A Large Scale Genetic Analysis of c-Myc-regulated Gene Expression Patterns
2003; Elsevier BV; Volume: 278; Issue: 14 Linguagem: Inglês
10.1074/jbc.m210462200
ISSN1083-351X
AutoresBrenda O’Connell, Ann Cheung, Carl P. Simkevich, Wanny Tam, Xiaojia Ren, Maria K. Mateyak, John M. Sedivy,
Tópico(s)Fungal and yeast genetics research
ResumoThe myc proto-oncogenes encode transcriptional regulators whose inappropriate expression is correlated with a wide array of human malignancies. Up-regulation of Myc enforces growth, antagonizes cell cycle withdrawal and differentiation, and in some situations promotes apoptosis. How these phenotypes are elicited is not well understood, largely because we lack a clear picture of the biologically relevant downstream effectors. We created a new biological system for the optimal profiling of Myc target genes based on a set of isogenic c-myc knockout and conditional cell lines. The ability to modulate Myc activity from essentially null to supraphysiological resulted in a significantly increased and reproducible yield of targets and revealed a large subset of genes that respond optimally to Myc in its physiological range of expression. The total extent of transcriptional changes that can be triggered by Myc is remarkable and involves thousands of genes. Although the majority of these effects are not direct, many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. Myc-activated functions are indicative of a physiological state geared toward the rapid utilization of carbon sources, the biosynthesis of precursors for macromolecular synthesis, and the accumulation of cellular mass. In contrast, the majority of Myc-repressed genes are involved in the interaction and communication of cells with their external environment, and several are known to possess antiproliferative or antimetastatic properties. The myc proto-oncogenes encode transcriptional regulators whose inappropriate expression is correlated with a wide array of human malignancies. Up-regulation of Myc enforces growth, antagonizes cell cycle withdrawal and differentiation, and in some situations promotes apoptosis. How these phenotypes are elicited is not well understood, largely because we lack a clear picture of the biologically relevant downstream effectors. We created a new biological system for the optimal profiling of Myc target genes based on a set of isogenic c-myc knockout and conditional cell lines. The ability to modulate Myc activity from essentially null to supraphysiological resulted in a significantly increased and reproducible yield of targets and revealed a large subset of genes that respond optimally to Myc in its physiological range of expression. The total extent of transcriptional changes that can be triggered by Myc is remarkable and involves thousands of genes. Although the majority of these effects are not direct, many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. Myc-activated functions are indicative of a physiological state geared toward the rapid utilization of carbon sources, the biosynthesis of precursors for macromolecular synthesis, and the accumulation of cellular mass. In contrast, the majority of Myc-repressed genes are involved in the interaction and communication of cells with their external environment, and several are known to possess antiproliferative or antimetastatic properties. c-Myc-estrogen receptor fusion protein bromodeoxyuridine trifunctional enzyme carbamoyl phosphate synthetase, aspartate transcarbamylase, dihydroorotase glyceraldehyde phosphate dehydrogenase 4-hydroxytamoxifen quantitative real time reverse transcription PCR The Myc protein is a member of the basic region/helix-loop-helix/leucine zipper (b/HLH/Zip) family of transcriptional regulators and is capable of exerting both transactivation and transrepression activities (1Oster S.K. Ho C.S. Soucie E.L. Penn L.Z. Adv. Cancer Res. 2002; 84: 81-154Google Scholar, 2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar). Transactivation is mediated by binding as an obligate heterodimer with the b/HLH/Zip factor Max to the consensus sequence CA(C/T)GTG (the E box) (3Luscher B. Larsson L.G. Oncogene. 1999; 18: 2955-2966Google Scholar). Transrepression is less well understood (4Claassen G. Hann S.R. Oncogene. 