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• W2022149730 abstract "We have investigated the basis for the striking difference between the broad DNA sequence selectivity of the c-Myb transcription factor minimal DNA-binding domain R2R3 in vitro and the more restricted preference of a R2R3VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyces cerevisiae transactivation system. We show that sequence discrimination in yeast is highly dependent on the expression level of Myb effector protein. Full-length c-Myb and a C-terminally truncated protein (residues 1–360) were also included in the study. All of the tested Myb proteins displayed very similar DNA binding properties in electrophoretic mobility shift assays. Only minor differences between full-length c-Myb and truncated c-Myb(1–360) were observed. In transactivation studies in CV-1 cells, the MRE selectivity was highest at low expression levels of Myb effector proteins. However, the discrimination between MRE variants was rapidly lost with high input levels of effector plasmid. In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the results obtained in the yeast system. These data suggest that the MRE selectivity of c-Myb is an intrinsic property of only the R2R3 domain itself and that the transactivation response of a specific MRE in vivo may be highly dependent on the expression level of the Myb protein in the cell. We have investigated the basis for the striking difference between the broad DNA sequence selectivity of the c-Myb transcription factor minimal DNA-binding domain R2R3 in vitro and the more restricted preference of a R2R3VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyces cerevisiae transactivation system. We show that sequence discrimination in yeast is highly dependent on the expression level of Myb effector protein. Full-length c-Myb and a C-terminally truncated protein (residues 1–360) were also included in the study. All of the tested Myb proteins displayed very similar DNA binding properties in electrophoretic mobility shift assays. Only minor differences between full-length c-Myb and truncated c-Myb(1–360) were observed. In transactivation studies in CV-1 cells, the MRE selectivity was highest at low expression levels of Myb effector proteins. However, the discrimination between MRE variants was rapidly lost with high input levels of effector plasmid. In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the results obtained in the yeast system. These data suggest that the MRE selectivity of c-Myb is an intrinsic property of only the R2R3 domain itself and that the transactivation response of a specific MRE in vivo may be highly dependent on the expression level of the Myb protein in the cell. The c-Myb protein is a transcription factor encoded by the c-myb proto-oncogene (reviewed in Refs. 1Lüscher B. Eisenman R.N. Genes Dev. 1990; 4: 2235-2241Crossref PubMed Scopus (213) Google Scholar, 2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 3Weston K. Curr. Opin. Genet. Dev. 1998; 8: 76-81Crossref PubMed Scopus (179) Google Scholar). A short N terminus is followed by a highly conserved DNA-binding domain, a centrally located transactivation domain, and more C-terminally located negative regulatory domains. The DNA-binding domain is well characterized, comprising the three imperfect repeats, R1, R2, and R3. The R2 and R3 repeats alone are sufficient for sequence-specific DNA binding (4Gabrielsen O.S. Sentenac A. Fromageot P. Science. 