J Biol Chem, Vol. 274, Issue 31, 21986-21994, July 30, 1999

Sequence Selectivity of c-Myb in Vivo
RESOLUTION OF A DNA TARGET SPECIFICITY PARADOX^*

Kristin Brevik Andersson, Tone Berge, Vilborg Matre, and Odd S. Gabrielsen

From the Department of Biochemistry, University of Oslo, N-0316 Oslo 3, Norway

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The c-Myb protein is a transcription factor encoded by the c-myb proto-oncogene (reviewed in Refs. 1-3). 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 (4). This DNA-binding motif is highly conserved throughout evolution in both animal and plant kingdoms (5).

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 p48^v-myb (AMV-derived) and p135^gag-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 (6). 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 (7). 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 (8).

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  and  chains, CD4, ADA, and lck (2, 6). However, it has been difficult to establish a direct link between candidate genes, growth control, and oncogenic transformation (9). 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 (10). Other promising candidate c-myb target genes, such as tom-1 (11) and the adenosine 2B receptor (12), 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)).¹ The Myb consensus sequence was first deduced by isolation of chicken genomic DNA fragments bound by v-Myb on filters (13) and from comparison of putative Myb binding sites within the SV40 enhancer (14). Polymerase chain reaction-based binding-site selection methods with Myb proteins resulted in minor extensions of the MRE consensus sequence (15, 16). 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 R₂ subdomain (17-19). 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 complex in vitro (17). A more detailed analysis of the second half-site revealed a flexible sequence requirement, possibly caused by a flexible structure in R₂ (20-22). 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 -galactosidase reporter plasmids in Saccharomyces cerevisiae (23). 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Reagents-- CV-1, COS-1, and K-562 cells were obtained from ATCC. COS-1 and CV-1 cells were maintained in Dulbecco's modified Eagle's medium. K-562 cells were maintained in RPMI 1640 medium. All media were supplemented with 10% fetal calf serum, 20 mM glutamine, 1% penicillin and streptomycin. Cells were grown in 5% CO₂ in a humidified atmosphere.

Constructions and Plasmids-- The mammalian expression plasmid pCIneo-R₂R₃VP16 encoding the chicken minimal DNA-binding domain R₂R₃ (residues 89-192) fused to the herpes simplex virus VP16 transactivation domain (residues 413-488) was constructed as follows: the relevant fragment was amplified by polymerase chain reaction (forward primer, 5'-gcgaagcttatgGAACTTATCAAAGGTCCATGG; reverse primer, 5'-gcgggatccttactaACCGTACTCGTCAATTCCAAG) from the yeast plasmid pDBD₁₁R₂R₃ (23), subcloned as a HindIII-BamHI fragment into pBluescriptII SK⁺ and inserted as a XhoI-NotI fragment into pCIneo (Promega). The expression plasmids encoding full-length murine c-Myb, (pEQP2-CMV-c-Myb, (24)), truncated murine c-Myb (residues 1-360), (pEQP2-CMV-c-Myb(1-360)),² and AMV v-Myb (pCB6⁺CMV-AMV)² proteins were a kind gift from Bernhard Lüscher. All the expression plasmids contained the CMV promoter for directing transcription and the SV40 ori for replication in SV40 large T antigen⁺ cells.

The mammalian luciferase reporter constructs pGL2/tk/3xGG, pGL2/tk/3xGT, pGL2/tk/3xTG, and pGL2/tk/3xTT were made by inserting double-stranded oligos with triple copies of the variant Myb recognition element sequences as follows, in reverse orientation into the SmaI site of pGL2/tk (25): 3xGG, 5'-GCATTATAACGGTCTTTAACGGTCTTTAACGGTCTTTTAGCGCC-3'; 3xGT, 5'-GCATTATAACGTTCTTTAACGTTCTTTAACGTTCTTTTAGCGCC-3'; 3xTG, 5'-GCATTATAACTGTCTTTAACTGTCTTTAACTGTCTTTTAGCGCC-3'; 3xTT, 5'-GCATTATAACTTTCTTTAACTTTCTTTAACTTTCTTTTAGCGCC-3'. Details on these variants of the mim-1 A site are described in Ref. 23.

The low-copy (centromeric) yeast expression plasmids pDBD₁₁ (26) and pDBD₁₁R₂R₃ (23) express proteins under the control of the GAL1-10 promoter. The high copy yeast expression plasmids pDBD₁₁HK and pDBD₁₁HK/R₂R₃ were constructed by ligating the 3.1-kilobase pair AatII-PvuII fragment of pGAD424SphI (containing the 2µ ori) to the AatII-SpeI(blunted) fragments of pDBD₁₁ and pDBD₁₁R₂R₃ (3.5 and 3.8 kilobases, respectively). The plasmid pGAD424SphI was made by excision of a SphI fragment and religation of pGAD424 (CLONTECH).