1999; 18: 2925-2933Google Scholar, 5Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunsky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nature Cell Biol. 2001; 3: 392-399Google Scholar). In either mode Myc is a weak transcriptional regulator, exerting most of its effects within the 2–5-fold range. In a general sense, the up-regulation of Myc strongly enforces proliferation and growth, antagonizes cell cycle withdrawal and differentiation, and in some situations promotes apoptosis (6Obaya A.J. Mateyak M.K. Sedivy J.M. Oncogene. 1999; 18: 2934-2941Google Scholar, 7Prendergast G.C. Oncogene. 1999; 18: 2967-2987Google Scholar, 8Schmidt E.V. Oncogene. 1999; 18: 2988-2996Google Scholar). In agreement, the down-regulation of Myc results in the attenuation of both cell division and cell growth as well as protection against some apoptotic processes (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google Scholar, 10Mateyak M.K. Obaya A.J. Sedivy J.M. Mol. Cell. Biol. 1999; 19: 4672-4683Google Scholar, 11Johnston L.A. Prober D.A. Edgar B.A. Eisenman R.N. Gallant P. Cell. 1999; 98: 779-790Google Scholar, 12Adachi S. Obaya A.J. Han Z. Ramos-Desimone N. Wyche J.H. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 4929-4937Google Scholar, 13Trumpp A. Refaeli Y. Oskarsson T. Gasser S. Murphy M. Martin G.R. Bishop J.M. Nature. 2001; 414: 768-773Google Scholar). Despite extensive research, the specific mechanisms by which these highly evident biological end points are achieved are not well understood. This is largely because a comprehensive list of biologically relevant Myc target genes has not yet been defined. A wide variety of techniques have been employed in the hunt for Myc targets, ranging from differential expression screens, promoter analysis, and informed guesswork (14Cole M.D. McMahon S.B. Oncogene. 1999; 18: 2916-2924Google Scholar, 15Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Google Scholar, 16Greasley P.J. Bonnard C. Amati B. Nucleic Acids Res. 2000; 28: 446-453Google Scholar) to the modern methods of microarray profiling, serial analysis of gene expression, and chromatin immunoprecipitation (17Coller H.A. Grandori C. Tamayo P. Colbert T. Lander E.S. Eisenman R.N. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3260-3265Google Scholar, 18Guo Q.M. Malek R.L. Kim S. Chiao C. He M. Ruffy M. Sanka K. Lee N.H. Dang C.V. Liu E.T. Cancer Res. 2000; 60: 5922-5928Google Scholar, 19Nesbit C.E. Tersak J.M. Grove L.E. Drzal A. Choi H. Prochownik E.V. Oncogene. 2000; 19: 3200-3212Google Scholar, 20Boon K. Caron H.N. van Asperen R. Valentijn L. Hermus M.C. van Sluis P. Roobeek I. Weis I. Voute P.A. Schwab M. Versteeg R. EMBO J. 2001; 20: 1383-1393Google Scholar, 21Schuhmacher M. Kohlhuber F. Holzel M. Kaiser C. Burtscher H. Jarsch M. Bornkamm G.W. Laux G. Polack A. Weidle U.H. Eick D. Nucleic Acids Res. 2001; 29: 397-406Google Scholar, 22Schuldiner O. Benvenisty N. Oncogene. 2001; 20: 4984-4994Google Scholar, 23Menssen A. Hermeking H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6274-6279Google Scholar). This search has been complicated by several factors. First, the weak transcriptional effects of Myc present significant experimental challenges. Second, by all recent indications the total set of Myc targets may be very large. Third, not all E boxes are bound by Myc, and transient transfection studies do not adequately reflect regulation in a chromosomal context. Fourth, comparing tumor cells expressing amplified Myc with nonderegulated counterparts is complicated by the nonisogenic nature of the cells. A widely used approach has been to compare cell lines engineered to overexpress ectopic Myc with parental cells (17Coller H.A. Grandori C. Tamayo P. Colbert T. Lander E.S. Eisenman R.N. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3260-3265Google Scholar, 19Nesbit C.E. Tersak J.M. Grove L.E. Drzal A. Choi H. Prochownik E.V. Oncogene. 