The Expression of the Developmentally Regulated Proto-oncogenePax-3 Is Modulated by N-Myc

N-Myc is a member of the Myc family of transcription factors that have been shown to play a pivotal role in cell proliferation and differentiation. In this report, we have investigated the relationship between N-Myc and the developmental control gene Pax-3. Using transient transfection assays, we show that the Pax-3 promoter is activated by both N-Myc-Max and c-Myc-Max. Moreover, we show that Myc regulation ofPax-3 promoter activity is dependent upon a noncanonical E box site in the 5′ promoter region of Pax-3. In addition, we show that ectopic expression of both N-Myc and c-Myc leads to increased expression of Pax-3 mRNA. Furthermore, we show that Pax-3 mRNA expression is cell cycle-regulated and that the 5′ promoter region of Pax-3 (bp −1578 to +56) can direct cell cycle-dependent gene expression with kinetics similar to that of the endogenous transcript. Site-directed mutagenesis of the E box site within the Pax-3 promoter significantly altered the pattern of expression through the cell cycle. These results suggest that the Myc family of transcription factors may modulate Pax-3 expression in vivo. N-Myc is a member of the Myc family of transcription factors that have been shown to play a pivotal role in cell proliferation and differentiation. In this report, we have investigated the relationship between N-Myc and the developmental control gene Pax-3. Using transient transfection assays, we show that the Pax-3 promoter is activated by both N-Myc-Max and c-Myc-Max. Moreover, we show that Myc regulation ofPax-3 promoter activity is dependent upon a noncanonical E box site in the 5′ promoter region of Pax-3. In addition, we show that ectopic expression of both N-Myc and c-Myc leads to increased expression of Pax-3 mRNA. Furthermore, we show that Pax-3 mRNA expression is cell cycle-regulated and that the 5′ promoter region of Pax-3 (bp −1578 to +56) can direct cell cycle-dependent gene expression with kinetics similar to that of the endogenous transcript. Site-directed mutagenesis of the E box site within the Pax-3 promoter significantly altered the pattern of expression through the cell cycle. These results suggest that the Myc family of transcription factors may modulate Pax-3 expression in vivo. N-Myc is a member of the Myc family of transcription factors (c-Myc, N-Myc, L-Myc, B-Myc, and S-Myc) that are characterized by a basic DNA binding domain and dimerization domain composed of a helix-loop-helix and leucine zipper. Members of this family have been shown to play a pivotal role in cell proliferation and terminal differentiation. The forced expression of c-Myc promotes progression into S phase and inhibits differentiation and entry into a quiescent state (1Freying S.O. Dang C.V. Lee W.M.F. Cell Growth Differ. 1990; 1: 339-343PubMed Google Scholar). Furthermore, the deregulation of myc gene expression has been implicated in the pathogenesis of several tumor types. The human NMYC gene is frequently amplified in neuroblastoma, a childhood cancer of neural crest origin (2Schwab M. Altato K. Klempnauer K.H. Varmus H.E. Bishop J.M. Gibert F. Brodeur G. Goldstein M. Trent J. Nature. 1983; 316: 245-248Crossref Scopus (1054) Google Scholar, 3Siamon D.J. Boone T.C. Seeger R.C. Keith D.E. Chazin V. Lee H.C. Souza I.M. Science. 1986; 232: 768-772Crossref PubMed Scopus (85) Google Scholar).NMYC amplification in this cancer is associated with rapid tumor progression, advanced stages, and poor prognosis. N-myc shares many of the properties of c-myc, although unlike c-myc, whose expression appears to be ubiquitous, N-myc is primarily expressed during early embryogenesis (4Hirning U. Schmid P. Schutz A.W. Rettenberger G. Hameister H. Mech. Dev. 1991; 33: 119-126Crossref PubMed Scopus (86) Google Scholar). In mice, N-myc expression is highest around E9.5, where expression is observed in early neural crest lineages, limb buds, and developing central nervous system (5Kato K. Kanamori A. Wakamatsu Y. Sawai S. Kondoh H. Dev. Growth Differ. 1991; 33: 29-36Crossref Scopus (24) Google Scholar). The expression of N-myc then declines in these tissues upon the onset of differentiation. The importance of N-myc in embryogenesis is demonstrated by the finding that homozygous N-myc null mice die around embryonic day 11.5 with abnormalities in the limb buds and in the central and peripheral nervous systems (6Sawai S. Shimono A. Wakamatsu Y. Palmes C. Hanaoka K. Kondoh H. Development. 