Yeast -galactosidase reporter plasmids with Myb responsive element variants 3xGG, 3xGT, 3xTG, and 3xTT were made by inserting double-stranded oligos with triple copies of the variant Myb recognition element sequences (see above) 3xGG, 3xGT, 3xTG, and 3xTT, respectively, upstream of a HIS3 minimal promoter and an open reading frame encoding a His3p-lacZ fusion protein.³

Expression of c-Myb Proteins-- COS-1 cells were transfected exactly as described (24) with 30 µg of pCIneo-R₂R₃VP16, pEQP2-CMV-c-Myb, or pEQP2-CMV-c-Myb(1-360) on 100-mm plates (seeded with 3 × 10⁵ cells the day before). After 36 h, cells were washed once in phosphate-buffered saline on ice before lysis in 500 µl modified Buffer F (10 mM Tris-HCl, pH 7.0, 50 mM NaCl, 5 µM ZnCl₂, 1% Triton X-100) (24) supplemented with 1× Complete Protease Inhibitor^TM (Roche Molecular Biochemicals) and centrifugation for 30 min at 4  °C. Aliquots were frozen in liquid N₂ and stored at 80  °C. The minimal DNA-binding domain of chicken c-Myb, R₂R₃, was expressed in Escherichia coli and purified as described previously (4).

Electrophoretic Mobility Shift Assay-- DNA binding was monitored by EMSA. The sequences of the double-stranded oligo for the mim-1 A site (27) and variant MRE GG, GT, TG, and TT oligos are as in Ref. 23, except that all variant oligos were extended at the 3'-end with nucleotides TGG in order to improve annealing efficiency (see Table I). Variant MRE oligos were labeled to the same specific activity and purified as described (23). The mim-1 A site oligo was labeled and purified as described (23). Cold double-stranded oligos for competition were similarly annealed, filled-in and PAGE-purified.

Myb proteins in whole-cell lysates were titrated by EMSA and standardized to the same amount of Myb DNA binding activity on the mim-1 A site oligo, due to effects of c-Myb on the transactivation level of the internal standard plasmid (see below on transactivation in mammalian cells). The variation of the DNA-binding activity (on mim-1 A) between extracts with the different Myb proteins within the same transfection series was within ±50% in lysate volume; typically 1-3 µl of COS-1 cell extract was used per binding reaction. COS-1 cell lysates with various Myb proteins were adjusted to equal volumes with Buffer F before incubation in Buffer H (20 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, pH 8.0, 10% glycerol), with final NaCl concentration 60 or 75 mM. Where indicated, binding reactions with recombinant R₂R₃ (25-50 fmol) were included. 5'--³²P-Labeled DNA oligo probe (10 or 30 fmol) was added, and the binding mixture (total volume 20 µl) was incubated for 10 min at 25 °C before electrophoresis or competition with cold oligos. Binding reactions were run on 5% 0.5× TBE PAGE gels and 0.5× TBE buffer at 4  °C and 15 V/cm. Autoradiography was performed by standard procedures, and relative amounts of protein-DNA complexes were quantified by densitometric scanning. For comparison of band intensities, one of the protein-DNA complexes per series was arbitrarily designated as 100%.

Transactivation Assays in Mammalian Cells-- CV-1 cells were transfected by polyethyleneimine (28) or by CaPO₄ precipitation with precipitate parameters as described (29). Cells were seeded the day before at 1.5 × 10⁵ cells/plate in 60-mm plates. Cells were transfected with 4 µg of pGL2/tk/3xMRE variant reporter plasmid and the amounts of expression plasmid indicated in the figures. In transfections with polyethyleneimine, the total amount of expression plasmid input was kept constant at 5 µg by adding appropriate amounts of the pCIneo plasmid. Polyethyleneimine was added to 9 equivalents (= 2430 nmol pr total 9 µg DNA/plate). Cells were lysed and scraped in 400 µl of reporter lysis buffer (Promega) or lysed in 400 µl of passive lysis buffer (in cotransfections with pRL-CMV).

In preliminary experiments, 25 ng pRL-CMV (Renilla reniformis luciferase, Promega) or 0.5 µg of pCMV--galactosidase (Promega) per plate were included as internal controls of transfection efficiency. However, we consistently observed effects on the transactivation level of the internal control plasmid depending on the amount of input c-Myb expression plasmid (data not shown). All transactivation results are presented without standardization to internal control plasmids.

