identification of novel targets difficult. Nuclear run-on Indeed, only a few of the known targets have been identified by differential screening techniques, the majority of Myc-regulated Cells from two 15 cm dishes were resuspended in 8 ml ice-cold genes being postulated by the presence of E-box sequences buffer A (10 mM Tris–HCl, pH 7.5, 5 mM MgCl ,10mM (30). In the present study, we describe a computerized NaCl, 0.5 mM DTT), incubated on ice for 20 min, centrifuged, screening strategy so as to facilitate initial identification of resuspended in 2 ml buffer A and lysed by rapid pipetting of potential Myc-regulated genes, and characterize two novel the suspension. Nuclei were recovered by centrifugation at Myc targets, nucleolin and BN51. 2000 r.p.m. for 5 min, washed once in 2 ml buffer B (20 mM Tris pH 7.5, 10 mM MgCl , 140 mM KCl, 20% glycerol, 0.5 mM DTT), resuspended in buffer B supplemented with MATERIALS AND METHODS 0.5 mMATP,0.5 mM CTP, 0.5mMGTP,5 MUTP and Computer-based analysis of sequences in the GenBank 200 Ci [ - P]UTP, and incubated at 30 C for 15 min. The databases reaction was stopped with 1 ml ice-cold buffer C (10 mM Tris–HCl, pH 7.5, 50 mM MgCl , 500 mM NaCl, 2 mM CaCl ). RNase-free The program (cpg)was writteninCandusedthe GCG 2 2 DNase (200 U) was added to the mixture, followed by incubation package library functions (Wisconsin Package v.8, Genetics at room temperature for 5 min. The reaction mix was then Computer Group, Madison, WI) for the input of parameters, passed twice through a QIAshredder™ unit (Qiagen) and RNA nucleotide query sequences and GCG formatted database was extracted using the RNeasy™ midi-prep system (Qiagen), access (EMBL). With each perfect match for VCACGTGB in according to the manufacturer’s instructions. Prehybridization any nucleotide sequence entry of the database, the program of filters was performed in TESS2D (20 mM TES, pH 7.4, computed three values from within windows of determined 2 Denhardt’s solution, 400 mM NaCl, 2.6 mM EDTA and length around the detected match. First, the average GC 0.07% SDS) supplemented with 0.4 mg/ml yeast RNA at 65 C content expressed as the global percentage of G and C in the for 4 h. The eluant (200 l) from the RNeasy™ preparation of window, secondly, the CpG/GpC ratio and, finally, observed/ nuclear run-on RNA was added to 1 ml of TESS2D and expected CpG, defined as (no. CpG/length)/[(no. C no. G)/ hybridization allowed to take place for 24 h at 65 C. The blots length]. If the 5 -or3 -end of the sequence did not permit were then washed three times for 30 min in 1 SSC, 0.1% SDS application of the predetermined window length, the computation at 65 C. Run-on signals were quantified with a phosphorimager. was made on the available sequence. The searches presented here used the following criteria: a window size of 100 nt in both Reverse northern and northern blotting directions and a GC content 65% (in both directions from the hit). Total cellular RNA was isolated using the RNeasy™ mini prep Induction of Myc-ER cells kit (Qiagen). An aliquot of 1 g of RNA was labeled by random 32 32 priming using a mixture of [ - P]dATP and [ - P]dCTP. The We used Myc-ER clone 3, a Rat1 cell line constitutively labeled probes were isolated by spin filtration using Sephadex expressing a Myc–ER fusion protein (7,10). Cells were grown to ~40% confluence in phenol red-free DMEM medium G-50. Hybridization of slot-blotted DNAs was performed as (Gibco BRL no. 11880-028) supplemented with 10% charcoal- for nuclear run-on, apart from the use of denatured salmon sperm DNA as non-specific competitor. Northern blotting was stripped fetal calf serum to prevent activation of Myc–ER. Subconfluent cultures were serum-starved for 24 h prior to performed by standard methods using random primed DNA activation of Myc–ER by addition of 200 nM trans-4-hydroxy- probes. All signals were quantified with a phosphorimager. 