1993; 117: 1445-1455Crossref PubMed Google Scholar). Most notably, the N-myc-deficient embryos showed a great reduction in the number of mature neurons, especially those derived from the neural crest such as sensory and sympathetic neurons. These defects occurred despite compensatory c-Myc increases (7Stanton B.R. Perkins A.S. Tessarollo L. Sassoon D.A. Parada L.F. Genes Dev. 1992; 6: 2235-2247Crossref PubMed Scopus (310) Google Scholar), suggesting a unique role for N-Myc in development. To function, N-Myc, like c-Myc, must heterodimerize with Max proteins. Max proteins also contain a basic DNA binding domain and a helix loop helix and leucine zipper dimerization motif (8Blackwood M.E. Eisenman N.R. Science. 1991; 251: 1211-1217Crossref PubMed Scopus (1480) Google Scholar). This heterodimerization is required for sequence specific DNA binding as well as for biological function. Myc-Max heterodimers recognize the core sequence CA(C/T)GTG, termed the E box Myc sequence. In both yeast and mammalian cells, Myc-Max complexes are capable of activating reporter gene constructs containing concatamerized E box Myc sites (9Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar,10Luscher B. Larsson L.G. Oncogene. 1999; 18: 2955-29668Crossref PubMed Scopus (165) Google Scholar). In addition, Max proteins can also heterodimerize with Mad proteins that negatively regulate cell growth. Max-Mad complexes bind to the same E box motif as the Myc-Max complexes, but in contrast to Myc-Max complexes, they repress transcription (11Ayer D.E. Kretzner L. Eisenman R.N. Cell. 1993; 72: 211-232Abstract Full Text PDF PubMed Scopus (623) Google Scholar). Mad family members repress transcription through their association with the Sin3 proteins, which in turn recruit histone deacetylases to the complex (12Ayer D.E. Lawrence Q.A. Eisenman R.N. Cell. 1995; 80: 767-776Abstract Full Text PDF PubMed Scopus (526) Google Scholar). The central member of the Myc/Max/Mad network is Max, which is very stable. In contrast, the expressions of both Myc and Mad proteins are highly regulated. The myc genes are actively transcribed in dividing cells, but little expression can be detected in quiescent or differentiated cells. By comparison, the Mad genes are usually expressed in resting or differentiated cells with little expression in dividing cells (13Chin L.N. Schreiber-Agus I. Pelicer K. Chen H. Lee M. Dudast C. Crdon-Cardo C. DePinto R.A. Proc. U. S. A. 1995; PubMed Scopus Google Scholar). number of genes for c-Myc have been of the genes that are by c-Myc are in the and of and such as C. G. Proc. U. S. A. 1993; PubMed Scopus Google and Cell. 1995; PubMed Scopus Google Scholar), or in cell control such as K. Chen Nature. Scopus Google Scholar), D.J. Proc. U. S. A. PubMed Scopus Google Scholar), and B. J. V. Cell Growth Differ. 1993; Google Scholar). Pax-3 is a member of a family of transcription factors C. U. B. 1991; PubMed Scopus Google that have been shown to play a role in early of the Pax-3 gene has been shown to to a of developmental abnormalities neural a of limb and in neural cell Scholar, M. P. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, C. V. J. 1990; Google Scholar, M. M. P. 1993; PubMed Scopus Google Scholar, A. J. 1993; Google Scholar). like is expressed during early embryogenesis within neural crest lineages, limb buds, and the developing nervous The expression of Pax-3 in these tissues is to cells and is upon differentiation G. U. J. P. J. 1992; Scopus Google Scholar, 1998; PubMed Scopus Google Scholar). the of expression of N-myc and we have the relationship between N-Myc and and in this we that Pax-3 is a direct of N-Myc and and cells in containing and cell and in and To cell N-Myc, cells and with N-Myc expression with of which the by the transfection with of cells These then and in containing Cell by cells with 1995; PubMed Scopus Google and for The the in to a binding domain of the The is activated by the of the but is to cells in and for to Myc To to the for This results in the of the from the to the In transient transfection assays, and cells and as 1991; Full Text PDF PubMed Scopus Google Scholar). The transfection by cells with to the of C. DNA Scholar). of the a and then to of the to cell cells in for the cells or with containing and for the the as by 1983; PubMed Scopus Google Scholar), and the of the of by a to a and with in for in in and a of N-Myc or c-Myc and in then in containing with a of to for then and the of the to of by the with and from from and by as by 1998; PubMed Scopus Google Scholar), with and and the gene and and by and and for the gene S by the with a reporter and the with a To the as 1998; PubMed Scopus Google Scholar), and amplified by direct of the of in a to the with to of of in a containing in for for by a amplification of for and for the sequence in and to the of the gene as by J. PubMed Scopus Google