2000; 19: 3200-3212Google Scholar, 21Schuhmacher M. Kohlhuber F. Holzel M. Kaiser C. Burtscher H. Jarsch M. Bornkamm G.W. Laux G. Polack A. Weidle U.H. Eick D. Nucleic Acids Res. 2001; 29: 397-406Google Scholar,23Menssen A. Hermeking H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6274-6279Google Scholar). However, it is questionable to what extent this approach can detect genes that respond optimally to physiological changes in Myc expression. In an attempt to circumvent the latter problem, some time ago we generated c-myc null cells that were derived by gene targeting from an immortalized but otherwise nontransformed rat fibroblast cell line (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google Scholar). To date, the c-myc−/− cells have been used in two limited profiling experiments that examined the expression of 4,400 rat (18Guo Q.M. Malek R.L. Kim S. Chiao C. He M. Ruffy M. Sanka K. Lee N.H. Dang C.V. Liu E.T. Cancer Res. 2000; 60: 5922-5928Google Scholar) and 6,355 mouse (24Watson J.D. Oster S.K. Shago M. Khosravi F. Penn L.Z. J. Biol. Chem. 2002; 277: 36921-36930Google Scholar) cDNAs and expressed sequence tags in spotted glass slide microarray formats. To create a new biological system for the optimal profiling of Myc target genes, we have reconstituted c-myc−/− cells with the conditionally active, tamoxifen-specific c-Myc-estrogen receptor fusion protein (MycER)1 (25Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Google Scholar). These new cell lines allow the modulation of Myc activity from essentially null to supraphysiological. To achieve maximum consistency in expression profiling we sought a simple experimental regimen in which the only changing parameter was the expression of c-Myc and in which a change in c-Myc status elicited and clear and significant change in phenotype. We chose to use randomly cycling, exponential phase cultures, and we developed conditions such that cells experienced a constant environment and were in a balanced, steady state of growth for significant periods of time. Under these conditions c-myc null cells displayed a pronounced phenotype, a 2–3-fold reduction in macromolecular synthesis accompanied by a commensurate slowing of the cell cycle (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google Scholar). Most importantly, we showed that under these conditions both c-myc+/+ and c-myc−/−cultures cycled uniformly, namely, that there were no cohorts of differentially cycling or noncycling cells within a given culture (26Obaya A.J. Kotenko I. Cole M.D. Sedivy J.M. J. Biol. Chem. 2002; 277: 31263-31269Google Scholar). Expression profiling using a total of 81 Affymetrix GeneChip arrays was performed in three experiments (Fig. 1). First, we compared c-myc+/+ (TGR), c-myc−/− (HO), and c-myc−/− cells reconstituted with a constitutive c-myc transgene (HOmyc3). This revealed the total number of genes that respond to a sustained loss of c-Myc under exponential growth conditions. Second, c-myc−/− cells reconstituted with the conditional c-mycER transgene (HOmycER) were stimulated with 4-hydroxytamoxifen (OHT), and data were collected during a 16-h time course. This revealed the kinetics of the responses to Myc activation. Finally, the time course of induction with OHT was performed in the presence of cycloheximide, revealing a subset of direct transcriptional targets of c-Myc. All experiments, including the growth of cells and preparation of RNA, were performed on three separate occasions (independent biological replicates), and all data were subjected to a statistical analysis of significance. TGR-1 is a hprt– subclone of the Rat-1 cell line (27Prouty S.M. Hanson K.D. Boyle A.L. Brown J.R. Shichiri M. Follansbee M.R. Kang W. Sedivy J.M. Oncogene. 1993; 8: 899-907Google Scholar). HO15.19 (referred to as HO) is a homozygous c-myc null derivative of TGR-1 generated by gene targeting (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google Scholar). HOmyc3 was derived from HO15.19 by constitutively expressing murine c-myccDNA using a retroviral vector (10Mateyak M.K. Obaya A.J. Sedivy J.M. Mol. Cell. Biol. 1999; 19: 4672-4683Google Scholar). HOmyc3 cells express c-Myc protein at three to four times the level seen in TGR cells. HOMycER12 and HOMycER104 were derived in the same fashion to express MycER (25Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Google Scholar). MycER is a hybrid protein consisting of the entire c-Myc polypeptide at its N terminus and the ligand (estrogen) binding domain of the human estrogen receptor at the C terminus. In the MycER construct used here the estrogen binding domain has been mutated to be specific for the agonist OHT. Retroviral vectors were packaged in BOSC cells (28Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Google Scholar), and supernatants were used to infect HO15.19 cells. Colonies were selected with 120 ॖg/ml hygromycin (Calbiochem), ring cloned, and expanded into clonal cell lines. The mRNA encoding the MycER protein is thus expressed constitutively from the viral long terminal repeat promoter, and the activity of this promoter is not affected by OHT. OHT is instead believed to elicit a conformational change in MycER which allows the protein to become biologically active. All cultures were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 107 calf serum (Hyclone) at 37 °C in an atmosphere of 57 CO2, except BOSC cells, which were supplemented with 107 fetal bovine serum (Hyclone). Great care was taken that cultures were cycling asynchronously and were in rapid and exponential phase of growth (26Obaya A.J. Kotenko I. Cole M.D. Sedivy J.M. J. Biol. Chem. 2002; 277: 31263-31269Google Scholar). Briefly, cells were always split at subconfluent densities (<507) and at relatively low dilution (1:10 for c-myc+/+ and 1:4 for c-myc−/− cells). Cultures can thus be maintained continuously at densities of 10–507 confluence (to avoid any contact inhibition), and the relatively frequent passaging (every 3–4 days) and medium changes maintain a rapid growth rate. This regimen was followed for a minimum of two passages before cells were harvested for other experiments. MycER was activated with 200 nm OHT (Sigma). Dose-response studies showed that 200 nm OHT is saturating for the activation of MycER. OHT was dissolved in absolute ethanol at 1 mm and stored at −80 °C. Mock-treated cultures received vehicle (ethanol) at a final concentration of 0.027. Protein synthesis was inhibited with 20 ॖg/ml cycloheximide (Sigma) which was added 30 min before the addition of OHT. BrdUrd labeling and flow cytometry were performed as described previously (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google Scholar), except that the Vectastain Elite ABC™ and Novared™ kits (Vector Laboratories) were used for histochemical staining. The c-myc, full-length MycER (25Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Google Scholar), and a deletion mutant of MycER missing amino acids 106–143 (29Marhin W.W. Chen S. Facchini L.M. Fornace Jr., A.J. Penn L.Z. Oncogene. 1997; 14: 2825-2834Google Scholar) cDNAs were cloned into the HpaI site of the pLXSH retroviral vector (30Miller A.D. Miller D.G. Garcia J.V. Lynch C.M. Methods Enzymol. 1993; 217: 581-599Google Scholar) using standard procedures (31Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Total RNA for Northern hybridization and microarray analysis was isolated using TriZol reagent (Invitrogen). Total RNA for quantitative real time PCR (qPCR) was isolated using the RNaqueous-4PCR kit (Ambion). Northern hybridization was performed using the formaldehyde gel method, and 32P-labeled probes were synthesized using the random oligonucleotide labeling method from gel-purified restriction or PCR fragment templates as described previously (32Wei W. Hemmer R.M. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 6748-6757Google Scholar). qPCR was performed using the Applied Biosystems Prism 7700 Sequence Detector and software. Primers were designed using Primer Express software (Applied Biosystems) for amplification of 100-bp fragments. cDNA was generated using the TaqMan reverse transcription kit and amplified using the SYBR green PCR and reverse transcription PCR kits (Applied Biosystems). Amplification efficiencies were determined by serial dilution of template cDNA for each gene. All samples were run in triplicate. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the internal standard. GAPDH was used because microarray profiling showed that the signals for six distinct GAPDH probe sets were equivalent between TGR, HO, and HOmyc3 cell lines under our exponential phase culture conditions. Protein samples were prepared by lysing whole cells in radioimmune precipitation assay buffer (33Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar) supplemented with protease inhibitors. Immunoblotting was performed as described previously (10Mateyak M.K. Obaya A.J. Sedivy J.M. Mol. Cell. Biol. 1999; 19: 4672-4683Google Scholar, 34Hanson K.D. Shichiri M. Follansbee M.R. Sedivy J.M. Mol. Cell. Biol. 1994; 14: 5748-5755Google Scholar). The following antibodies were used: c-Myc (Upstate Biotechnology, cat. 06-340), neomycin phosphotransferase II, (5 Prime – 3 Prime, Inc., cat. 7-511721), and actin (Sigma, cat. A5316). Horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunoresearch. Signals were visualized using the ECL reagent (Amersham Biosciences). Target cRNA was prepared and hybridized (45 °C, 16 h) to GeneChip rat U34 arrays according to the manufacturer's directions (Affymetrix). Hybridized arrays were washed and stained using the GeneChip fluidics station 400 and scanned using the Agilent GeneArray scanner. Signals were analyzed using Microarray Suite 5.0 software (Affymetrix). Data were normalized using a set target intensity of 1,500, published to a data base using MicroDB 3.0, and analyzed in Data Mining Tool 3.0 software (Affymetrix). Analysis of each cell line and/or condition was based on three biological replicates (RNAs prepared from independent experiments performed at different times). The replicates were used to calculate the means and standard deviations for the expression values of all probe sets for each cell line and/or condition. Probe sets were considered present if they received a present call in two of the three biological replicates. Pairwise comparisons between cell lines and/or conditions were made using Student's t test (p < 0.05). Myc-influenced expression patterns were assigned to four categories: 1) activated by Myc; 2) repressed by Myc; 3) activated by overexpression of Myc; and 4) repressed by overexpression of Myc (for examples, see Fig. 4). Probe sets were categorized based on the following criteria. The ratio of the average signal intensity between HOmyc3 and HO and/or between TGR and HO was ≥2.0. The ratio of the average signal intensity between HOmyc3 and HO and/or between TGR and HO was ≤0.50. HO/Myc3 average intensity values were significantly greater than those of TGR and those of HO (p < 0.05), TGR and HO average intensity values were not statistically different (p < 0.05), and the ratio of average intensity values between TGR and HO was less than 1.4. 14 probe sets were moved from category 1 to category 3 based on visual inspection (in these cases, the ratio of the average intensity values between HO and HOmyc3 was greater than or equal to twice the ratio of average intensity values between HO and TGR. The HOMyc3 average intensity values were significantly less than those of TGR and those of HO (p < 0.05), TGR and HO average intensity values were not statistically different (p < 0.05), and the ratio of average intensity values between TGR and HO was greater than 0.7. Nine probe sets were moved from category 2 to category 4 based on visual inspection (in these cases, the ratio of average intensity values between HO and HOmyc3 was less than or equal to half the ratio of average intensity values between HO and TGR). Probe sets were considered responsive to OHT if, at any given time point, they displayed statistically significant (p< 0.05) differences between OHT and vehicle-treated replicates, and the fold change between the means was ≥1.5. Of the 535 probe sets on the U34A chip which were identified in the initial comparison of TGR, HO and HOmyc3 cell lines (experiment 1), 142 probe sets were OHT-responsive by the above criteria. An additional 460 probe sets satisfied the OHT inducibility test (experiment 2, Fig. 1) but failed the 2-fold induction limit set in the comparison of TGR, HO, and HOmyc3 cells (experiment 1). 76 probe sets were recovered from this list and designated as c-Myc targets if the inducibility in the TGR, HO, and HOmyc3 comparison (experiment 1) was ≥1.5. The resultant 218 (142 + 76) MycER-responsive probe sets out of the total 611 (535 + 76) probe sets represent 180 nonredundant genes. 