448 Nucleic Acids Research, 2000, Vol. 28, No. 2 Plasmid constructions Such a strategy is dependent upon defining the nucleotide sequence preferences of Myc/Max dimers. We have previously To generate the reporter pNucL14 (Fig. 2A), the promoter, identified the extended consensus VCACGTGB (where V = A, exon 1, intron1and the first 8bpofexon2wereclonedin C or G and B = T, C or G), as the optimal E-box site for Myc/Max, pGL3basic (Promega). Digestion with NcoI and subsequent both in vitro and in vivo (37). This E-box sequence contains a treatment with S1 nuclease followed by religation destroyed central CpG dinucleotide. CpG pairs throughout mammalian the natural translation start codon located in exon 1. The genomes are generally methylated at position N7 of cytosine, product of this reaction was confirmed by sequencing. To except in small regions of the genome (~0.3–1.5 kb) called generate the mutants pNucL14mut1, pNucL14mut2 and CpG islands. In vitro Myc/Max dimers fail to recognize the E-box pNucL14mut1/2 we first subcloned the EcoRV–HindIII fragment if the central CpG dinucleotide is methylated (38). By inference, of pNucL14 into pBluescript to generate pBSnuc. This most functional E-boxes should be located within CpG islands. construct was used for PCR mutagenesis of the nucleolin gene In fact, a feature that characterizes several Myc target genes, by mutating the respective CACGTG sequences to TACGTG. including ODC, cad and LDH-A, is the location of the E-boxes Subcloning the various mutants back into pNucL14 using within CpG islands. Furthermore, CpG islands are characterized EcoRV and HindIII then produced each reporter construct. To by a distinct sequence composition (39; see below) and are generate pGL3-BN51 (Fig. 3A), a genomic DNA fragment generally located at the 5 -end of genes, frequently encompassing spanning the promoter, exon 1, intron 1 and part of exon 2 of the promoter, exon 1 and intron 1. We thus designed a screen BN51 was recovered from plasmid pBN51eco (36) with XbaI based on three main steps: (i) computer-based identification of and AflIII. This fragment was subcloned in pGL3-basic VCACGTGB motifs located within potential CpG islands (Promega) cleaved with NheIand NcoI. The AflIII + NcoI ligation among mammalian genomic or cDNA sequences; (ii) large-scale allowed fusion of BN51 and luciferase on the natural BN51 experimental analysis of the computer-selected population to start codon. Mutagenesis of the E-box (pGL3-BN51-mut1, identify candidate Myc-regulated genes; (iii) detailed charac- Fig. 3A) was performed by PCR using native Pfu polymerase terization of individual genes. (Stratagene) in a modified buffer (30 mM Tricine pH 8.4, 2mM MgCl , 5 mM 2-mercaptoethanol, 0.01% Thesit). We Computer-based identification of putative Myc target amplified a 500 bp fragment of intron 1 encompassing the NarI sequences and AflIII sites and engineered new EcoRI and BamHI sites on We developed a program that analyses the base composition of each side of this fragment. The PCR product was cloned into DNA sequences surrounding E-boxes, permitting the selection pBluescript and sequenced (pBS-BN51). The mutant BN51 of potential CpG islands. The criteria adopted were a G+C fragment was recovered (NarI–AflIII) and subcloned in content >65% (compare with <50% in bulk genomic DNA) pGL3basic (XbaI–AflIII) in a three-way ligation together with and a ratio of CpG to GpC >0.6 (compare with <0.5 in bulk the XbaI–NarI fragment from pBN51eco. cDNAs expressing genomic DNA) (39–41). Our first search (June 1995, used as a humanMyc,Mad1orUSF were subclonedin the expression basis for this work) resulted in the selection of 977 E-boxes vector BJ3 (13). within potential CpG islands from a total of 42 971 E-box- Transient transfections and reporter gene assays containing sequences identified in the databases (Table 1). This represents 2.27% of the E-box-containing sequences and 293T cells were transiently transfected with a standard calcium 0.33% of the total number of sequences analyzed. Included in phosphate DNA precipitation procedure. Plasmid quantities the selected population were the E-boxes of known Myc target used in the transfections were 10 ng of nucleolin or BN51 genessuchasODC and cad. Not all E-box-containing CpG reporters driving expression of firefly luciferase (FL), 10 ng of islands will correspond to bona fide Myc-regulated genes. In aCMV-driven Renilla-luciferase (RL) construct (Promega) order to identify those that are indeed regulated by Myc in and 0.1 g of BJ3 derivatives (empty BJ3 vector or BJ3 cultured cells, the selected genes were requested from original expressing Myc, Mad1 or USF). Rat1 fibroblasts were trans- sources: 316 were obtained and analyzed experimentally as fected with Superfect™ (Qiagen AG) with 0.1 gofreporter, described below. 