K-562 cells were washed once in RPMI 1640 medium without additions. Cells were resuspended at 2 × 10⁷ cells/ml. Aliquots (500 µl) were added to 10 µg of pGL2/tk reporter plasmid, transferred to 4-mm-gap electroporation cuvettes, and pulsed once at 310 V and 960 microfarads (optimized) in a Bio-Rad Gene Pulser giving a pulse decay of t_1/2  20 ms. Cells were diluted in RPMI 1640 medium with additions. After 36 h, cells were washed once in phosphate-buffered saline, resuspended in reporter lysis buffer, snap-frozen in liquid N₂, and thawed. Debris was pelleted by centrifugation.

All lysates were analyzed for luciferase activity using either a luciferase assay kit or dual luciferase assay kit (when pRL-CMV was cotransfected) as described by the manufacturer (Promega).

Transactivation Assays in Yeast-- Transactivation assays in the yeast strain INVSC1 (Invitrogen) were performed in galactose-containing medium as described (23) with the control plasmids pDBD₁₁ or pDBD₁₁HK and the c-Myb R₂R₃VP16 expression plasmids pDBD₁₁R₂R₃ and pDBD₁₁HK/R₂R₃. The yeast reporter plasmids contained triple copy GG, GT, TG, and TT oligos (see above) inserted 5' of the reporter gene (see above). Induction of -galactosidase activity was assayed with o-nitrophenyl -D-galactopyranoside (Sigma) as substrate (30).

Western Blot-- Cell lysates from COS-1-transfected cells were run on a 15% discontinuous SDS-PAGE gel and electroblotted onto a nitrocellulose filter. Filters were blocked in BLOTTO, and Myb proteins were detected by the murine Mab 5E11 (31), followed by horseradish peroxidase-conjugated donkey anti-mouse antibody (Amersham Pharmacia Biotech). Yeast strain INVSCI transformed with Myb expression plasmid and the 3xGG MRE reporter plasmid were grown in selective media. Equal amounts of cells were lysed with glass beads and 5% trichloroacetic acid (32), run on 15% SDS-PAGE gels, and blotted onto polyvinylidene difluoride membranes. R₂R₃VP16 fusion protein was detected by the murine monoclonal anti-VP16 antibody 2GV4 (33) and horseradish peroxidase-conjugated donkey anti-mouse antibody. Horseradish peroxidase activity was detected by ECL or ECL⁺, as described by the manufacturer (Amersham Pharmacia Biotech).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The MRE Selectivity of R₂R₃VP16 in Vitro and in Vivo in Mammalian Cells-- We have previously demonstrated that recombinant c-Myb R₂R₃ can bind equally well to MRE variants containing a guanine in positions 5 and/or 6 of the consensus core MRE TAACNG as analyzed by EMSA. In sharp contrast to this low selectivity in vitro, transactivation of the same MRE variants as 3xMRE reporter plasmids in S. cerevisiae by R₂R₃VP16 fusion protein was highly selective (23). We first asked whether this striking in vitro/in vivo difference in MRE discrimination could be attributed to the fusion of the VP16 transactivation domain to the c-Myb minimal DNA-binding domain in the in vivo experiment. A CMV-based expression plasmid for chicken c-Myb minimal DNA-binding domain R₂R₃ fused to the VP16 transactivation domain, R₂R₃VP16, was transfected into COS-1 cells, and whole-cell lysates were used as the protein source for EMSA.

The DNA binding capacity of R₂R₃VP16 expressed in mammalian cells was compared with R₂R₃ produced in E. coli by EMSA. The set of MRE variant oligos used in this study are listed in Table I with the nomenclature referring to the bases in nucleotide positions 5 and 6 in the MRE core consensus sequence. COS-1 cell lysate with R₂R₃VP16 or recombinant R₂R₃ was bound to MRE oligo GG, GT, TG, or TT (Fig. 1). The oligos were labeled to equal specific activity, thus yielding directly comparable intensities of DNA-protein complexes. R₂R₃VP16 bound the GG, GT, and TG oligos (Fig 1, lanes 2-4) with very similar intensities (relative binding of the oligos was 100, 94, and 89%, respectively, by scanning densitometry). Recombinant R₂R₃ also bound the GG, GT, and TG oligos with very similar intensities (100, 120, and 120% respectively) (Fig. 1, lanes 6-8, and Ref. 23). Neither R₂R₃VP16 lysate (Fig. 1, lane 5) nor recombinant R₂R₃ (23) was able to bind to the TT MRE variant oligo. This comparison shows that the C-terminal fusion of the VP16 transactivation domain to c-Myb R₂R₃ does not have any major effect on the MRE-selectivity. These data also exclude the possibility that the expression system for the Myb protein (i.e. E. coli versus mammalian cells) might affect the DNA binding properties of R₂R₃.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   DNA binding selectivity of R2R3VP16. COS-1 cell lysate with c-Myb R2R3VP16 (2 µl, lanes 2-5) or recombinant R2R3 (320 fmol, lanes 6-8) was bound to oligo GG, GT, TG, or TT (10 fmol each for R2R3VP16 and 220 fmol each for recombinant R2R3), as indicated. Probes were labeled to equal specific activity. Complexes were incubated for 10-15 min at 25  °C before analysis by EMSA. Protein-DNA complexes are marked with arrows.

