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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. 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 microarray To a new biological system for the optimal profiling of Myc target we have c-myc−/− cells with the c-Myc-estrogen receptor fusion protein Nucleic Acids Res. Scholar). new cell lines the of Myc activity from essentially null to To in expression profiling we a experimental in which the the expression of and in which a in elicited and clear and significant in We to and we that cells a and were in a state of growth for significant of these c-myc null cells a a in macromolecular by a of the cell cycle (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google Scholar). we that these both and that were of or cells within a A.J. I. M.D. Sedivy J.M. J. Biol. Chem. 2002; 277: Scholar). profiling a total of in experiments First, we c-myc−/− and c-myc−/− cells with a c-myc This revealed the total of genes that respond to a of growth Second, c-myc−/− cells with the conditional were with 4-hydroxytamoxifen and were a time This revealed the of the to Myc the time of with in the of a subset of transcriptional targets of the growth of cells and of were on biological and all were to a analysis of is a of the cell line S.M. M. Sedivy J.M. Oncogene. 8: Scholar). to as is a c-myc null of generated by gene targeting (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google Scholar). derived from by expressing a M.K. Obaya A.J. Sedivy J.M. Mol. Cell. Biol. 1999; 19: 4672-4683Google Scholar). cells protein to the in cells. and were derived in the to Nucleic Acids Res. Scholar). is a protein of the its and the binding of the human receptor the In the used the binding has been to be specific for the were in cells D. Proc. Natl. Acad. Sci. U. S. A. and were used to cells. were with and cell lines. The the protein is expressed from the and the activity of this promoter is not by is to a in which the protein to biologically were in with in an of which were with that were and were in rapid and of growth A.J. I. M.D. Sedivy J.M. J. Biol. Chem. 2002; 277: Scholar). cells were and for and for c-myc−/− can be of and the and changes a rapid growth This for a of two cells were for with studies that is for the of in and a of with which the of and were as (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google that the and were used for The Nucleic Acids Res. and a of S. A.J. Penn L.Z. Oncogene. 1997; Scholar) cDNAs were the of the Scholar) J. A Scholar). for and microarray analysis for quantitative real time PCR the the and were the from or PCR as Sedivy J.M. Mol. Cell. Biol. 2001; 21: Scholar). the and were for of generated the reverse transcription and amplified the PCR and reverse transcription PCR were by serial of for were in phosphate dehydrogenase used as the used because microarray profiling that the for were and cell lines were by cells in D. A Scholar) with as M.K. Obaya A.J. Sedivy J.M. Mol. Cell. Biol. 1999; 19: 4672-4683Google Scholar, M. Sedivy J.M. Mol. Cell. Biol. Scholar). The were and were from were the and to rat to the were and the and the were were a set target of to a and in of cell line based on biological from experiments The were used to the and for the expression of all for cell line were present a present in two of the biological cell lines were expression were to by by by of and by of Myc were based on the The of the and and The of the and and were significantly of and of and were not and the of and less were from to based on these the of the and or to the of and The were significantly less of and of and were not and the of and were from to based on these the of and less or to the of and were to time significant and and the the the on the which were in the of and cell lines were by the the but the set in the of and cells were from this list and as targets the in the and The of the total genes. of the genes in cells the in cells. the were by the Myc activity in in cells but the and the or the protein is capable of Myc activity in the of we this to Myc for were the of were the in cells were and Myc-activated or Myc-repressed the in cells. that were to to respond to of Myc to in cells not in and expressed a of in cells less of the in were to be Myc targets in expression with and with time were significant and a of were as indirect targets of Myc in expression with were not from the and in expression time with and were not significant To the of on expression the of the and cells were with for for the time and and to microarray of the total on the were by by a factor of in either or cells. of the are on the list of genes in I. However, these were by in cells and not in cells. The genes by in cells are and The expression of these genes in a of and cells in the of because of their in cells may be the the of may to be indirect Myc to Myc expression gene of to or the in expression evident or the of that the gene to Myc to with in the of used to indirect mode of indirect in cell line as by are the in target gene expression and c-myc−/− cells expressed as the of to in target gene expression and cells expressed as the of by in target gene expression c-myc−/− and c-myc−/− cells expressed as the of to by genes in carbon phosphate to dehydrogenase protein protein to of to to chromatin to of to to receptor protein transcription factor transcription cell cycle to receptor for gene growth factor protein growth to factor to to C. to C. to human to protein to human by protein of protein protein protein to and tumor cell cell to human protein to protein receptor receptor for A to human receptor receptor factor factor to to C. to human to human protein to of to or the in expression evident or the of that the gene to Myc The to with in the of used to indirect mode of indirect in cell line as by are the in target gene expression and c-myc−/− cells expressed as the of to in target gene expression and cells expressed as the of by in target gene expression c-myc−/− and c-myc−/− cells expressed as the of to genes in in a new The Nucleic Acids Res. Scholar) the with in the of resulted in an of and in the a and of c-myc−/− cells in the the of the parental cells. of a an of Myc not The are likely to be the of of Myc activity elicited by the in the of of both were and cell lines. were for derived from a and derived from a cells expressed of protein cells were to the of the is known to its we examined the expression of the which is by of the c-myc gene targeting and of the c-myc promoter by the of both and cells with the expression of the In contrast, of by not in the or cell lines expression of Myc the cell cycle and the of cells in (9Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048Google we of and The of cells in increased from to in cells and from to in cells a with lines with The of cells in with The cell lines were for the of growth, and in in the for and c-myc−/− cells as the of were used as not the of or cells not We examined the expression of several known target genes in cells in the and of of resulted in the up-regulation of and down-regulation of the expression of essentially or The has been for these genes by comparing and cells growth A. Mateyak M.K. K. Obaya A.J. Adachi S. Sedivy J.M. M.D. Dev. Scholar). In on the expression of or in either or cells We that cells in the of c-myc−/− cells and that of a In for experiments in