Scholar). The Pax-3 promoter to amplified from DNA by the and and The amplified into and The Pax-3 promoter from with and and into to or into promoter constructs by and or The then and to to to to and to then to the constructs to expression of a reporter The expression containing N-myc and a of H. The from M. and into The and a from G. The E box sequence in the Pax-3 promoter from to mutagenesis the and The sequence of by for in by to for and and The the Pax-3 E box Myc sequence a E box Myc sequence E box sequence and a E box Myc sequence the by 1983; PubMed Scopus Google Scholar). as 1998; PubMed Scopus Google Scholar). a and of which with the to the of the To the of the also for with of specific to the of the cell cells and in of and and for DNA by L. Cell a The of cells in by cells the with and the of cells by cells with cell cells a of then by the of for then for and and for DNA c-Myc and N-Myc mRNA To Pax-3 expression is by N-Myc, the 5′ promoter region of the Pax-3 gene (bp −1578 to +56) amplified by from DNA and This region of the Pax-3 promoter has been shown by C. Development. Google to be for the ofPax-3 expression in vivo. the Pax-3 promoter (bp −1578 to +56) then of the reporter gene in the The Pax-3 promoter then into the cell with expression containing the of the N-myc gene or the of the activity transient with a that of the Pax-3 promoter with N-Myc or c-Myc to the of Pax-3 promoter N-Myc c-Myc in Pax-3 promoter activity and of the Pax-3 promoter with N-Myc and Max or c-Myc and Max to a in Pax-3 promoter In contrast, of Max with to a in Pax-3 promoter activity and In addition, the of expression for N-Myc-Max and increased in a expression of the Pax-3 promoter increased suggesting that members of the Myc family of transcription factors can activity in The expression of by the of of N-Myc-Max or c-Myc-Max. shown that the Myc family of transcription factors is capable of in transient transfection assays, we investigated this regulation dependent the direct binding of the Myc proteins to sites within the Pax-3 To a of Pax-3 promoter constructs derived from the promoter (bp −1578 to +56) promoter constructs to direct gene and we the of N-Myc-Max and to that of the promoter constructs activated in the of both N-Myc and Max and c-Myc and Max, from the to which activated by N-Myc-Max or This that the between and are for Myc regulation of Pax-3 of this region of the Pax-3 Myc E box binding site of the transcription site of this site from to the of N-Myc and Max or c-Myc and Max to promoter activity To N-Myc-Max or heterodimers can bind to the E box site from the Pax-3 cell from cells with expression containing N-Myc, c-Myc, and Max with a containing the Pax-3 E box and DNA binding by A. K. M. B. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In cells with expression little binding to the Pax-3 E box site In cells with E box binding that in control cells observed cells with N-Myc and Max, complex with a very complex In cells with both N-Myc and Max, the complex by both N-Myc and Max suggesting that this E box complex N-Myc-Max binding to the Pax-3 E box sequence. The by Max, suggesting that this E box binding Max binding The E box complex also observed in from cells with expression which is with this complex Max also that c-Myc, like N-Myc, bind to the E box motif with Max, binding These suggest that Max and N-Myc-Max and heterodimers can bind to the Pax-3 E box motif in To the binding of N-Myc-Max and for the Pax-3 E box the E box Myc sequence the E box site from or a E box In these the Pax-3 E box as a with from cells, with N-Myc-Max or and with a or of The of a of the Pax-3 E box site binding by and with N-Myc-Max and The E box motif and the E box site from gene to N-Myc-Max and although both the E box and E box site in binding N-Myc-Max containing a Pax-3 E box site to N-Myc-Max or binding to the These that both N-Myc-Max and heterodimers can bind to the E box motif in although N-Myc-Max heterodimers to bind to the Pax-3 E box site with c-Myc-Max. shown that N-Myc-Max and heterodimers can bind to the Pax-3 E box motif in cells, the Pax-3 E box sequence for binding to Myc-Max heterodimers in the cell To from the cell These cells in the of but upon the cells cell and into a mature cells but upon the ofPax-3 from dividing cells with a containing the Pax-3 E box site and by E observed Moreover, this binding E box binding by of containing the E box site but by containing a E box sequence. Furthermore, the of N-Myc and Max the of c-Myc also binding the c-Myc is specific for c-Myc and with members of this family this that in dividing cells, the E box site within the Pax-3 promoter is by a of N-Myc-Max and that cells both