0.1 gofCMV-RLand 1 g of BJ3 derivatives. All transfections were performed in 12-well tissue culture plates. Transfected Experimental screens of candidate Myc target genes cells were incubated in DMEM (Gibco BRL catalog no. Upon Myc–ER activation, Myc target genes are induced, as are 31966-021) with 10% fetal calf serum and harvested 40 h post- their transcription rates and mRNA levels (8). These two transfection. Cells were lysed and luciferase activities were parameters were evaluated for our collection of computer- measured with the Dual Luciferase assay (Promega) using a identified clones by nuclear run-on and reverse northern Lumac Biocounter M2500. RL activity was used to normalize blotting, respectively (see Materials and Methods). The screen FL activity within each sample. consisted of 316 genes, as well as internal controls for genes previously shown to be induced (ODC, PT and cdc25a) and RESULTS not induced (36B4, tubulin, actin and GAPDH) by Myc. Plasmids were replica slot-blotted on duplicate nylon filters and probed Principle of the screen in parallel with radiolabeled nuclear run-on RNA or first strand Owing to their low level of induction, identification of novel cDNA probes, derived from parallel cultures of Rat1 Myc–ER Myc targets by differential screening strategies has been very cells,beforeand4hafter activationwith OHT.Each signal limited in its success. We therefore developed an alternative was normalized to that of the 36B4 gene under the same conditions. approach, aimed at identifying potential targets for which Table 2 gives the fold induction for various genes upon Myc–ER sequence information was already available in the databases. activation, as described below. 36B4 encodes acidic ribosomal Nucleic Acids Research, 2000, Vol. 28, No. 2 449 Table 1. Number of sequences selected and screened by the program cpg Species Sequences analyzed Sequences with E-boxes Sequences with E-boxes in CpG islands Primate 36 442 12 775 (35%) 473 (1.29%) Rodent 24 375 11 324 (46%) 142 (0.58%) Mammalian 6483 2200 (34%) 130 (2.01%) EST 224 784 12 327 (5.5%) 124 (0.06%) Vertebrate 7544 4345 (58%) 108 (1.43%) Total 299 628 42 971(14%) 977 (0.33%) The number of sequences fulfilling the requirement of an E-box located within a potential CpG island was determined on the June 1995 EMBL release. The fraction of each population selected is shown in parentheses. Table 2. Run-on and reverse northern analysis identifies a number of potential targets of Myc Gene Nuclear run-on Reverse northern 1 36B4 1.00 l.07 0.19 (n =8) 2 Tubulin n.d. l.14 0.21 (n =5) 3 Actin n.d. 1.14 0.28 (n =8) 4 ODC 2.77, 1.l1 2.30 1.11 (n =6) 5 -Prothymosin n.s. 1.37 0.83 (n =8) 6 cdc25a n.s. 1.06 0.31 (n =8) 7 Nucleolin 2.56, 2.01 2.98 1.86 (n =7) 8BN51 2.16 0.78 (n =4) 2.53 0.71 (n =7) 9 Cyclin D2 8.33, 1.91 2.09 0.46 (n =4) 10 Nucleolar protein p120 7.97, 2.41 3.27 1.54 (n =6) 11 wee I kinase 1.91, 1.88 3.20 1.29 (n =4) Figure 1. Run-on and reverse northern analysis of nucleolin and BN51 gene 12 a05 2.42, 0.95 1.94 0.87 (n =7) expression in Rat1 Myc–ER cells. Serum-starved Rat1 Myc–ER cells were treated with OHT for 4 h (+) or left untreated (–) prior to analysis of gene 13 HSC70 1.34,1.03 2.95 0.41 (n =4) expression by (A)run-onor (B) reverse northern analysis (see Materials and 14 c02 n.s. 1.94 0.54 (n =4) Methods). The genes analyzed are indicated at the top. 15 f11 n.s. 3.66 0.89 (n =4) 16 pl(mcm3) n.s. 1.96 0.37 (n =4) 17 c-met n.s. 2.34 0.47 (n =4) Six of the candidate genes showed increases in run-on 18 Topoisomerase I n.s. 2.02 0.98 (n =6) signals (Table 2, rows 7–13), although the results obtained with this technique were prone to variability. Figure 1A shows 19 Nucleolar antigen Ki67 n.s. 2.00 0.44 (n =4) an example of run-on analysis for nucleolin and BN51. The Rows 1–3 show the internal controls, rows 4–6 positive controls and rows 7–19 genes that had shown no response to Myc–ER in the nuclear genes identified as potential targets of Myc. Serum-starved Rat1 Myc–ER run-on also showed no changes by reverse northern. However, cells were treated with OHT for 4 h or left untreated prior to analysis of gene those genes that had shown an apparent increase in transcription expression by run-on or reverse northern blotting. All values were calculated relative to the expression of 36B4. n.d., not determined; n.s., no signal. levels also showed increases in mRNA levels (Table 2, rows 7–13, and Fig. 