We next performed transactivation assays in mammalian cells with R₂R₃VP16 and 3xMRE reporter plasmids to evaluate whether the high selectivity in yeast also could be observed in mammalian cells or whether this selectivity was a property only of the yeast system. CV-1 cells were transfected with pGL2/tk/3xMRE reporter plasmids and increasing amounts of R₂R₃VP16 expression plasmid (Fig. 2). There was no significant difference in the ability of R₂R₃VP16 to discriminate between MRE variants GG, GT, and TG in mammalian cells, regardless of the input level of effector plasmid. This finding contrasts our earlier observations in S. cerevisiae (23). This poor selectivity of R₂R₃VP16 for MRE in CV-1 cells is in keeping with results from in vitro-based selection strategies for defining the Myb MRE (13-16). One might therefore tentatively conclude that the high sequence selectivity of Myb R₂R₃VP16 in S. cerevisiae was an artifact of the yeast system. However, there are important biological differences between the two transactivation systems that might affect the sensitivity of the assay. In the mammalian CV-1 cell system, only about 20-25% of the population was transfected (as judged by cells expressing green fluorescent protein, data not shown). In this type of transient transfection assay, there is no control over the number of plasmids taken up per cell, i.e. no control of gene dosage (effector) or percentage of cells harboring both effector and reporter plasmids. Also, the R₂R₃VP16 protein was expressed from the very strong CMV promoter, which presumably results in a very high expression level of Myb protein per transfected cell. In the yeast system, the cell population is homogeneous, with all cells containing both effector and reporter plasmids. The Myb R₂R₃VP16 protein was expressed from the Gal1-10 promoter at a supposedly physiological level from a low copy number (CEN-ARS) plasmid. The gene dosage and expression level of effector protein is therefore well controlled in the yeast system. We hypothesized that the differences in sequence selectivity between yeast and mammalian cell system might in part be attributed to the expression level of the effector protein.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   The transactivation of ability of R2R3VP16 on MRE variant recognition elements in CV-1 cells. CV-1 cells were transfected with expression construct for R2R3VP16 at the indicated concentrations (µg) and pGL2/tk reporter plasmids (4 µg) containing triple copies of variant Myb recognition elements (3xGG, 3x GT, 3xTG, and 3xTT). Fold relative transactivation was calculated relative to the pGL2/tk/3xGG reporter with empty expression plasmid (pCIneo). Results are expressed as mean fold induction of luciferase activity and S.E. (n = 3).