N-Myc and c-Myc proteins In contrast, Mad or E box binding in cells, little binding to the E box site observed mRNA expression during the cell cycle. cells by for The cells then from the cell by the of and cells and the of for the of N-Myc and c-Myc expression by and mRNA expression by and for the of cells in or phase the shown that N-Myc and c-Myc can promoter we the that Pax-3 expression may be by members of the Myc family of transcription factors in vivo. a gene of N-Myc, then expression of Pax-3 mRNA be to be altered in cell the expression ofPax-3 mRNA in a of cell containing a NMYC gene and or amplified NMYC gene and (2Schwab M. Altato K. Klempnauer K.H. Varmus H.E. Bishop J.M. Gibert F. Brodeur G. Goldstein M. Trent J. Nature. 1983; 316: 245-248Crossref Scopus (1054) Google Scholar). as shown in that cell expressed of NMYC with the highest of NMYC detected in and cell These are the cell with amplified NMYC that of with of NMYC with and cells the highest of Pax-3 and cell ofPax-3 and the of detected in the cell with a NMYC gene and To this between N-Myc and Pax-3 mRNA expression the cell with expression containing a N-myc cell by transfection and for that in the cells with the the of N-Myc that observed in control cells which with the expression mRNA also increased in the cell N-Myc, with the highest of detected in the cell and These cell also the highest of N-Myc These results suggest that the ectopic expression of N-Myc leads to in endogenous Pax-3 mRNA we have shown that c-Myc can also bind to the Pax-3 E box with N-Myc-Max we investigated Pax-3 mRNA increased in cell despite we to cell cell a of c-Myc, and these cell then to c-Myc by the of to in Pax-3 mRNA that in the activated cell Pax-3 mRNA the that Pax-3 expression is by members of the Myc family of transcription factors in vivo. Pax-3 mRNA expression also where the expression of Myc proteins is to be c-Myc and are to during the cell A. A. G. J. PubMed Scopus Google we the of Myc expression and the ofPax-3 mRNA during the of the cell in the cell To cells by for The cells then from this cell by the of to the cells. and the of to the cells and for DNA by for c-Myc and N-Myc expression by and for Pax-3 mRNA by that in cells, expression of N-Myc or c-Myc be detected within of the of to the cells, of N-Myc and c-Myc and the expression of both N-Myc and c-Myc addition, through S phase and mRNA also through the cell cycle. expression of Pax-3 mRNA detected in the within of the of of Pax-3 mRNA and by addition, of Pax-3 mRNA of Pax-3 mRNA then as the of the cells S phase the in the in N-Myc and c-Myc expression by which be with by N-Myc or To members of the Myc family of transcription factors are for the cell cycle-dependent expression of Pax-3 the 5′ promoter region (bp −1578 to +56) of the Pax-3 gene of the reporter gene in the cells with this with and by The cells then from their cell by the of and and and for and DNA as shown in that the expression of addition, expression through S This pattern of cell expression with the of expression of endogenous Pax-3 mRNA during the cell and that of the in the cell expression of Pax-3 are within the region −1578 to of the Pax-3 In contrast, observed in the expression of as by Y. Cell. 1993; PubMed Scopus Google or in expression To the E box Myc sequence of promoter is required for the cell expression of the reporter the Pax-3 promoter the E box site of the gene in and the expression of this with the promoter through the of the cell cycle. we that the of activity in the cells with the that observed in cells with the suggesting that the E box site in the promoter has Pax-3 expression in cells. also that the expression of the promoter during the cell both the of and the of expression in cells with the promoter with the In cells with the a in activity occurred the of with the onset of S This with a in activity the in cells with the These results suggest that the E box binding site of the transcription site in promoter role both in the of Pax-3 expression in cells and in the of Pax-3 expression in the of the cell cycle. In this report, we have the developmental control as a of the Myc family of transcription have shown in transient that both N-Myc and c-Myc the activity of promoter by and The of the Pax-3 promoter by the of N-Myc and c-Myc with their dimerization The of N-Myc or c-Myc to promoter in the of Max the that cells endogenous Max, which may heterodimers with the Myc proteins. The transfection of Max, in the of N-Myc or c-Myc, in contrast, to a reduction in Pax-3 promoter Max proteins have been to with the Sin3 proteins C. Cell. 1993; PubMed Scopus Google Scholar), Max inhibits activity by for the Pax-3 E box site with endogenous Myc-Max The network of has been shown to modulate gene expression through a of E box which have the core sequence (9Facchini L.M. Penn L.Z. FASEB J. 1998; 12: 633-651Crossref PubMed Scopus (333) Google Scholar, B. Larsson L.G. Oncogene. 