1B). Furthermore, a number of the genes that gave no signal in nuclear run-on were detectable by reverse northern. Of these, seven were induced upon Myc–ER activation phosphoprotein P0 and is not modulated by Myc, estradiol, (Table 2, rows 13–19). In summary, 316 clones were tested, tamoxifen, the Rb–E2F pathway or upon cell cycle entry (42–44; 123 gave a detectable signal with run-on and/or reverse Table 2, row 1). Actin and tubulin were also not induced northern, 110 were invariant upon Myc–ER activation and 13 (Table 2, rows 2 and 3). GAPDH was consistently induced by were induced in several experiments (Table 2, rows 7–19). We Myc–ER by a factor of 2.5 and was therefore no longer used as also attempted to see whether the sequences surrounding these a reference. Of the known Myc target genes that yielded a potential novel targets allowed, in conjunction with those of detectable signal in run-on and/or reverse northern, ODC was already established targets, the definition of a more precise induced ~2-fold, PT to a lesser extent and cdc25A not at all (Table 2, rows 4–6). consensus sequence surrounding the E-box. However, no 450 Nucleic Acids Research, 2000, Vol. 28, No. 2 additional similarity could be found beyond the VCACGTGB of our original search. At this stage, the 13 genes must still be considered as potential Myc target genes and should be studied in more detail to confirm their response to Myc. In particular: (i) direct and more quantitative analysis of mRNA levels should confirm induction of these genes by Myc–ER with, if possible, induction observed in the presence of the protein synthesis inhibitor cycloheximide, demonstrating that the gene is a direct tran- scriptional target of Myc; (ii) E-boxes in the regulatory regions of these genes should confer Myc-dependent transcriptional activation in transient transfection assays. Of the 13 genes identified, two, nucleolin and BN51, were studied in further detail and met these criteria. RNA was prepared from Rat1 Myc–ER cells before and after induction with OHT, and in the presence or absence of cycloheximide, and analyzed by northern blotting. Six hours following Myc–ER activation, nucleolin and BN51 mRNAs were induced ~2-fold (data not shown), consistent with the reverse northern data (Table 2). Although modest, these levels of induction by Myc–ER are similar to those observed for other Myc-induced genes in this system. The effect of cyclo- heximide on Myc–ER-induced transcription could not be tested, since expression of these genes was induced by cycloheximide alone, a phenomenon that has already been seen for other targets of Myc (45). Nucleolin and BN51 are induced by Myc in an E-box-dependent manner Nucleolin genes and/or cDNAs have been cloned from human (46,47), mouse (48), rat (49) and hamster (50), and related Figure 2. Myc activates the nucleolin promoter via E-boxes located in intron 1. proteins have been identified in budding and fission yeasts (A) Structure of the nucleolin reporter gene constructs. The mouse nucleolin promoter, exon 1, intron 1 and the first 8 nt of exon 2 were fused to the 5 -end (51,52). All the mammalian genes contain a first intron that of the luciferase gene in pGL3-basic to generate the reporter construct overlaps with a highly conserved CpG island (53). The mouse pNucL14. The ATG in exon 1 was deleted (see Materials and Methods). In gene contains five E-boxes spanning 150 bp of intron 1, within pNucL14-mut1 and pNucL14-mut2, two groups of E-boxes located in intron the CpG island (Fig. 2A). The first two and the fourth E-boxes 1 were mutated as shown (black dots). All E-boxes were mutated in are conserved in the human and hamster genes. We generated pNucL14-mut1/2. The reporters were transiently transfected into (B)Rat1 and (C) 293T cells, together with plasmids expressing Myc, Mad or USF, as a reporter construct containing the promoter, exon 1, intron 1 indicated. Luciferase activities are expressed relative to pNucL14 alone. The and the first 8 bp of exon 2 of mouse nucleolin driving expression data are averaged from three independent experiments. of luciferase (pNucL14, Fig. 2A). The nucleolin ATG in exon 1 was mutated. When transfected into either 293T or Rat1 cells, pNucL14 showed high basal transcriptional activity, comparable to a CMV–luciferase reporter (data not shown). E-boxes mutated. Mutation of CACGTG to TACGTG eliminates Co-transfection