Effect of Copy Number on MRE Selectivity in Yeast-- In S. cerevisiae, the expression level of a protein can easily be manipulated by using low (CEN-ARS) or high (2 µ) copy number expression plasmids. We exploited this feature by examining the effects of the copy number of the Myb expression plasmid on the transactivation level of 3xMRE-HIS3-lacZ fusion reporter plasmids. At a low expression level of Myb R₂R₃VP16, a high degree of selectivity for MRE sequence in terms of transactivation level was evident (Fig. 3A). Transactivation of the 3xGG reporter was strong, whereas the 3xGT and 3xTG variants were transactivated only marginally above the background level, and the 3xTT variant was not transactivated at all. In contrast, when R₂R₃VP16 was expressed from a high copy plasmid, the high degree of selectivity between MRE sequence variants was lost, with the 3xGT and 3xTG variant reporter plasmids being transactivated only slightly less than the 3xGG variant (Fig. 3B). The expected difference in the expression levels of Myb R₂R₃VP16 from the low copy and high copy vectors (Fig. 3C) was verified by Western blotting and detection by an anti-VP16 transactivation domain antibody (2GV4) (33). Notably, the 3xGG MRE reporter plasmid was transactivated to the same level regardless of the copy number of the effector plasmid, implying that R₂R₃VP16 expressed at a low level was sufficient to saturate the MREs on this reporter plasmid. The less optimal GT and TG MRE variant reporters were maximally transactivated when R₂R₃VP16 was overexpressed. This suggested that overexpression of R₂R₃VP16 leads to occupation of suboptimal DNA-binding sites and high transactivation, giving the impression that those DNA-binding sites were optimal for the R₂R₃VP16 protein. These data show clearly that the sequence selectivity of R₂R₃VP16 in S. cerevisiae is highly dependent on the protein expression level. These results also show that very subtle differences in DNA-binding affinity that are too small to be clearly detected in vitro may be amplified to give large differences in transcriptional output in vivo.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Effects of copy number on Myb transactivation in yeast. Low copy (A) or high copy (B) expression plasmids for R2R3VP16 (pDBD11R2R3 and pDBD11HK/R2R3, respectively) and yeast reporter plasmids containing the triple copies of the indicated variants of the MRE were cotransformed into S. cerevisiae INVSCI cells. Transactivation was measured as -galactosidase activity. Background is the transactivation level on the 3xGG reporter plasmid with the empty expression plasmids pDBD11 (A) or pDBD11HK (B). Data are presented as mean -galactosidase units and S.E. (n = 4). A Western blot (C) shows the expression level of R2R3VP16 as detected by the anti-VP16 antibody 2GV4 (33) in cells cotransformed with the 3xGG reporter and the following plasmids as indicated: N, pDBD11; LC, pDBD11R2R3; HC, pDBD11HK/R2R3.

Analysis of the DNA Binding Properties of Full-length Myb Protein Expressed in COS Cells-- The relevance of using the S. cerevisiae transactivation system for evaluating optimal DNA-binding sites for c-Myb requires that Myb proteins have very similar properties in yeast and in mammalian cells. The high selectivity for the GG MRE variant in yeast raised the question of whether R₂R₃VP16 (and R₂R₃) accurately reflected the MRE selectivity of full-length c-Myb. We wished to characterize the selectivity of full-length c-Myb on a selected set of variant MRE sequences. Truncated c-Myb protein was also included, because there have been conflicting reports on whether the DNA binding activity of c-Myb is affected by C-terminal truncations (16, 34-36). COS-7 cells have been successfully used for high expression levels of full-length murine c-Myb (24). We expressed c-Myb(FL) (full-length murine c-Myb) and c-Myb(1-360) (murine c-Myb residues 1-360) proteins in COS-1 cells using SV40-dependent replicative plasmids with CMV-directed gene expression. Whole-cells lysates were made under mild denaturing conditions, and the content of Myb proteins was analyzed by Western blotting. As shown in Fig. 4, lysates from cells transfected with c-Myb(1-360) (lane 1) and c-Myb(FL) (lane 2) contained readily detectable amounts of Myb proteins with expected molecular masses of 45 and 75 kDa, respectively. We did not observe major breakdown fragments of c-Myb, as reported for other expression systems (34, 35, 37). Some weak extra bands representing possible proteolytic fragments were detected. Because the Myb proteins were detected by an anti-Myb DBD antibody, these fragments would be expected to have DNA binding activity. However, we did not observe additional shifted bands in the EMSA (see Fig. 5), suggesting that these minor bands would not interfere in our analysis.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Expression of full-length Myb proteins in COS-1 cells. Expression plasmids encoding c-Myb(1-360) or c-Myb(FL) proteins were transfected into COS-1 cells, and whole-cell lysates were made as described under "Materials and Methods." Equal amounts of lysates (25 µl) were run on SDS-PAGE gels. Myb proteins were detected by Western analysis using the 5E11 monoclonal anti-Myb antibody (31) and detection by ECL. M, molecular mass markers; lane 1, c-Myb(1-360) lysate; lane 2, c-Myb(FL) lysate; lane 3, 50 fmol of recombinant R2R3. Detected proteins are marked with arrows.

View larger version (61K): [in this window] [in a new window]   Fig. 5.   Sequence selectivity of Myb proteins in vitro. Standardized COS-1 cell lysates with c-Myb(1-360) (lanes 1-4) or c-Myb(FL) (lanes 5-8) were bound to oligos GG, GT, TG, or TT as indicated. Probes were labeled to equal specific activity. Recombinant R2R3 protein was included as an internal control (lane 9). Complexes were incubated for 10-15 min at 25  °C before analysis by EMSA. Protein-DNA complexes are marked with arrows.