1999; 18: 2955-29668Crossref PubMed Scopus (165) Google Scholar). 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This is with that show that N-Myc and c-Myc expression is to cells. in cells, we binding to the E box This may be to the very activity of Mad in cells or to the that complexes are into many complexes in cells, which be detected A. K. M. B. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This is the that with Sin3 and Sin3 to a number of proteins such as histone deacetylases and by I. R.C. L. R.A. Genes Dev. PubMed Google have demonstrated a of between the N-Myc and c-Myc proteins. to show that mice in which the been with N-myc into and between mice and the of mice with the of mice also and of observed in a of These observed between mice and mice suggest that between N-Myc and c-Myc This is with the that although is sequence between N-Myc and c-Myc, are unique to we that N-Myc-Max complexes with to the Pax-3 E box site c-Myc-Max. in DNA binding between and N-Myc-Max has been by and M.E. Proc. U. S. 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S. 1999; Google the sequence for regulation of these genes have to be The finding that Myc-Max heterodimers can bind to the E box site in promoter in the that Pax-3 expression in may be by members of the Myc family of transcription In of this we have shown that expression in a number cell The ofPax-3 mRNA expression in cell with amplified NMYC gene is a number of cell have been and be to the of this and the that Pax-3 to the pathogenesis of by Pax-3 mRNA in a of is to mRNA has also been in neural crest 1999; Google Scholar, J. Google Scholar), where has been shown to be for tumor cell J. Google Scholar). amplification of Myc in these with increased Pax-3 expression to be In we also detected of in a of cell to cell from a promoter Cell the of c-Myc, Moreover, the of by to mRNA This that the ectopic expression of c-Myc, like N-Myc, can increased expression ofPax-3 mRNA. Pax-3 is in gene of the Myc family of transcription then Pax-3 expression be in cell mRNA also be where expression of Myc is mRNA expression during the cell as N-Myc and c-Myc expression Moreover, a in c-Myc and N-Myc expression observed the of early in a in Pax-3 mRNA observed This between the in Myc expression and Pax-3 expression is with the that Pax-3 is a gene for the Myc family of transcription factors and with that show that the expression of Myc genes such Cell. 1995; PubMed Scopus Google are also like Pax-3 in Using reporter we also to show that of the required cell expression are within the 5′ region of the Pax-3 promoter from −1578 to of the E box site within the significantly altered the pattern of expression of the reporter in cells. In cells with the a in activity observed S Furthermore, the of the observed with the suggesting that Myc binding proteins play role in the cell cycle-dependent expression of Pax-3. also observed that the of expression of the the promoter in the cells. This finding that the E box motif in promoter is by factors in cells. E box binding factors may the Mad family of proteins, which have been shown to be highly expressed in cells and may play a role in the of Pax-3 expression in cells. these suggest that Pax-3 cell expression is through the noncanonical E box motif in the promoter of that show that myc J. Cell. PubMed Scopus Google Scholar, K. C. A. H. Oncogene. PubMed Scopus Google cell cycle-regulated expression are required to Myc in the cell cycle-dependent expression of Pax-3. is the of Myc regulation ofPax-3 Pax-3 has been to play in cell in cell The finding that Pax-3 expression is by the Myc and the that Pax-3 expression is cell cycle-regulated a role for Pax-3 in early proliferation during The between Pax-3 and the cell is by A. Oncogene. 1998; 18: Scopus Google have shown that Pax-3 with the gene of the tumor gene A. Oncogene. 1998; 18: Scopus Google Scholar). The family of proteins, which and are of cell In quiescent cells, is and with the transcription This inhibits the transcription of which are for cell progression a Genes Dev. 1998; 12: PubMed Scopus Google Scholar). cells are with is and which is then to genes and cell Pax-3 also with the of and this association between Pax-3 and as with inhibits the of Pax-3 to both Pax-3 mRNA expression and to be cell suggesting a role for Pax-3 in cell progression in cell proliferation and differentiation The expression containing N-myc and Max a of H. the a from M. and a from G.