of a Myc expression construct (BJ3–myc3) all binding and responsiveness to Myc/Max complexes activated the reporter ~4-fold in Rat1 and 293T cells (Fig. 2B (13,39,54). pNucL14-mut1 had reduced basal activity and C). A Myc mutant deficient in DNA binding (MycR392/ compared with the wild-type construct and was neither activated 394/396A; 13) had no effect (data not shown). In contrast to by Myc nor repressed by Mad1 (Fig. 2B and C). pNucL14-mut2 Myc, a construct expressing Mad1 repressed pNucL14 activity had a basal level close to that of the native reporter and was (Fig. 2B and C). The reporter was also activated by USF, still induced by Myc and repressed by Mad1 (Fig. 2B and C). which also recognizes and transactivates from E-box elements, pNucL14-mut1/2 behaved very similarly to pNucL14mut1. albeit to a lesser extent. The pNucL14 reporter was also induced by USF. In Rat1 cells, A deletion mutant of the reporter was made in which the this response was largely dependent upon the first three E-boxes, region encompassing the five E-boxes was deleted (pNucL8). although a residual response of pNucL14-mut1 and mut1/2 to This reporter had a decreased basal transcriptional activity USF was observed (Fig. 2B). In 293T cells, the response to (40% of that of the native reporter) and was unresponsive to USF was largely independent of all E-boxes (Fig. 2C). Altogether, co-transfection of Myc or Mad expression plasmids (data not our data show that E-boxes 1, 2 and/or 3 of intron 1 are responsible shown). To further address which of the E-boxes were responsible for activation of the nucleolin promoter by Myc, its repression for mediating the effects of Myc and Mad we constructed three by Mad and are limiting for the basal activity of the promoter. series of point mutations. pNucL14-mut1 had the first three CACGTG motifs mutated to TACGTG, pNucL14-mut2 had In contrast, E-boxes 4 and 5 play a minimal role, if any. Given the last two E-boxes mutated and pNucL14-mut1/2 had all five that E-box 3 is not conserved in all species (see above), we Nucleic Acids Research, 2000, Vol. 28, No. 2 451 was significantly induced by USF. Thus, BN51 appears to be a Myc-specific target, as has been previously shown for cad and PT. DISCUSSION We have shown here that the application of a computer-based analysis of DNA databases using restrictive sequence- dependent criteria permits the selection of potential Myc target genes. Furthermore, several criteria establish that the genes for BN51 and nucleolin are bona fide Myc targets: (i) BN51 and nucleolin gene transcription (as assessed by run-on) and mRNA levels (as assessed by reverse northern) are induced upon activation of Myc–ER in Rat1 cells; (ii) Myc activates transcription from the BN51 and nucleolin promoters via one and three (probably two) E-boxes, respectively; (iii) in either case, the E-boxes are located in the first intron and within a CpG island, as defined by the initial conditions of our screen. Consistent with our identification of BN51 as a Myc target, BN51 transcription is induced after serum stimulation of quiescent fibroblasts, but this induction is abolished by cycloheximide (36). Thus, BN51 is a ‘delayed early’ serum response gene that must be induced by the product of an ‘immediate early’ gene, such as c-myc (reviewed in 2). Nucleolin expression is enhanced in tumors and in proliferating cells, is low in serum-deprived cells, increases upon cell cycle re-entry (55–58) and is induced by interleukin-2 in T cells (59). In human neuroblastoma cells, nucleolin and N-myc expression decrease concomitantly during differentiation (60). Nucleolin is an abundant nucleolar and multifunctional protein involved in rRNA processing and ribosome assembly Figure 3. Myc activates the BN51 promoter via a single E-box located in (61–65 and references therein). The human BN51 gene was intron 1. (A) Structure of the BN51–reporter gene constructs. The human cloned by virtue of its ability to suppress G arrest in a temperature- BN51 promoter, exon 1, intron 1 and the first 6 nt of exon 2 were fused to the sensitive hamster cell line (tsBN51) (66). BN51 encodes a 5 -end of the luciferase gene in pGL3-basic (pGL3-BN51). The natural BN51 subunit of RNA polymerase (pol) III homologous to the ATG in exon 2 was fused in-frame to luciferase. The E-box located in intron 1 was mutated to TACGTG (pGL3-BN51-mut). During