75 of the 180 genes (417) identified in HO/mycER12 cells satisfied the same statistical criteria in HOmycER104 cells. Of the remaining 105 genes, 49 (277) were already deregulated by the high basal Myc activity in HOmycER104 cells, 36 (207) behaved qualitatively similarly in HOmycER104 cells but failed the t test, and 20 (117) failed the fold change test or behaved anomalously. Because the MycER protein is capable of eliciting low Myc activity even in the absence of OHT, we also asked whether this 舠leakiness舡 could mask potential responses to Myc activation if, for example, probe sets were already maximally induced/repressed before the addition of OHT. Probe sets were considered leaky if the average intensity values in HOmycER12 cells were statistically different (p < 0.05) and greater (for Myc-activated genes) or smaller (for Myc-repressed genes) than the average intensity values in HO cells. Probe sets that were leaky, nonresponsive to OHT, unable to respond to elevated levels of Myc (nonresponsive to OHT in HOmycER104 cells and/or not overexpressed in HOmyc3 versus TGR cells), and expressed above a threshold intensity value of 500 in TGR cells comprised less than 107 of the probe sets identified in experiment 2. Probe sets were considered to be direct Myc targets if differences in expression between samples treated with cycloheximide plus OHT and those treated with cycloheximide alone at any time point were statistically significant (p < 0.05) and had a magnitude of ≥1.5. Probe sets were classified as indirect targets of Myc if differences in expression between samples treated with cycloheximide alone were not statistically different (p< 0.05) from the untreated control and if differences in expression at any time point between samples treated with cycloheximide plus OHT and cycloheximide alone were not statistically significant (p < 0.05). To assess the effect of OHT alone on RNA expression (in the absence of the MycER transgene) TGR and HO cells were treated with OHT for 16 h (or vehicle for the same time period), and RNA was extracted and subjected to microarray analysis. 288 of the total 8,799 probe sets on the U94A chip were affected by OHT by a factor of ≥2 in either TGR or HO cells. 2 of the OHT-affected probe sets are on the list of 180 genes reported in Table I. However, these probe sets were affected by OHT only in HO cells and not in TGR cells. The genes affected by OHT alone in HO cells are α-mannosidase II (M24353) and cytosolic Na/K-transporting ATPase, B subunit (AA859920). The expression of these genes was clearly affected in a comparison of TGR, HO, and HOmyc3 cells in the absence of OHT; however, because part of their response in HOmycER cells may be the result the effect of OHT alone, further examination may reveal them to be indirect Myc targets.Table IGenes responsive to Myc expression AccessionEncoded gene productFunctionE/M/L1-aKinetics of response to MycER activation: early (E), middle (M), or late (L) if the change in expression was first evident at 2–4 h, 8 h, or 16 h after the addition of OHT, respectively. O designates that the gene responded to Myc overexpression only.Fold changeD/I/X1-bThe response to MycER activation with OHT in the presence of cycloheximide was used to evaluate direct versus indirect mode of action: D, direct target; I, indirect target; X, inconclusive target.ER1041-cResponse confirmed in HOmycER104 cell line indicated as (+).qPCR1-dResponse confirmed by qPCR. Values given are the -fold change in target gene expression between c-myc+/+(TGR) and c-myc−/− (HO) cells expressed as the ratio of TGR to HO qPCR values.T/H1-eFold change in target gene expression between c-myc+/+ (TGR) and c-myc−/−(HO) cells expressed as the ratio of TGR divided by HO average intensities.M/H1-fFold change in target gene expression between Myc-reconstituted c-myc−/− (HOmyc3) and c-myc−/− (HO) cells expressed as the ratio of HOmyc3 to HO average intensities.Genes activated by MycMetabolic enzymes D26393Hexokinase, type IIGlycolysisE1.41.6D*M767671-g* designates genes identified in previous screens.Fatty acid synthaseFatty acid biosynthesisE1.31.5X*D10853Phosphoribosyl pyrophosphate amidotransferasePurine biosynthesisE1.92.6X AI008131S-Adenosylmethionine decarboxylasePolyamine biosynthesisE1.71.5X J03588Guanidinoacetate