the of we examined the of on the expression of the protein of the protein and and protein is known to be with a in the range of Kim EMBO J. 1999; 18: the of the protein the of not This the of as a to the of Myc to targets that respond within a time from of and and expression profiling rat cell line on and of the of biological to the rat in significant and cells and cells. an expression differential of the of and and the of to were to their of were as by Myc, as by Myc, as by of Myc, and as by of of are in The of expression whose biological to c-myc is not To the of the microarray analysis, we examined the expression of and genes In of the the microarray the most of the known rat genes it used in of cells were with either or and were and in The time the time of in total The time on to biological for a total of the expressed on the in the and in were to in of some present on the the to to genes or expressed sequence were to their of as or the in expression evident or the of within these genes were to general are in The cell line in a time of with and a of the and we to which of the genes and expressed sequence tags that we as may be of cells were with either or and were and in The time not either the time in biological of genes were as all were in the Myc-activated In we genes and genes that to indirect is well that can strongly gene expression. effects have the of significantly the of Myc on the expression of target genes and the of all time all of the genes that the of a or indirect target significant or from In these we do not that a clear a and an indirect target can be of time in are in and the for all genes are in I. The the most extensive of the rat genes and expressed sequence The set is the most comprehensive analysis of gene expression to In we expressed by or that of all were expressed in of the is to Myc within the differential expression the of the rat can be to be in this cell However, the differential expression is to the of all genes. The significantly increased yield of genes achieved in this is the of ability to modulate Myc expression from to This is by the that a (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 which on ectopic expression in a cell targets from a total of genes studies used and very for is clear that a subset of Myc targets can respond to Myc the majority of in the range of physiological expression. the effects of Myc on gene expression, it is that of expressed to Myc in the cell of the genes have been in of the is the time of genes because effects on the of the However, Myc-repressed genes of the set and of the total of expressed that the extent to which Myc-repressed genes are in the time is likely to be The most likely for the large of genes is that to the of Although the of targets with be the that many genes respond that Myc are likely to for the the list of genes many indirect targets, that indirect effects can be rapid to in the time The changes that the of to cells are and is not that the extensive of cellular which in to of Myc activity or several cell is clear that the majority of these changes are because of all that are expressed in c-myc−/− cells to cells are to a of expression in the cell Although the of with in the of has been used to and indirect of Myc, that this has limited The are the of the protein and the of significant changes in gene expression by the were significantly by the were and direct, were and and were and genes were is that the of the the of and the effects of are on the of many genes. is that by Myc on interaction with as and that this interaction or the activity of the is by Myc-activated the of and indirect targets and are this not for genes to by we can targets the genes. of were on the and the set of of the rat we can Myc targets in a rat fibroblast growth Although and experimental expression profiling, are several it may be the total of genes. First, a gene may not be by Myc all growth the of Myc of cells significantly to the regulation of genes that may respond growth Second, some genes may be to respond to Myc a specific of the cell Third, cell or cell effects are likely to be Fourth, some genes are or not all by the and genes in profiling have functions The on the Myc-activated list of are involved in carbon and of these have been (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, 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, P.J. Mol. Cell. Biol. 1997; Scholar, H. Kim S. R. M. Y. D. Lee L.A. Dang C.V. J. Biol. Chem. 2000; Scholar). is is the of that the and of as biosynthesis of in carbon as in and in most In to and many of the genes are and their is correlated with rapid growth and on the Myc-activated list are functions that protein synthesis, involved in the and of the of and and factors. genes in this of which have been The expression of in c-myc−/− but in that are likely to be indirect However, because many were in it is that protein genes can respond to very Myc as in tumor cells 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). The of several protein functions on the Myc-activated both and is with an increased for protein these are the and and all of which have been as Myc have been to have important roles in the of apoptosis. The have effects S. S. D. T. Y. R. P. R. A. EMBO J. 1999; 18: Scholar, A. J. B. S. EMBO J. 1999; 18: Scholar). In contrast, and as Myc-repressed in analysis, have been to as of apoptosis their ability to from J.M. C. P. L. C. S. G. C. Cell Biol. 2000; Scholar) and the of M.C. F. J. Biol. Chem. 2001; We a of the as a Myc-repressed In a general sense, the majority of Myc-activated functions are indicative of a physiological state geared toward the rapid utilization of carbon and the biosynthesis of precursors for the of and with the up-regulation of the for protein and these changes the accumulation of cellular which is to cellular Myc the expression of cell cycle the as to which or cell are the effectors. The of genes that and cell growth, as well as the of of Myc targets in this it very that all these effects are that both and cell cycle functions may be of an enzyme involved in carbon or a cell cycle both the growth of c-myc−/− cells S. B. O. I. A. Sedivy J.M. M.D. Mol. Cell. Biol. 2002; Scholar, H. C. M. Obaya A.J. Mateyak M.K. Kohlhuber F. Dang C.V. Sedivy J.M. Eick D. B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: Scholar). The Myc-repressed genes in to the Myc-activated and protein functions are and the list is by genes involved in cell and and In the latter has not been as This list genes involved in of as the protein the the protein and D. In involved in and the and the were to be by In a general sense, a significant of Myc-repressed genes are involved in the interaction and communication of cells with their external is to that several of these targets have been to possess tumor and antimetastatic properties. genes involved in and cellular inappropriate Myc expression may a for tumor cell Although be to and indirect targets and to the functions of the and this has significantly of the of Myc on cellular and has revealed a of for The total extent of transcriptional changes that can be triggered in to Myc activity is and it be that many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. 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O’Connell et al. (Fri,) studied this question.
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