construction of the RPC53 subunit of Saccharomyces cerevisiae RNA pol C (the reporters, we resequenced the exon 1–intron 1 region of the BN51 gene, pol III counterpart in yeast; 67–69). RNA pol III activity is revealing several errors in the published sequence (GenBank accession no. temperature sensitive in extracts of tsBN51 cells, suggesting L15301) (73). These included the omission of 81 nt at the 5 -end of intron 1 that loss of BN51/pol III function is directly responsible for G and several nucleotide substitutions. In the published sequence there were two 1 arrest (68). Consistent with this notion, tsRPC53 mutants of E-boxes, one in intron 1 and one spanning the exon 1–intron 1 boundary. Resequencing demonstrated that the latter E-box does not exist (GenBank S.cerevisiae also arrest in G at the restrictive temperature (67). accession no. for the corrected sequence AF142779). The reporters were transiently It will be interesting to address whether Myc is rate limiting for transfected in (B) Rat1 and (C) 293T cells, together with plasmids expressing RNA pol III transcription in vivo. RNA pol III is involved in Myc, Mad or USF, as indicated. Luciferase activities are expressed relative to transcription of the 5S rRNA and tRNAs. While RNA pol I pGL3-BN51 alone. The data are averaged from three independent experiments. transcribes the 45S precursor of the 28S, 18S and 5.8S rRNAs, the maturation of these rRNAs is defective in tsBN51 cells at the restrictive temperature (69). Recent data suggest that Myc function is critical not only for cell surmise that E-boxes 1 and 2 are critical for regulation by Myc cycle progression, but also for cell growth, i.e. the accumulation of and Mad1. cell mass in both Drosophila (70) and mammalian cells The human BN51 gene contains a single E-box within intron 1 (discussedin6). Deletionofbothc-myc alleles by homologous (Fig. 3A legend). We constructed a reporter gene (pGL3-BN51) recombination in a diploid clone of Rat1 fibroblasts impaired, containing the BN51 promoter, exon 1, intron 1 and the first but surprisingly did not abrogate, cell proliferation (71). These 6 bp of exon 2, allowing the use of the natural BN51 start –/– Rat1 myc cells grew and proliferated very slowly, with codon to initiate translation of luciferase. This reporter showed prolonged G and G phases and decreased rates of total RNA 1 2 a significant level of basal activity (~20% of CMV–luciferase), and protein accumulation (as well as protein degradation), wasinduced by Myc ~3-fold, and repressedbyMad1inboth whilst maintaining a normal cell size. This is the phenotype Rat1 and 293T cells (Fig. 3B and C). Mutation of the E-box expected if the rates of cell proliferation and cell growth are (pGL3-BN51mut) abolished the response to both Myc and concomitantly reduced. We speculate that two of the main –/– Mad (Fig. 3B and C). Neither pGL3-BN51wt nor pGL3-BN51mut defects in Rat1 myc cells are decreases in RNA pol III 452 Nucleic Acids Research, 2000, Vol. 28, No. 2 activity and in the production of ribosomes, leading to the discussions that took place in the course of this work. Our observed generalized defects in cellular protein synthesis. thanks also go to Dr Phil Shaw and Prof. Susanna Cotecchia for helpful comments on the manuscript. We would also like to It was recently reported that most known Myc-induced thank Prof. Susanna Cotecchia for her consideration during the genes, with the exception of cad, were not down-regulated in –/– preparation of this manuscript. P.J.G. was supported by Rat1 myc cells (72). It should be recalled, however, that those fellowships from EMBO and the European Community. B.A. cells were derived from immortalized Rat1 cells and were selected was the recipient of a START fellowship and of a research in culture to grow without myc. Thus, Myc-dependent genes that grant from the Swiss National Science Foundation. are essential for cell proliferation might have been re-expressed in –/– Rat1 myc cells, owing to genetic or epigenetic alterations that might have occurred either before of after disruption of c-myc.It REFERENCES will be important to address whether BN51, nucleolin or other 1. Downs,K.M., Martin,G.R. and Bishop,J.M. 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Nucleic Acids Research – Oxford University Press

Published: Jan 15, 2000

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