1991; 253: 1140-1143Crossref PubMed Scopus (123) Google Scholar). This DNA-binding motif is highly conserved throughout evolution in both animal and plant kingdoms (5Rosinski J.A. Atchley W.R. J. Mol. Evol. 1998; 46: 74-83Crossref PubMed Scopus (209) Google Scholar).A large body of evidence suggests strongly that c-Myb is involved in regulating cell growth and differentiation in hematopoietic cells. In chickens, retroviral v-Myb proteins p48v- myb(AMV-derived) and p135gag-myb-ets (E26-derived) both elicit myeloid leukemia. In mice, the majority of retroviral insertions in the c-myb gene result in N-terminal truncations of the c-Myb protein and deregulated expression, leading to myeloid leukemia (6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholar). The abrogation of fetal liver hematopoiesis in mice with a c-myb null mutation confirmed a vital role for c-Myb in the development of hematopoietic cells (7Mucenski M.L. McLain K. Kier A.B. Swerdlow S.H. Schreiner C.M. Miller T.A. Pietryga D.W. Scott Jr., W.J. Potter S.S. Cell. 1991; 65: 677-689Abstract Full Text PDF PubMed Scopus (872) Google Scholar). A recent study showed that c-Myb is expressed in human primitive hematopoietic stem cells in the fetal aorta region, prior to colonization and definitive hematopoiesis in the fetal liver, confirming an important role for c-Myb in the development of hematopoietic cells (8Labastie M.C. Cortes F. Romeo P.H. Dulac C. Peault B. Blood. 1998; 92: 3624-3635Crossref PubMed Google Scholar).Much effort has been put into identifying relevant target genes for c-Myb action, and binding sites for c-Myb have been identified in an increasing number of gene promoters, including mim-1, neutrophile elastase (ELA2), cdc-2, c-myc,bcl-2, T cell receptor δ and γ chains, CD4, ADA, and lck (2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholar). However, it has been difficult to establish a direct link between candidate genes, growth control, and oncogenic transformation (9Gonda T.J. Int. J. Biochem. Cell Biol. 1998; 30: 547-551Crossref PubMed Scopus (38) Google Scholar). A notable exception is that of v-Myb being able to transactivate the GBX2 gene promoter and up-regulate transcription of the transcription factor GBX2. GBX2 in turn regulates transcription of the chicken growth factor cMGF, thereby creating an autocrine regulatory loop (10Kowenz-Leutz E. Herr P. Niss K. Leutz A. Cell. 1997; 91: 185-195Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Other promising candidate c-myb target genes, such as tom-1 (11Burk O. Worpenberg S. Haenig B. Klempnauer K.H. EMBO J. 1997; 16: 1371-1380Crossref PubMed Scopus (60) Google Scholar) and the adenosine 2B receptor (12Worpenberg S. Burk O. Klempnauer K.H. Oncogene. 1997; 15: 213-221Crossref PubMed Scopus (24) Google Scholar), have been identified in differential screens, but promoter analysis data are not yet available.Several approaches have been used to define the consensus core DNA-binding site for c-Myb, YAACNG (Myb recognition element (MRE)). 1The abbreviations used are: MRE, Myb recognition element; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; DBD, DNA-binding domain.1The abbreviations used are: MRE, Myb recognition element; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; DBD, DNA-binding domain. The Myb consensus sequence was first deduced by isolation of chicken genomic DNA fragments bound by v-Myb on filters (13Biedenkapp H. Borgmeyer U. Sippel A.E. Klempnauer K.H. Nature. 1988; 335: 835-837Crossref PubMed Scopus (433) Google Scholar) and from comparison of putative Myb binding sites within the SV40 enhancer (14Nakagoshi H. Nagase T. Kanei-Ishii C. Ueno Y. Ishii S. J. Biol. Chem. 1990; 265: 3479-3483Abstract Full Text PDF PubMed Google Scholar). Polymerase chain reaction-based binding-site selection methods with Myb proteins resulted in minor extensions of the MRE consensus sequence (15Howe K.M. Watson R.J. Nucleic Acids Res. 1991; 19: 3913-3919Crossref PubMed Scopus (96) Google Scholar, 16Weston K. Nucleic Acids Res. 1992; 20: 3043-3049Crossref PubMed Scopus (64) Google Scholar). Mutational analysis confirmed by NMR structural data has revealed that the Myb MRE is bipartite. The first half-site (YAAC) has the majority of specific contacts to R3, and the less well defined second half-site has mainly specific contacts with the R2subdomain (17Ording E. Kvavik W. Bostad A. Gabrielsen O.S. Eur. J. Biochem. 1994; 222: 113-120Crossref PubMed Scopus (31) Google Scholar, 18Ogata K. Morikawa S. Nakamura H. Sekikawa A. Inoue T. Kanai H. Sarai A. Ishii S. Nishimura Y. Cell. 1994; 79: 639-648Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 19Tanikawa J. Yasukawa T. Enari M. Ogata K. Nishimura Y. Ishii S. Sarai A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9320-9324Crossref PubMed Scopus (133) Google Scholar). The first half-site of the MRE is absolutely required for DNA-binding. Sequence substitutions in the second half-site mainly affected the half-life of the protein-DNA complexin vitro (17Ording E. Kvavik W. Bostad A. Gabrielsen O.S. Eur. J. Biochem. 1994; 222: 113-120Crossref PubMed Scopus (31) Google Scholar). A more detailed analysis of the second half-site revealed a flexible sequence requirement, possibly caused by a flexible structure in R2 (20Myrset A.H. Bostad A. Jamin N. Lirsac P.N. Toma F. Gabrielsen O.S. EMBO J. 1993; 12: 4625-4633Crossref PubMed Scopus (127) Google Scholar, 21McIntosh P.B. Frenkiel T.A. Wollborn U. McCormick Klempnauer K.-H. Feeney J. Carr M.D. Biochemistry. 1998; 37: 9619-9629Crossref PubMed Scopus (24) Google Scholar, 22Jamin N. Gabrielsen O.S. Gilles N. Lirsac P.N. Toma F. Eur. J. Biochem. 1993; 216: 147-154Crossref PubMed Scopus (34) Google Scholar). In particular, base changes were readily accommodated in the highly conserved G6 position of the MRE, as long as a G was present in position 5. Bacterially expressed R2R3 protein bound the MRE variants TAACGG, TAACGT, and TAACTG with very similar binding affinities as measured by electrophoretic mobility shift assay (EMSA). In contrast, these sequences conferred strikingly different transactivation activities (GG ≫ TG > GT and nonfunctional TT) in a strictly Myb DBD-dependent effector/reporter system, using a R2R3VP16 fusion protein and MRE β-galactosidase reporter plasmids inSaccharomyces cerevisiae (23Ording E. Bergholtz S. Brendeford E.M. Jamin N. Gabrielsen O.S. Oncogene. 1996; 13: 1043-1051PubMed Google Scholar). This suggests that optimal MRE sequences defined by in vitro binding studies may not directly reflect their importance and/or activity in vivo. The present study investigates in detail the basis for this in vivo/in vitro paradox. We have compared the MRE selectivity of recombinant R2R3 and R2R3VP16 in EMSAs and investigated the effect of Myb effector expression level on MRE preference in S. cerevisiae. The MRE selectivity of full-length and a C-terminally truncated Myb protein was evaluated both in vitro and in vivo in mammalian cells. We show that the MRE target sequence selectivity is an intrinsic property of the R2R3 domain, independent of other regions in the protein. However, more importantly, the apparent MRE target selectivity in vivo is highly affected by the Myb protein expression level both in yeast and in mammalian cells.DISCUSSIONWe have investigated the basis for the paradox between the broad MRE sequence selectivity of c-Myb R2R3 in vitro and the highly restricted preference of R2R3VP16 protein for MRE target sequence in a S. cerevisiae transactivation system (23Ording E. Bergholtz S. Brendeford E.M. Jamin N. Gabrielsen O.S. Oncogene. 1996; 13: 1043-1051PubMed Google Scholar). We have shown that this striking difference could not be attributed to fusion of the VP16 transactivation domain to R2R3 used in the in vivo studies. We found only subtle differences in the MRE preference of R2R3VP16 compared with recombinant R2R3 in vitro, implying that the addition of the VP16 transactivation domain did not functionally affect the neighboring DBD. We also investigated the DNA binding properties of longer c-Myb proteins, and found that the in vitro MRE selectivity of c-Myb(FL) and C-terminally truncated c-Myb(1–360) was very similar to that of both R2R3VP16 and R2R3. In general, only minor differences were found between the in vitro DNA binding properties of c-Myb(FL) and C-terminally truncated c-Myb(1–360) proteins. However, we observed that c-Myb(FL) consistently bound slightly less well to the TG oligo (MBS-I-like) than the GG oligo (mim-1 A-like), compared with the other expressed Myb proteins. This difference was also reflected in the transactivation data, where a MRE TG variant reporter plasmid was transactivated more poorly than a MRE GG variant (see below).Several studies have reported that a C-terminal truncation of c-Myb resulted in an increase in the DNA binding activity (34Ramsay R.G. Ishii S. Gonda T.J. Oncogene. 1991; 6: 1875-1879PubMed Google Scholar, 35Tanaka Y. Nomura T. Ishii S. FEBS Lett. 1997; 413: 162-168Crossref PubMed Scopus (7) Google Scholar, 38Ramsay R.G. Ishii S. Gonda T.J. J. Biol Chem. 1992; 267: 5656-5662Abstract Full Text PDF PubMed Google Scholar). It has been suggested that this increased DNA binding also could contribute to the transforming phenotype of v-Myb (34Ramsay R.G. Ishii S. Gonda T.J. Oncogene. 1991; 6: 1875-1879PubMed Google Scholar). Other studies have demonstrated that truncation of the C-terminal tail did not increase the DNA binding capacity of c-Myb tested in EMSAs on themim-1 A binding site (GG-type) (24Oelgeschlager M. Krieg J. Luscher-Firzlaff J.M. Luscher B. Mol. Cell. Biol. 1995; 15: 5966-5974Crossref PubMed Scopus (73) Google Scholar, 36Krieg J. Oelgeschlager M. Janknecht R. Luscher B. Oncogene. 1995; 10: 2221-2228PubMed Google Scholar), an MRE sequence motif derived from λ DNA (TG-type) (41Howe K.M. Reakes C.F. Watson R.J. EMBO J. 1990; 9: 161-169Crossref PubMed Scopus (131) Google Scholar), or a mim-1A-related sequence motif (GG-type) in the c-myc promoter (42Zobel A. Kalkbrenner F. Guehmann S. Nawrath M. Vorbrueggen G. Moelling K. Oncogene. 1991; 6: 1397-1407PubMed Google Scholar). Our data with c-Myb proteins expressed in mammalian cells agree with the latter studies in that there is no major difference in the DNA binding activity between full-length and C-terminally truncated c-Myb. However, we do find subtle differences that are dependent on the MRE target site. Taken together, our results suggest that the ability to discriminate between MRE sequences is an intrinsic property of the c-Myb DNA-binding domain only, independent of other domains in the c-Myb protein.A clue to resolution of the in vitro/in vivo DNA target specificity paradox was the observation that in the yeast system, the strong MRE sequence selectivity in vivo was highly dependent on the physiological expression level of Myb effector protein. The ability of c-Myb proteins to transactivate selected MRE target sites in mammalian cells was also found to be strongly dependent on the expression level of c-Myb effector protein, both in a effector/reporter system (CV-1) and in an intrinsic c-Myb-expressing cell line (K-562). At a low input of Myb effector in CV-1 cells and in c-Myb+ K-562 cells, transactivation of MRE reporter plasmids was dependent on the position 5 and 6 nucleotides (see TableI) in the order GG > GT ∼ TG, in accordance with results from the yeast system, with the c-Myb effector being expressed at a physiological level. At high expression levels of Myb effector proteins, this discrimination between MRE sequences was lost, both in mammalian cells and in the S. cerevisiae system. At very high input levels of c-Myb proteins in CV-1 cells, the 3xTT MRE reporter plasmid was transactivated to a high level, even though this MRE was not bound by Myb proteins in EMSAs. This effect was particularly prominent for full-length c-Myb. Our findings suggest that activation of target genes by c-Myb may greatly depend on the expression level of Myb proteins in the cell, a notion also suggested by others (38Ramsay R.G. Ishii S. Gonda T.J. J. Biol Chem. 1992; 267: 5656-5662Abstract Full Text PDF PubMed Google Scholar).It has been difficult to define bona fide target genes for c-Mybin vivo. Several in vitro methods have therefore been employed to predict MRE target sequences. An important question is whether these in vitro methods are able to accurately predict in vivo Myb target sites. Several in vitro studies have demonstrated that the minimal Myb DNA-binding domain R2R3 is able to accommodate a number of changes in the DNA MRE second half-site (14Nakagoshi H. Nagase T. Kanei-Ishii C. Ueno Y. Ishii S. J. Biol. Chem. 1990; 265: 3479-3483Abstract Full Text PDF PubMed Google Scholar, 15Howe K.M. Watson R.J. Nucleic Acids Res. 1991; 19: 3913-3919Crossref PubMed Scopus (96) Google Scholar, 16Weston K. Nucleic Acids Res. 1992; 20: 3043-3049Crossref PubMed Scopus (64) Google Scholar, 18Ogata K. Morikawa S. Nakamura H. Sekikawa A. Inoue T. Kanai H. Sarai A. Ishii S. Nishimura Y. Cell. 1994; 79: 639-648Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 23Ording E. Bergholtz S. Brendeford E.M. Jamin N. Gabrielsen O.S. Oncogene. 1996; 13: 1043-1051PubMed Google Scholar). In binding site selection studies with random oligos, the MRE consensus sequence was determined as YAACKGHA (15Howe K.M. Watson R.J. Nucleic Acids Res. 1991; 19: 3913-3919Crossref PubMed Scopus (96) Google Scholar) or YAACKGHH (16Weston K. Nucleic Acids Res. 1992; 20: 3043-3049Crossref PubMed Scopus (64) Google Scholar). In these studies, the (T/C)AACGG sequence was prevalent in selected single MRE-site oligos, 70% (15Howe K.M. Watson R.J. Nucleic Acids Res. 1991; 19: 3913-3919Crossref PubMed Scopus (96) Google Scholar) and 38–70% (16Weston K. Nucleic Acids Res. 1992; 20: 3043-3049Crossref PubMed Scopus (64) Google Scholar), depending on the Myb protein used. Efforts have also been made at thermodynamic measurements (43Oda M. Furukawa K. Ogata K. Sarai A. Nakamura H. J. Mol. Biol. 1998; 276: 571-590Crossref PubMed Scopus (93) Google Scholar) and free energy matrixes (44Deng Q.L. Ishii S. Sarai A. Nucleic Acids Res. 1996; 24: 766-774Crossref PubMed Scopus (30) Google Scholar) as aids in predicting MRE sites in vitro. However, these methods were based only on the MBS-I (TG-like) version of the MRE, which we find a suboptimal Myb binding site in vivo. In well characterized Myb target gene promoters (TableII), Myb binding sites with a G in both positions 5 and 6 is also strongly represented. In our study, the bona fide Myb MRE site TAACGG from the mim-1 promoter (27Ness S.A. Marknell A. Graf T. Cell. 1989; 59: 1115-1125Abstract Full Text PDF PubMed Scopus (374) Google Scholar) was highly preferred in transactivation assays compared with the MBS-I-like sequence TAACTG, both in yeast and in mammalian cells with moderate expression of Myb effector protein. Taken together, there is good correlation between Myb MRE sites in Myb target genes and the preferred MRE sites in both mammalian cells (low effector input level) and in our model in vivo transactivation system in S. cerevisiae.Table IIDefined Myb recognition elements in gene promotersMRE consensus1234 YAAC5678NG9aBase positions in the MRE consensus sequence are numbered 1 to 9.Ref.mim-1AgcattaTAACGGtt ttttagcgc2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar,6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholarc-mycagagtTAACGGtt tt2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholarbcl-2tgcCAACGGgg aaa2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarCD13/APNtccTAACGGac cggc2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarTCRδcatTAACGGtt gg2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholartom-1cctTAACGGa11Burk O. Worpenberg S. Haenig B. Klempnauer K.H. EMBO J. 1997; 16: 1371-1380Crossref PubMed Scopus (60) Google ScholarNeutrophile elastaseatgCAACGGcc tcc2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarCD4caaCAACTGgg gg2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarSV40 enhancer (MBS-I)cacccTAACTGac acacat2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarTCRγggttTAACTGctc2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarADAccacCAACTGcc at2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarlckggcAAACCGcc ac2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google ScholarEOS47atCAACAGct a2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholarcdc-2ctccTAACCCta agt2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholara Base positions in the MRE consensus sequence are numbered 1 to 9. Open table in a new tab A general problem in the analysis of gene promoters is that putative transcription factor binding sites are present at a much higher frequencies than the expected number of the target genes for that particular transcription factor. This paradox is partially resolved by the concerted action of several transcription factors supplying the specificity of the particular gene promoter activity. Another possibility, illustrated in this study, is that the activity of transcription factor binding sites may be more stringent in vivo than suggested by in vitro analysis methods. A general weakness of transient transfection studies in mammalian cells is the lack of control of the test gene expression level on a per cell basis, and conditions may easily become nonphysiological, as demonstrated in this study.Our model transactivation system in S. cerevisiae has several promising features. First, our data demonstrate that c-Myb R2R3VP16 fusion protein expressed in yeast has DNA binding properties very similar to full-length c-Myb and is able to mimic the MRE sequence selectivity of full-length mammalian c-Myb in the yeast effector/reporter system. The R2R3VP16 fusion protein should therefore be a suitable model protein for measuring the DNA binding activity of c-Myb on putative Myb target DNA sequences independently of other domains in the c-Myb protein. Second, the system exploits features of yeast plasmids such as control of copy number (gene dosage), the use of metabolic markers to ensure the presence of effector and reporter plasmids in a homogenous yeast population (in contrast to cell-line-dependent variations and wide range of transfection efficiencies in mammalian cells), and the plasmids being in a physiological chromatin-packaged state (45Morse R.H. Science. 1993; 262: 1563-1566Crossref PubMed Scopus (70) Google Scholar). In summary, the advantage of the yeast system is that the expression level of the effector protein (transcription factor) is well controlled, thus avoiding overexpression and misleading transactivation of suboptimal target DNA-binding sites, as demonstrated in this study. Our yeast effector/reporter system may easily be applied as an in vivotool for evaluating putative DNA-binding sites for other monomeric transcription factors. The c-Myb protein is a transcription factor encoded by the c-myb proto-oncogene (reviewed in Refs. 1Lüscher B. Eisenman R.N. Genes Dev. 1990; 4: 2235-2241Crossref PubMed Scopus (213) Google Scholar, 2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 3Weston K. Curr. Opin. Genet. Dev. 1998; 8: 76-81Crossref PubMed Scopus (179) Google Scholar). A short N terminus is followed by a highly conserved DNA-binding domain, a centrally located transactivation domain, and more C-terminally located negative regulatory domains. The DNA-binding domain is well characterized, comprising the three imperfect repeats, R1, R2, and R3. The R2 and R3 repeats alone are sufficient for sequence-specific DNA binding (4Gabrielsen O.S. Sentenac A. Fromageot P. Science. 1991; 253: 1140-1143Crossref PubMed Scopus (123) Google Scholar). This DNA-binding motif is highly conserved throughout evolution in both animal and plant kingdoms (5Rosinski J.A. Atchley W.R. J. Mol. Evol. 1998; 46: 74-83Crossref PubMed Scopus (209) Google Scholar). A large body of evidence suggests strongly that c-Myb is involved in regulating cell growth and differentiation in hematopoietic cells. In chickens, retroviral v-Myb proteins p48v- myb(AMV-derived) and p135gag-myb-ets (E26-derived) both elicit myeloid leukemia. In mice, the majority of retroviral insertions in the c-myb gene result in N-terminal truncations of the c-Myb protein and deregulated expression, leading to myeloid leukemia (6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholar). The abrogation of fetal liver hematopoiesis in mice with a c-myb null mutation confirmed a vital role for c-Myb in the development of hematopoietic cells (7Mucenski M.L. McLain K. Kier A.B. Swerdlow S.H. Schreiner C.M. Miller T.A. Pietryga D.W. Scott Jr., W.J. Potter S.S. Cell. 1991; 65: 677-689Abstract Full Text PDF PubMed Scopus (872) Google Scholar). A recent study showed that c-Myb is expressed in human primitive hematopoietic stem cells in the fetal aorta region, prior to colonization and definitive hematopoiesis in the fetal liver, confirming an important role for c-Myb in the development of hematopoietic cells (8Labastie M.C. Cortes F. Romeo P.H. Dulac C. Peault B. Blood. 1998; 92: 3624-3635Crossref PubMed Google Scholar). Much effort has been put into identifying relevant target genes for c-Myb action, and binding sites for c-Myb have been identified in an increasing number of gene promoters, including mim-1, neutrophile elastase (ELA2), cdc-2, c-myc,bcl-2, T cell receptor δ and γ chains, CD4, ADA, and lck (2Ness S.A. Biochim. Biophys. Acta. 1996; 1288: F123-F139Crossref PubMed Scopus (97) Google Scholar, 6Wolff L. Crit. Rev. Oncog. 1996; 7: 245-260Crossref PubMed Scopus (61) Google Scholar). However, it has been difficult to establish a direct link between candidate genes, growth control, and oncogenic transformation (9Gonda T.J. Int. J. Biochem. Cell Biol. 1998; 30: 547-551Crossref PubMed Scopus (38) Google Scholar). A notable exception is that of v-Myb being able to transactivate the GBX2 gene promoter and up-regulate transcription of the transcription factor GBX2. GBX2 in turn regulates transcription of the chicken growth factor cMGF, thereby creating an autocrine regulatory loop (10Kowenz-Leutz E. Herr P. Niss K. Leutz A. Cell. 1997; 91: 185-195Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Other promising candidate c-myb target genes, such as tom-1 (11Burk O. Worpenberg S. Haenig B. Klempnauer K.H. EMBO J. 1997; 16: 1371-1380Crossref PubMed Scopus (60) Google Scholar) and the adenosine 2B receptor (12Worpenberg S. Burk O. Klempnauer K.H. Oncogene. 1997; 15: 213-221Crossref PubMed Scopus (24) Google Scholar), have been identified in differential screens, but promoter analysis data are not yet available. Several approaches have been used to define the consensus core DNA-binding site for c-Myb, YAACNG (Myb recognition element (MRE)). 1The abbreviations used are: MRE, Myb recognition element; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; DBD, DNA-binding domain.1The abbreviations used are: MRE, Myb recognition element; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; DBD, DNA-binding domain. The Myb consensus sequence was first deduced by isolation of chicken genomic DNA fragments bound by v-Myb on filters (13Biedenkapp H. Borgmeyer U. Sippel A.E. Klempnauer K.H. Nature. 1988; 335: 835-837Crossref PubMed Scopus (433) Google Scholar) and from comparison of putative Myb binding sites within the SV40 enhancer (14Nakagoshi H. Nagase T. Kanei-Ishii C. Ueno Y. Ishii S. J. Biol. Chem. 1990; 265: 3479-3483Abstract Full Text PDF PubMed Google Scholar). Polymerase chain reaction-based binding-site selection methods with Myb proteins resulted in minor extensions of the MRE consensus sequence (15Howe K.M. Watson R.J. Nucleic Acids Res. 1991; 19: 3913-3919Crossref PubMed Scopus (96) Google Scholar, 16Weston K. Nucleic Acids Res. 1992; 20: 3043-3049Crossref PubMed Scopus (64) Google Scholar). Mutational analysis confirmed by NMR structural data has revealed that the Myb MRE is bipartite. The first half-site (YAAC) has the majority of specific contacts to R3, and the less well defined second half-site has mainly specific contacts with the R2subdomain (17Ording E. Kvavik" @default.
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