In preliminary transactivation assays, we consistently observed that the input level of Myb expression vector affected the transactivation level of the internal control plasmid(s). We therefore omitted the use of an internal control plasmid as a method to normalize cell lysates with respect to transfection efficiencies. We found a good correspondence between the relative amounts of c-Myb(FL) and c-Myb(1-360) proteins present in the lysates and DNA binding activities. For example, the c-Myb(1-360) lysate in Fig. 4 contained twice as much Myb protein and twice as much DNA binding activity on the mim-1 A oligo (data not shown) as an equal volume of c-Myb(FL) lysate. Before EMSA, we therefore chose to standardized the relative amounts of Myb proteins present in the COS-1 lysates from the same transfection by equal DNA binding activity to a mim-1 A site oligo (see under "Materials and Methods").

We first investigated the ability of c-Myb(FL) and c-Myb(1-360) proteins to bind to selected MRE variant oligos. To allow direct comparison, standardized lysates (see above) containing c-Myb(1-360) or c-Myb(FL) were bound to GG, GT, TG, or TT oligos and analyzed by EMSA (Fig. 5). Both c-Myb(1-360) (Fig. 5, lanes 1-4) and c-Myb(FL) (lanes 5-8) bound with similar intensities to the GG, GT, and TG MRE variant oligos. The relative intensities for c-Myb(1-360) and c-Myb(FL) bound to GG, GT, and TG were 100, 91, and 78%, and 100, 77, and 70%, respectively, as measured by densitometric scanning (arbitrary units, see under "Materials and Methods"). A 20-30% reduction in the binding of c-Myb(1-360) and c-Myb(FL) to the GT and TG oligos relative to GG was consistently observed. Also, c-Myb(FL) bound slightly more weakly to the GT and TG oligos compared with c-Myb(1-360). This reduced binding affinity was also observed in other experiments (data not shown) and in those shown in Figs. 6 and 7. For the R₂R₃VP16 protein, this slight reduction in binding to GT and TG oligos was approximately 10% (Fig. 1, lanes 2 and 3). We concluded that there was no major shift in the sequence selectivity of c-Myb(FL) or c-Myb(1-360) compared with R₂R₃VP16 for the chosen MRE oligo subset.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Competition between Myb proteins for limiting amounts of probe. A, standardized COS-1 cell lysates with R2R3VP16, c-Myb(1-360), and c-Myb(FL) proteins were mixed before binding to serial dilutions of oligos GG, GT, TG, or TT at the indicated concentrations. Probes were labeled to equal specific activity. Complexes were incubated for 10-15 min at 25  °C before EMSA. Protein-DNA complexes are marked with arrows. B, densitometric scan of the autoradiogram in A. All of the protein-DNA complexes were calculated as a percentage of the amount of R2R3VP16/GG complex.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   Stability of Myb/DNA complexes. A, standardized COS-1 cell lysates with R2R3VP16, c-Myb(1-360) and c-Myb(FL) were mixed before binding to a radioactive mim-1 oligo (30 fmol/lane). Complexes were incubated at 25  °C for 15 min before competition with 25-fold molar excess cold GG, GT, TG, or TT oligos (750 fmol/lane) for times as indicated. Samples were then immediately loading on an EMSA gel. Lane 1 is without COS-1 lysate. Protein-DNA complexes are marked with arrows. B, densitometric scan of the autoradiogram in A. All of the protein-DNA complexes were calculated as percentage of the amount of R2R3VP16/mim-1 A complex.

We next examined whether subtle differences between the DNA binding capacity of the three Myb proteins could be detected by allowing them to compete with each other for decreasing amounts of oligo. We hypothesized that a difference in the binding affinity between the three proteins would result in a difference in the ability to bind the MRE oligo under DNA-limiting conditions. Standardized lysates with each of the three Myb proteins were mixed and bound to serial dilutions of GG, GT, or TG oligo, and analyzed by EMSA (Fig. 6). There was no striking differences in ability of the three Myb proteins R₂R₃VP16, c-Myb(1-360), and c-Myb(FL) to compete with each other for limiting amounts of the same MRE oligo (Fig. 6). The amounts of protein-DNA complex for each protein and oligo combination were calculated relative to the amount of R₂R₃VP16/GG complex (Fig. 6B). The rate of decreasing protein-DNA complexes with decreasing oligo concentration was overall very similar (Fig. 6B). However, some differences were found. In general, all three Myb proteins formed less protein-DNA complexes with the GT (35-65%) and TG (45-65%) oligos relative to the GG. Also, c-Myb(FL) bound less well than c-Myb(1-360) to the GT and TG oligos (approximately 10-20% less). This subtle difference between c-Myb(FL) and c-Myb(1-360) is also reflected in Fig. 5, as shown above. A binding reaction with the TT oligo (Fig. 6A, lane 15) was included as a negative control, demonstrating the absence of nonspecific protein-DNA complexes in the binding reactions. This experiment suggests that a subtle difference in binding affinity of c-Myb(FL) and also c-Myb(1-360) could be ranked in the order GG > TG  GT.