methyltransferaseCreatine synthesisE2.22.8X+*M58040Transferrin receptorIron transportE6.07.6X5.6 S70011Sideroflexin 1 (mitochondrial)Iron transportE6.69.9X+ J05571S-adenosylmethionine synthetaseOne carbon transferEO1.11.6X AA799700Selenophosphate synthetase 2Selenocysteine synthesisEO1.22.5X+ AI059508TransketolasePentose phosphate pathwayM1.93.0X+ AA859981Myo-inositol monophosphatase A2Phosphoinositide biosynthesisM1.82.4X+*AB007768CADPyrimidine biosynthesisM4.06.1X+2.1 U07201Asparagine synthetaseAmino acid biosynthesisM1.71.8X AI230228Similar to phosphoserine aminotransferaseAmino acid biosynthesisM2.85.1X D10262Choline kinasePhospholipid biosynthesisMO1.02.4X*L25387Phosphofructokinase CGlycolysisL3.65.0I D13921Acetyl-CoA acetytransferase (mitochondrial)Carbon utilizationL1.41.5X+ AA891785Isocitrate dehydrogenase (mitochondrial)Energy metabolismL2.32.0X+ AA799466Adenylate kinase 2Energy metabolismL1.41.8X+*X13527Fatty acid synthetase (acyl carrier protein)Fatty acid biosynthesisL1.72.2X+ AA799779Dihydroxyacetonephosphate acyltransferaseLipid biosynthesisL1.31.7X+ U57042Adenosine kinasePurine salvageL2.22.2XRibosome biogenesis AF025424RNA polymerase I (127 kDa subunit)rRNA synthesisE1.51.7X+ AA799724RNA polymerase I (RPA16 subunit)rRNA synthesisE1.61.9I+*AA998882Nucleolar phosphoprotein Nopp140Nucleolar assemblyM1.75.0D+3.6 AA892562Nucleolar protein NAP57rRNA processingM1.51.8I*J04943Nucleoplasmin-related protein B23Ribosome biogenesisM1.82.4I*U21719DEAD-box RNA helicase DDX21, nucleolarrRNA processingL2.83.9D+ AA891759Similar to DEAD-box RNA helicaserRNA processingL1.41.7X+*M55017NucleolinRibosome biogenesisL2.22.6D+1.4Protein synthesis AI031019eIF-2BαTranslation initiation factorE1.41.5D+ U38253eIF-2BγTranslation initiation factorE1.72.0D*AA875205eIF3, subunit 9Translation initiation factorE1.62.1I+*M98327tRNA-valine synthetaseAminoacylation of tRNAM1.92.5D*AA891689L38Mitochondrial ribosomal proteinM2.02.6I+ AA866234Highly similar to EF-TuTranslation elongation factorMO1.32.0I+*J02648elF2-αTranslation initiation factorL1.61.5X+ AA891553elF3, subunit 7Translation initiation factorL1.82.1I U62635L23-related proteinRibosomal proteinL3.42.6XProtein folding*U75392BAP-37Mitochondrial chaperoneE2.02.6X+*AA858640Hsp60Mitochondrial chaperoninM2.02.4D*AI170613Hsp10Mitochondrial chaperoninM1.62.2X+*AI169631ProhibitinMitochondrial chaperoneM1.62.3X+3.2*AA799645TCP-1 γ subunitCytosolic chaperoninM1.51.8X+*U62940GrpE stress-inducibleMitochondrial chaperoneL1.31.9IProtein degradation M61142MetalloendopeptidaseProtein degradationE2.52.6X+ U38379γ-Glutamyl hydrolaseProtein degradationE2.21.8X+ M19647Kallikrein-1Serine proteaseM1.82.4X AB012759Prolyl endopeptidaseProtein degradationM1.41.7X AA891445SKD3Clp/Hsp 104 ATPase familyM1.62.9I+ AI176351Tripeptidylpeptidase IIProtein degradationL2.12.5XProtein glycosylation, transport, ion transport AF087431Glucosidase 1Protein glycosylationE1.61.8D+ AB006451Inner membrane translocase TIM23Protein transport (mitochondrial)E1.21.6I+ AF061242Inner membrane translocase subunit TIM9BProtein transport (mitochondrial)EO1.12.7I D13985Chloride channelIon channelM1.52.5I AA859920Cytosolic Na/K transporting ATPase, B subunitIon pumpM2.01.8X+Vesicular trafficking AA875098ARF3Small G proteinE1.93.5X U40999UNC-119 (C. elegans) homologVesicular traffickingEO1.01.6X+ AA799879Moderately similar to synaptogyrin 1Synaptic vesicle proteinL2.24.0IDNA replication, repair, chromatin assembly*AJ222691DNA polymerase δ catalytic subunitDNA replicationM1.92.3X*AF062594Nucleosome assembly proteinChromatin assemblyM1.71.4D+ U60882Protein arginine N-methyltransferaseHistone H4 methylationM1.82.6I+*D44495APEX nucleaseDNA repairL2.84.2D4.8 AA894030Similar to tyrosyl-DNA phosphodiesteraseDNA repairL2.12.1I+Transcription D45254CNBPTranscription factorE1.72.2D AA875084Transducin-like enhancer of split 1Transcriptional corepressorE2.33.5X+*AB002406Tip49ATPase/DNA helicaseE1.82.2D+2.9 AA893611Moderately similar to MXI1Transcription factorEO1.22.3X AA799412Similar to estrogen-related receptor αTranscription factorEO0.92.1X*X62875HMG I/YTranscription factorM1.01.9D AI229637Myb-binding protein (p160)Transcriptional
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