The stability and/or apparent affinity of the three different Myb proteins for the MRE variant oligos was also estimated by comparing the ability of these oligos to compete out a mim-1 A oligo from preformed protein-DNA complexes. Standardized COS-1 lysates with each of the three Myb proteins were mixed and bound to a radioactive mim-1 A site oligo, followed by competition with a 25-fold excess of nonlabeled oligos for the MRE variants GG, GT, TG, and TT. The MRE variant oligos showed a qualitative difference in the ability to compete out mim-1 A site oligo for all three forms of Myb protein (Fig. 7). The amounts of protein-DNA complex for each protein and oligo combination were calculated relative to the amount of R₂R₃VP16/mim-1 A (Fig. 7B). The exchange between radiolabeled oligo and competitor oligo appeared to be quite rapid for all three proteins, because there was no major difference between the 3 and 15 min time points. The GG oligo competed efficiently out the mim-1 A oligo bound to all three Myb proteins (complex intensities reduced by 40-70%, relative to complex bound to mim-1 A oligo), as expected, whereas the GT and TG variants were less efficient (10-40% reduction), and the TT was completely unable to compete out the bound mim-1 A oligo (lanes 11-13), thus reflecting the results in Figs. 5 and 6.

To summarize the EMSA experiments, we found only small differences between c-Myb(FL) and c-Myb(1-360) and R₂R₃VP16 proteins in the ability to bind to selected variants of the MRE, with an apparent DNA-binding affinity in the order GG > TG ~ GT. All three Myb proteins expressed in COS-1 cells exhibited DNA binding properties similar to previously characterized recombinant R₂R₃ (23). Because we were not able to quantitate the R₂R₃VP16 protein in the COS-1 lysates by anti-Myb antibody, we cannot formally prove that the DNA binding capacity of c-Myb(FL) and R₂R₃VP16 proteins are identical. However, the apparent properties of R₂R₃VP16 and c-Myb(FL) are very similar. More importantly, the DNA binding activity of c-Myb(FL) and the C-terminally truncated c-Myb(1-360) was very similar, in contrast to some reports (34, 35, 38).

Transactivation Ability of Myb Proteins on Variant MREs in Mammalian Cells-- We next tested the sequence selectivity of c-Myb proteins in transactivation studies in mammalian cells. CV-1 cells were transiently transfected with increasing amounts of expression plasmids for c-Myb(FL) or c-Myb(1-360) and pGL2/tk/3xMRE reporter luciferase plasmids (Fig. 8). In general, the MRE selectivity of c-Myb(FL) and c-Myb(1-360) seemed to be very similar. For both c-Myb(FL) (Fig. 8A) and c-Myb(1-360) (Fig. 8B), some preference for MRE variant (GG > GT ~ TG) was evident at a low input of expression construct (1 µg). This result shows the same direction of selectivity as R₂R₃VP16 expressed at a low copy number in yeast (Fig. 3A), although less pronounced. However, at increasing input of effector plasmid, the preference of c-Myb(FL) and c-Myb(1-360) for MRE sequence was rapidly lost. Transactivation by c-Myb(1-360) was slightly higher than with c-Myb(FL), but the difference between the two is minor compared with other reports (39, 40) Surprisingly, the c-Myb(FL) and c-Myb(1-360) proteins had marked effects on transactivation of the pGL2/tk/3xTT reporter plasmid. Transactivation of the pGL2/tk/3xTT reporter plasmid increased rapidly with a high input of Myb expression plasmids. This effect cannot be due to specific binding to the TT MRE variant, because the EMSA (Figs. 1 and 5-7) clearly showed that the TT oligo was not bound by any of the expressed Myb proteins. This result clearly differs from the transactivation data with R₂R₃VP16, where transactivation from the TT reporter is minimal over the same range of input effector vector (Fig. 2). However, these results may not be directly comparable, as Myb FL and 1-360 contains the native c-Myb transactivation domain, whereas R₂R₃VP16 carries the herpes simplex virus VP16 transactivation domain. This difference in transactivation levels may reflect the observation that the c-Myb transactivation domain but not the VP16 TA domain could have effect(s) on transcription/transactivation independent of specific DNA binding. These data emphasize the importance of the c-Myb expression level as a major determinant of target site selectivity. At low expression levels, MRE selectivity is better or comparable to in vitro results. At high expression levels, the selectivity is worse than the in vitro results: fine discrimination between MRE sites is lost, and the negative control reporter (pGL2/tk/3xTT) is misleadingly transactivated to a high level.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   MRE selectivity of c-Myb proteins in CV-1 cells. CV-1 cells were transfected with c-Myb(FL) (A) or c-Myb(1-30) (B) expression constructs at the indicated concentrations (0-5 µg) and pGL2/tk/3xMRE reporter plasmids (4 µg) as indicated. Fold relative transactivation was calculated relative to the pGL2/tk/3xGG reporter with empty expression plasmid (pCIneo). Results are expressed as mean fold induction of luciferase activity and S.E. (A, n = 4; B, n = 3).

Finally, to assess the relevance of this conclusion, we evaluated the selectivity of c-Myb in naturally Myb-expressing cells. The c-Myb-expressing cell line K-562 was electroporated with pGL2/tk/3xMRE luciferase reporter plasmids or the basal level control pGL2/tk. In Fig. 9, a high selectivity for MRE variant sequences was observed with a strong preference for the GG sequence over the GT and TG MRE variants. The strong preference for the GG MRE oligo was evident in this cell type, very similar to the results obtained with a low copy number Myb effector plasmid in S. cerevisiae (Fig. 3A). This result further confirmed our hypothesis of the Myb expression level as a major determinant of Myb target site selectivity in vivo. We also included an AMV v-Myb expression construct in one experiment to test whether the transactivation response could be increased by a higher expression level of Myb protein or whether the transactivation levels with the 3xMRE reporter plasmids already were at a maximum. Addition of v-Myb increased the transactivation response further by 4-fold (Fig. 9, right column) compared with the endogenous transactivation level of pGL2/tk/3xGG, thus demonstrating that the observed transactivation level reflected the endogenous c-Myb protein level rather than the input of reporter plasmid DNA.

View larger version (42K): [in this window] [in a new window]   Fig. 9.   Discrimination between Myb recognition elements in Myb-expressing cells. K-562 cells were electroporated with pGL2/tk alone or with pGL2/tk/3xMRE reporter plasmids (10 µg) as indicated. Results are expressed as mean fold induction of luciferase activity over the pGL2/tk plasmid and S.E. (n = 4). On the right, 5 µg of expression plasmid for AMV v-Myb was cotransfected with the pGL2/tk/3xGG reporter plasmid (one experiment).

In conclusion, the transactivation data show that the target site selectivity of the Myb proteins c-Myb(FL) and c-Myb(1-360) is strongly influenced by the expression level of the Myb effector protein, both in S. cerevisiae and in mammalian cells. Furthermore, the expression level of Myb protein may severely affect the outcome of the effector/reporter transactivation assay.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We 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 (23). 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 (34, 35, 38). It has been suggested that this increased DNA binding also could contribute to the transforming phenotype of v-Myb (34). 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 the mim-1 A binding site (GG-type) (24, 36), an MRE sequence motif derived from  DNA (TG-type) (41), or a mim-1 A-related sequence motif (GG-type) in the c-myc promoter (42). 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 Table I) 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 (38).

It has been difficult to define bona fide target genes for c-Myb in 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 (14-16, 18, 23). In binding site selection studies with random oligos, the MRE consensus sequence was determined as YAACKGHA (15) or YAACKGHH (16). In these studies, the (T/C)AACGG sequence was prevalent in selected single MRE-site oligos, 70% (15) and 38-70% (16), depending on the Myb protein used. Efforts have also been made at thermodynamic measurements (43) and free energy matrixes (44) 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 (Table II), 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 (27) 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.

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 (45). 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 vivo tool for evaluating putative DNA-binding sites for other monomeric transcription factors.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Bernhard Lüscher for the murine Myb expression plasmids, to Dr. Joseph P. Lipsick for an aliquot of the 5E11 monoclonal antibody with the kind permission of Dr. Jonathan P. Sleeman, and to Dr. Pierre Chambon for the anti-VP16 transactivation domain antibody 2GV4.

	FOOTNOTES

^* This work was supported by the Norwegian Cancer Society, the Norwegian Research Council, and the Blix Foundation for Medical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Fellow of the Norwegian Cancer Society. To whom correspondence should be addressed: Institute for Experimental Medical Research, Ullevål Hospital, 0407 Oslo, Norway. Tel.: 47-2301-6800; Fax: 47-2301-6799; E-mail: k.b.andersson@ioks.uio.no.

² K. B. Andersson, T. Berge, V. Matre, and O. S. Gabrielsen, unpublished data.

³ T. Berge, K. B. Andersson, and O.S. Gabrielsen, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: MRE, Myb recognition element; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; DBD, DNA-binding domain.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES