(Received for publication, November 12, 1996)
From the Department of Biochemistry, University of Oslo, N-0316 Oslo 3, Norway
c-Myb is the founder member of a class of transcription factors with tryptophan-rich repeats responsible for DNA binding. Activated oncogenic forms of Myb are encoded by the avian retroviruses, avian myeloblastosis virus (AMV) and E26. AMV v-Myb encodes a truncated protein with 11 point mutations relative to c-Myb. The mutations in the DNA binding domain (DBD) were reported to impose distinct phenotypes of differentiation on transformed myeloid cells (Introna, M., Golay, J., Frampton, J., Nakano, T., Ness, S. A., and Graf, T. (1990) Cell 63, 1287-1297). The molecular mechanism operating has remained elusive since no change in sequence specificity has been found. We introduced AMV-specific point mutations in the minimal DBD of chicken c-Myb and studied their effect on structure and function of the purified protein. Fluorescence emission spectra and fluorescence quenching experiments showed that the AMV-specific point mutations had a significant effect on the conformation of the DBD, giving rise to a more compact structure, a change that was accompanied by a reduced sensitivity toward cysteine-specific alkylation and oxidation. The DNA binding properties were also altered by the AMV-specific point mutations, leading to protein-DNA complexes with highly reduced stability. This reduction in stability was, however, more severe with certain subtypes of binding sequences than with others. This differential behavior was also observed in an in vivo model system where DBD-VP16 fusions were coexpressed with various reporters. These findings imply that different subsets of Myb-responsive promoters may react differentially toward the AMV-specific mutations, a phenomenon that could contribute to the altered patterns of gene expression induced by the AMV v-Myb relative to wild type c-Myb.
The expression of the c-myb proto-oncogene is restricted to a limited range of differentiating cell types (for recent reviews, see Refs. 1 and 2). Myb's function is particularly important in early stages of hematopoiesis, where it plays a crucial but poorly defined role in maintaining the proliferative state of immature cells (reviewed in Refs. 3 and 4). High levels are expressed in immature cells of all lineages, and the expression is strongly down-regulated during terminal differentiation. Aberrant overexpression of c-Myb inhibits differentiation of hematopoietic precursor cells (reviewed in Ref. 5), while antisense oligonucleotides directed at c-myb inhibit their proliferation (6-8). Mice homozygous for a c-myb disruption die from multiple hematopoietic defects during embryonic development (9), and transgenic mice with T cell-specific expression of a dominant interfering allele of Myb display partially blocked thymopoiesis and diminished proliferation of mature T cells (10). The molecular mechanisms producing these phenotypes are poorly understood, in particular when it comes to Myb-regulated target genes with a role in proliferation and differentiation. Candidate target genes have recently been reviewed (2).
The c-myb gene encodes a 75-kDa sequence-specific DNA-binding transcription factor with at least three functional domains (2). The DNA binding domain (DBD)1 located near the amino terminus is a highly conserved tryptophan-rich region composed of three imperfect repeats (R1, R2, and R3), each related to the helix-turn-helix motif (11-13). Each repeat appears to have a distinct function. R3 is a fully folded domain mainly responsible for the sequence-specific recognition of the AAC core in the binding site (13, 14). R2 is more flexible and seems to undergo a conformational change upon binding to DNA, possibly to allow the protein to adapt to a range of flanking sequences (15-17) (see also Carr et al. (18)). R2 also contains a highly oxidizable cysteine implicated in redox control (16). An NMR-derived structure of a mouse R2R3-DNA complex was recently reported (14). The role attributed to R1 has been a stabilization of the complex through electrostatic interactions (19-21). An acidic transactivation domain is found centrally located in the protein, and a large carboxyl-terminal region appears to have a negative effect on Myb's transactivation and DNA binding functions. A recent report suggested that several subdomains may cooperate to form a functional transactivation domain, implying that the precise borders of this domain remain to be elucidated (22).
Distinct mechanisms of oncogenic activation seem to operate in the two isolated avian v-myb-containing retroviruses E26 and avian myeloblastosis virus (AMV) (reviewed in Refs. 1, 4, and 5). Both contain truncated v-myb oncogenes. E26 encodes an amino- and carboxyl-terminally truncated Myb protein that is fused to the v-Ets oncoprotein, while AMV encodes a truncated Myb protein that displays a number of specific amino acid substitutions relative to c-Myb. E26 is able to transform multipotent hematopoietic cells and causes erythroblastosis and a low level of concomitant myeloblastosis in chickens, while AMV transforms myelomonocytic cells and causes acute monoblastic leukemia.
The specific point mutations found in AMV v-Myb contribute to the establishment of the specific differentiation phenotype of AMV-transformed cells, probably by affecting sets of differentiation-specific genes. From experiments with hybrid viruses, the critical difference between the E26 and AMV viruses that causes the two distinct transformation phenotypes were mapped to the R2 repeat of the DNA binding domain (23). The AMV-specific point mutations also contribute to the oncogenic potential of the virus. Dini et al. (24) found that AMV v-myb caused a 10-fold higher frequency of leukemic transformation of primary hematopoietic cells in culture than did a similarly truncated c-myb without the mutations. Mutations in both the DNA binding and the transactivation domains seemed to cooperate in attaining a high transformation frequency (24).
Precisely how the AMV-specific point mutations causes such prominent phenotypic effects is poorly understood. Since the DBD of Myb is involved, the most obvious hypothesis would be to expect an altered DNA binding specificity leading to activation of a distinct set of genes. However, binding-site amplification experiments in vitro have not revealed significant differences in sequence recognition between c-Myb and v-Myb (25). An alternative hypothesis suggested by some investigators has been that the mutations modify interactions between Myb and other regulatory proteins (see Thompson and Ramsay (2)). Indirect support for this hypothesis was the finding that all three amino acid replacements are exposed on the surface of the DNA binding domain as judged from its structure (14). However, so far no direct evidence for this assumption has been reported, and no interacting proteins have been identified. In the present work we have exploited the possibility of significant effects intrinsic to R2. We show that the AMV-specific mutations alter the conformation of the flexible R2-domain and that this change is accompanied by altered DNA binding properties, altered redox sensitivity, and altered transactivation in a model yeast effector-reporter system. We also demonstrate that the quantitative alterations in DNA binding is more severe with some recognition sequences than with others, suggesting that the altered DNA binding properties act differentially and could thus affect subsets of genes differently.
The minimal DNA binding domain of the chicken c-Myb protein, R2R3, and two mutant derivatives were expressed in Escherichia coli using the T7 system (26), and proteins were purified as described previously (12).
MutagenesisSite-directed in vitro mutagenesis
was performed as described elsewhere (12, 27). Plasmids from positive
clones, identified by DNA sequencing, were used directly to transform
the E. coli BL21(DE3)LysS strain. The three AMV-specific
mutations introduced in R2 were I91N, L106H, and V117D as
illustrated in Fig. 1A. The R2R3[AMVHD] protein harbored the
L106H and V117D mutations, while the
R2R3[AMVNHD] protein harbored all
three alterations. Mutations were verified by amino acid sequencing of
the purified proteins through 32 steps. Automated Edman degradation was
performed on a 477A protein sequencer with an on-line 120A
phenylthiohydantoin amino acid analyzer from Applied Biosystems (Foster
City, CA).
Electrophoretic Mobility Shift Assay
DNA binding was monitored by the electrophoretic mobility shift assay (28), with the modifications described in Gabrielsen et al. (12). The basic duplex oligonucleotide probe used (Scheme 1)
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was based on the Myb recognition element (MRE) in the upstream region (site A) of the mim-1 gene (29). Derived variants with different configurations of Gs in positions 5 and 6 of the MRE consensus sequence were also used (Scheme 2)
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(30). Labeled duplex
oligonucleotides were obtained by end-labeling of a small complementary
oligonucleotide "MRE primer" (5-GGCGCTAAA-3
) using polynucleotide
kinase and [
-32P]ATP. After annealing and fill-in, all
probes got identical specific activities and therefore directly
comparable intensities in electrophoretic mobility shift assay. Labeled
duplex oligos were purified by polyacrylamide gel electrophoresis.
N-Ethylmaleimide (NEM) and azodicarboxylic acid bis(dimethylamide) (both from Sigma, the latter abbreviated as "diamide") were made fresh from powder dissolved in water. H2O2 was freshly diluted in water from a stock solution (30%, Aldrich). NEM alkylation reactions were stopped by addition of dithiothreitol to a final concentration of 10 mM. H2O2 oxidations were terminated by addition of 0.5 µl of 1 mg/ml catalase.
Protease Treatment of Myb R2R3Chymotrypsin solutions were made
fresh from powder dissolved in TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Purified Myb proteins were diluted in
TGE100-buffer (20 mM Tris-HCl, 10%
glycerol, 1 mM EDTA, 10 mM
-mercaptoethanol,
100 mM NaCl, pH 8.0) and incubated with protease for 15 min
at 37 °C. The reaction was stopped by addition of SDS loading
buffer. The samples were heated at 95 °C for 2 min and loaded onto a
10-20% gradient polyacrylamide gel containing sodium dodecyl sulfate
according to Laemmli (31).
Fluorescence experiments were
performed essentially as described previously (16). A Perkin-Elmer
LS-50B luminescence spectrometer and a Perkin-Elmer luminescence
spectroscopy cell of 120 µl were used for the fluorescence
experiments. The exitation wavelength was 295 nm, and exitation slit 15 nm. Emission spectra were recorded between 310 and 400 nm with an
emission slit of 5 nm, and a scanning speed of 500 nm/min. Each
recording was made as an average of three accumulated scans. Samples
were prepared from purified proteins in TE buffer (10 mM
Tris-HCl, 1 mM EDTA, 10 mM
-mercaptoethanol, pH 8.0) at a concentration of 2 µM. Fluorescence values
were corrected with respect to background fluorescence and the inner
filter effect of acrylamide. For denaturation, proteins were incubated
with 6 M guanidinium chloride for 15 min at room
temperature before recording.
The reporter plasmids used are described elsewhere (30). The c-Myb-R2R3[wt] and R2R3[AMVNHD] effector plasmids were derived from the centromeric plasmid pDBD11 (32), a yeast centromeric plasmid designed to express DNA binding domains fused to the strong transcriptional activator from the herpes simplex virus VP16 gene under the control of the yeast GAL1 promoter. The insert encoding the R2R3 domains were generated by polymerase chain reaction by which also proper restriction cleavage sites (HindIII and BglII) were generated.
A one-step transformation procedure (33) was used to transform the
yeast strain 12S16 (ura3, leu2, arg3) (34) with the different plasmid constructs. Transactivation obtained with
R2R3[wt]-VP16 and
R2R3[AMVNHD]-VP16 fusion proteins
induced by galactose was measured as -galactosidase units assayed
with o-nitrophenyl-
-D-galactopyranoside (Sigma) as substrate (35).
To assess whether the three
AMV-specific point mutations in R2 had any effect on the
structure of the minimal DNA binding domain of Myb
(R2R3), we first studied purified DBDs by
fluorescence spectroscopy taking advantage of the high content of
tryptophans in these domains conferring intrinsic fluorescent
properties to the proteins. Three subdomains were expressed in E. coli and purified, the chicken wild type
R2R3 protein (designated
R2R3[wt]) and two mutated
R2R3-harboring mutations found in
R2 of the AMV v-Myb protein. The protein designated
R2R3[AMVNHD] harbored all three AMV-specific mutations in R2 (I91N, L106H, and V117D),
while R2R3[AMVHD] harbored only
the two latter (L106H and V117D). Fig. 1 shows the locations of the introduced mutations relative to secondary structure elements of R2R3 (Fig. 1A) and
relative to its three-dimensional structure (Fig. 1B). First
we compared the fluorescence emission spectra of the two subdomains in
three states, guanidinium chloride-denatured, native, or DNA-bound
form, using the spectrum of denatured protein for normalization (Fig.
2). As described previously (16), the emission maximum
of R2R3[wt] was shifted toward a shorter
wavelength (340-344 nm) compared to that of the denatured protein (356 nm) due to the folding of the polypeptide. The folding was also
accompanied by a large reduction in quantum yield. Addition of DNA lead
to a further slight shift in emission maximum (337-339 nm) and also to
a further reduction in quantum yield. We have previously presented evidence that a DNA-induced conformational change in R2
contributes significantly to the decreased fluorescence of DNA-bound
R2R3[wt] (16).
A comparison of R2R3[wt], R2R3[AMVNHD], and R2R3 [AMVHD] revealed one clear distinction. While little difference was seen between the three proteins in DNA-bound form, R2R3[wt] and the two R2R3[AMV] proteins differed significantly in their free native state. Both R2R3[AMVNHD] and R2R3[AMVHD] gave lower quantum yields than R2R3[wt] (Fig. 2). No difference was observed between the double and triple mutant. The emission spectra of native R2R3[AMVNHD] and R2R3[AMVHD] were closer to the spectrum of the DNA-bound state than the emission spectrum of native R2R3[wt]. This suggested that the AMV-like DNA binding domain might have a more compact structure than its wild type homologue, and that the DNA-induced conformational change in R2R3[AMV] could be more modest than in R2R3[wt].
To obtain more direct evidence for this hypothesis, we performed
fluorescence quenching experiments to monitor if the AMV-specific mutations induced any alteration in the average exposure of tryptophans in the R2R3-protein. As seen from the
Stern-Volmer plot in Fig. 3, both
R2R3[AMVNHD] and
R2R3[AMVHD] were quenched
significantly less by the neutral quencher acrylamide than
R2R3[wt], supporting our hypothesis of a more
compact structure in the mutants relative to the wild type protein.
After binding to DNA, all three forms showed a similar tryptophan
exposure, indicating more similar conformations in the DNA-bound state.
Since a purified R3 domain was found to be less quenched
than R2R3[wt] (results not shown), the
tryptophans in R3 must be less exposed to solvent than the average of all six tryptophans in R2R3[wt].
Hence, the three tryptophans in wild type R2 must be
significantly more exposed to solvent than their R3
homologues in order for R2 to make a major contribution to
the increased slope of the Stern-Volmer plot of
R2R3[wt]. This exposure is then reduced upon
some critical mutations in R2, both the AMV-specific
mutations studied here and the previously reported C130V mutation
(designated C43V in Myrset et al. (16)). The L106H and V117D
mutations made the major contributions to this conformational effect,
since no significant differences were detected between the two proteins
that differed with respect to the I91N mutation. For this reason, in
some experiments below only R2R3[wt] and
R2R3[AMVHD] are compared.
If the difference in conformation between
R2R3[AMVHD] and
R2R3[wt] were sufficiently large, the two
proteins might be expected to display differences in their proteolytic
sensitivity. The two purified proteins were therefore subjected to
limited proteolysis by chymotrypsin. This protease was chosen since
none of the amino acid replacements in
R2R3[AMVHD] should affect the
specificity of the enzyme. The band pattern of proteolytic products
revealed distinct differences between
R2R3[AMVHD] and
R2R3[wt] as indicated by arrows in Fig.
4. Both the total number and the positions of specific
bands differed. This result strongly suggests that the AMV-specific
point mutations in R2 indeed have a specific effect on the
conformation of the DNA binding domain.
We also asked whether this conformational effect was specific to the
AMV mutations or whether the conformation of the wild type protein was
particularly sensitive to mutations in R2, such that any
mutations in this domain would have a high probability of changing the
conformation of R2. We analyzed a total of 15 different
point mutations distributed over the entire R2 domain by
running emission spectra on purified recombinant proteins and by
analyzing their DNA binding activity using the electrophoretic mobility
shift assay (data not shown). In the analysis, we found only two
classes of mutations. The first class displayed unaltered DNA binding
and no change in emission spectra, while the second displayed severely
reduced DNA binding accompanied by a significant red shift in
max as well as increased quantum yield in the emission spectra, suggesting a more open conformation and a defect in attaining the native active state. In no case did we observe spectra resembling the AMV emission spectra with significantly reduced quantum yield relative to the wild type protein. These results suggest that the
AMV-derived mutations have a rather specific effect on the structure of
the minimal DNA binding domain of Myb.
To assess if the altered
conformational state in the R2R3[AMV]
proteins had any effects on their DNA binding properties, we performed
a series of comparative quantitative DNA binding experiments with
R2R3[AMVNHD],
R2R3[AMVHD] and
R2R3[wt] using a sequence derived from the
strong A site upstream the mim-1 gene as a binding probe (29). A direct analysis of DNA binding using electrophoretic mobility
shift assay did not reveal striking differences between the three
proteins (Fig. 5A), consistent with previous
findings that the same consensus recognition sequence holds for both
c-Myb and AMV v-Myb (25). However, a clear difference was observed when
the stability of the three protein-DNA complexes were compared. Under
the specific conditions used to compare the decays of the complexes,
the R2R3[wt]-DNA complex dissociated slowly
and a substantial fraction resisted 20 min of competition with excess
unlabeled DNA. In contrast, both the
R2R3[AMV]-DNA complexes dissociated much more
rapidly. After 5 min of competition less remained of the two
R2R3[AMV]-DNA complexes than of the
R2R3[wt]-DNA complex after 20 min (Fig.
5B). These data indicated that the conformational change
induced by the AMV-specific mutations in R2 leads to a considerably reduced stability of the protein DNA complex.
To determine the specific to nonspecific binding constant ratios (Ks/Kn) (28), specific DNA complexes were titrated with increasing amounts of poly(dI-dC). As seen in Fig. 5C, the R2R3[AMVHD]-DNA complex was titrated at lower concentrations of poly(dI-dC) than the wild type protein. From the data shown a Ks/Kn ratio of 6 × 103 was calculated for the R2R3[AMVHD] protein compared to 6 × 104 for the wild type protein. Assuming that the nonspecific DNA affinity is not affected, the AMV-specific mutations in R2 causes a 10-fold reduction in the specific DNA binding constant.
The AMV-specific Mutations in R2 Alter the Relative Stability of Complexes Formed with Subtypes of MREsPrevious in vitro studies of preferred binding sequences found no evidence for any alteration of sequence specificity as a result of the AMV-specific mutations (25). We have recently shown that strong MRE sequences with different configurations of guanine bases in the binding site for R2 gave remarkably different levels of transactivation in vivo at low expression levels of c-Myb (30). In vitro, corresponding differences between the binding sites variants could be seen only in analyses of protein-DNA complex stabilities. Since this phenomenon is related to the sequence of the half-site recognized by R2, we asked whether the AMV-specific point mutations in R2 had any influence on the relative complex stabilities when bound to different MRE variants.
Using three different MRE variants (TAAC,
TAAC
, TAAC
) as binding probes, the
stabilities of the complexes formed with R2R3[AMVHD] and
R2R3[wt] were compared after competition with an excess of unlabeled MRE (Fig. 6). For all three
probes the R2R3[wt] protein formed more
stable complexes than
R2R3[AMVHD]. A substantial
fraction of all the R2R3[wt] complexes
resisted 5 min of competition, but differences in stabilities allowed
us to rank the complexes according to affinities in the order:
TAAC
> TAAC
> TAAC
(Fig.
6). This order parallels their ability to mediate transactivation
in vivo (see below). In contrast, the R2R3[AMVHD] protein formed much
less stable complexes. When bound to the TAAC
probe a
visible fraction of the complexes resisted 5 min of competition
(comparable to the R2R3[wt] protein in
complex with the TAAC
probe). However, no visible
complexes were seen even at the first time point (2 min of competition)
when the R2R3[AMVHD] protein was
bound to the TAAC
or to the TAAC
probes.
Thus, single-G MRE variants form highly unstable complexes with
R2R3[AMVHD], but are still
reasonably stable when bound by R2R3[wt].
This suggests that the destabilization of the protein-DNA complexes
caused by the AMV-specific mutations might be more severe with some MRE subclasses than with others.
The AMV-specific Mutations in R2 Alter the Redox Sensitivity of the DNA Binding Domain
The R2 repeat
that is mutated in the AMV v-Myb also harbors a highly conserved
redox-sensitive cysteine that was found to be essential for DNA
binding, transformation, and transcriptional transactivation (36, 37).
The same cysteine is located in a disordered flexible region of
R2 (15, 18). We have previously proposed that that this
cysteine could function as a molecular sensor for a redox regulatory
mechanism turning specific DNA binding on or off by controlling a
DNA-induced conformational change in R2 (16). Having found
that the AMV-specific mutations induced a more compact conformation in
R2, we asked if this could influence the redox sensitivity
of the conserved cysteine. First we titrated the two purified
R2R3 proteins with increasing concentrations of
the SH-specific alkylation reagent NEM. Specific DNA binding was
abolished for all three proteins at sufficiently high NEM concentrations, but the titration showed a clear difference between the
three variants with respect to NEM sensitivity since
R2R3[wt] was inactivated at significantly
lower concentrations of NEM than both
R2R3[AMVHD] and
R2R3[AMVNHD] (Fig.
7A). A corresponding difference was observed
when the time course of inactivation was measured (results not shown).
In both experiments a C130V mutant was unaffected, demonstrating the
specificity of the alkylation.
We next examined Myb inactivation by treating the proteins with the SH-specific oxidation reagent diamide. As shown in Fig. 7B, specific DNA binding of R2R3[wt] was lost at lower concentrations of the SH reagent than observed for R2R3[AMVHD], suggesting a reduced redox reactivity as a result of the point mutations in R2. A time course experiment led to the same conclusion (results not shown). Finally, Myb inactivation by oxidation of the proteins with H2O2 was monitored. Again, specific DNA binding of R2R3[wt] was lost at lower concentrations than observed for R2R3[AMVHD] (Fig. 7C). Thus, we conclude that the conformational change induced by the AMV-specific point mutations in R2, changes the reactivity of the conserved cysteine in this repeat, making the DNA binding domain less susceptible to inactivation through modulation of the redox state of the critical cysteine.
The Effects of the AMV-specific Mutations in Vivo Measured in a Yeast Effector-Reporter SystemTo assess the effects of altered
DNA binding properties in a model in vivo situation, fusions
between the DNA binding domains and the strong VP16 transactivation
domain were expressed in yeast. The fusion gene was under control of
the galactose-inducible GAL1-10 promoter in a centromeric low copy
number plasmid, the latter to better mimic expression levels in a
physiological in vivo situation. All reporters were high
copy number plasmids (2 µm) containing the E. coli lacZ
gene under control of the yeast CYC1 minimal promoter. Insertion of
three upstream MREs made each of them Myb-responsive. The reference
reporter pBP19 with 3 × TAACGGAAC inserted has been described
(34). Upon induction with galactose, the
R2R3[wt]-VP16 and the
R2R3[AMVNHD]-VP16 fusion proteins
were expressed to the same level as judged by Western blot analysis of
representative yeast extracts using a Myb-specific polyclonal antibody
(data not shown). Since the only difference between the two fusion
proteins were their DNA binding domains, the levels of induced
-galactosidase activity were taken as estimates of their in
vivo DNA binding. Both fusion proteins were found to bind the
pBP19 reporter plasmid and activate lacZ transcription.
Induction of R2R3[wt]-VP16 resulted in
979 ± 160
-galactosidase units, whereas only 395 ± 65
-galactosidase units were found with
R2R3[AMVNHD]-VP16 induced. These
results suggest that the destabilization of the DNA binding caused by the AMV-specific mutations observed with the TAACGG binding site in vitro, correlated with a corresponding reduced DNA
binding and transactivation in vivo.
We next
asked whether the AMV-specific point mutations in R2 had
any influence on the relative responses obtained with different subtypes of synthetic Myb-responsive promoters in vivo. For
this purpose, we employed reporter constructs containing three copies of MRE sequences having different configurations of Gs in positions 5 and 6 of the MRE consensus sequence; that is either 3 × TAAC, 3 × TAAC
, or 3 × TAAC
(designated "3×GG," "3×GT," or "3×TG reporters," respectively). A reporter with 3 × TAAC
was used as negative control. As illustrated in Fig.
8A, the 3×GG reporter gave the highest
levels of transactivation for both fusion proteins. The transactivation
obtained with the 3×GT and 3×TG reporters were generally much lower,
but still at a significant level when activated by the
R2R3[wt]-VP16 fusion protein, consistent with our previous report (30). In contrast, the
R2R3[AMVNHD]-VP16 fusion protein resulted in very low levels of transactivation for both
the 3×GT and 3×TG reporters. This suggests that the reduced DNA
binding seen with the R2R3[AMV] proteins,
relative to the wild type protein, is more severe for certain MRE
sequences than for others as already seen in in vitro data
in Fig. 6. This is illustrated in Fig. 8B where the
transactivation data for each effector are normalized to the 3×GG
reporter. If the reduction in DNA binding caused by the AMV-specific
point mutations were proportional for the different MREs, the three
synthetic promoters analyzed would have given pairwise equal
levels of transactivation when normalized as in Fig. 8B,
which is clearly not the case. In particular the 3×TG-reporter
responded very poorly to the
R2R3[AMVNHD]-VP16 fusion protein,
while it was reasonably activated by the
R2R3[wt]-VP16 fusion protein.
Thus, we conclude that, even if the AMV-specific point mutations do not directly alter the sequence specificity of the protein as demonstrated previously (25), these mutations seem to weaken the interaction with subclasses of the recognition sequence to a different extent. It is therefore conceivable that different subsets of Myb-responsive promoters react differentially to these mutations and that these quantitative effects contribute to the altered patterns of gene activation induced by AMV v-Myb relative to wild type c-Myb.
In this work we have investigated how the properties of Myb are affected by the point mutations that have been selected for in the DNA binding domain of AMV v-Myb. Despite striking phenotypic effects in vivo (23, 24), the mechanism of action of these mutations has remained elusive since no change in sequence specificity of the mutated protein was observed (25), and no alterations in protein-protein interactions with a hypothetical partner has been reported. To better understand which properties of the protein might have been altered, we performed a detailed biochemical analysis of the minimal DNA binding domain in mutated versus wild type forms.
Most of the properties analyzed were indeed altered. The AMV-specific mutations in the second repeat had significant effects on conformation, redox-sensitivity, and on quantitative aspects of the DNA binding properties of the protein. Even if the mutated protein binds the same range of sequences as the wild type, we found a sequence dependence in the magnitude of destabilization of the complexes in vitro and in DNA binding in vivo. These latter observations in particular offer a possible explanation of how the AMV-specific mutations can lead to a different spectrum of genes activated by AMV-Myb versus c-Myb.
The evidence for a conformational change comes primarily from fluorescence quenching experiments, which showed that the average exposure of the many tryptophans in the DNA binding domain is reduced in the two R2R3[AMV] mutants compared to that in R2R3[wt]. Since the introduced AMV-specific mutations are located in R2, it is reasonable that the conformation of this domain was most affected. The R2 domain seems to be flexible and temperature-sensitive, and it changes conformation upon binding to DNA (15-17, 38). This conformational flexibility might well be sensitive to mutations. Previously analysis of Cys mutations located in R2 also changed the conformation of the domain. However, when we screened a series of 15 additional mutations distributed over the entire R2, none were found with the same properties as R2R3[AMV], suggesting that the AMV-specific mutations have a particular effect on the structure.
It is noteworthy that all three point mutations in R2
represents changes from rather hydrophobic residues to more polar ones (I91N, L106H, and V117D). Since they are all located on the surface of
the protein (14), one possibility is that they might stabilize the
folded structure of the domain through solvation effects. In addition,
inspection of the structure reported by Ogata et al. (14)
revealed that two of the mutations (L106H and V117D) were close to each
other in space (Fig. 1B). If both were ionized, the
resulting electrostatic interaction between His+ and
Asp could also have a stabilizing effect on the AMV v-Myb
DNA binding domain. In accordance with this hypothesis all differences
observed in this work could be attributed to the L106H and V117D
mutations, since the two proteins
R2R3[AMVHD] and
R2R3[AMVNHD] behaved equally in
all comparative experiments.
R2 contains a single reactive cysteine that is exposed for modification in the free protein but protected in the tighter folded DNA-bound conformation (16). According to the structural model the cysteine is located in the interior of the protein in the DNA complex (14). The observation that the reactivity of this cysteine is reduced in the R2R3[AMV] proteins compared to the R2R3[wt] protein, suggests that the cysteine on average is in a less exposed conformation in R2R3[AMV]. It is possible that an unstable or nascent helix in the region around the cysteine (18) might be stabilized by a tighter conformation in other parts of R2. Alternatively, the reported cavity in R2 might be affected (17). The cysteine in R2 is highly conserved, but is not essential for DNA binding since hydrophobic substitutions may be introduced without reducing the DNA binding activity of the protein (16). Its high redox reactivity has led us and others to suggest that it might be conserved to keep the protein responsive toward a possible redox regulatory mechanism. The observation that the AMV-specific mutations make the protein less responsive toward Cys modification and oxidation suggests that the oncogenic version might remain partially active under conditions where the normal variant would be inactivated. This might contribute to its oncogenic potential under specific conditions.
Previous reports found no qualitative differences in DNA sequence recognition properties between AMV v-Myb and c-Myb (25). With respect to quantitative DNA binding properties AMV v-Myb subdomains were reported to bind stronger (19) or weaker (24) to MRE as judged by direct electrophoretic mobility shift assay using bacterial extracts or purified recombinant Myb. We therefore analyzed in more detail quantitative aspects of DNA binding using purified R2R3[wt], R2R3[AMVNHD], and R2R3[AMVHD]. Although the three minimal DNA binding domains seemed very similar when analyzed in a direct mobility shift assay, competition assays revealed striking differences in stabilities between the formed complexes. Our analysis clearly demonstrates that the AMV-specific mutations weakens the interaction with the mim-1-derived recognition sequence. This adds to our previous hypothesis that R2 plays a role in modulation of complex stability. In keeping with that different variants of the second half-site bound by R2 lead to different complex stabilities (20), we show here that the AMV mutant versions of R2 also displays altered complex stability.
The most interesting effect of the AMV-specific mutations was the
differential destabilization on certain MRE variants. Binding to a
TAAC variant of MRE was much more destabilized than a
complex with a TAAC
variant. The consequence of this
differential destabilization when analyzed in an in vivo
yeast system was that
R2R3[AMVNHD]-VP16 still
transactivated through a TAAC
variant of MRE, but gave very low level of transactivation through a TAAC
site. In
contrast R2R3[wt]-VP16 transactivated both
variants of synthetic promoters. Among the candidate target promoters
for Myb reported in the literature, the TG variants of binding sites
are found more often than the GG variants. It is therefore quite
possible that certain subclasses of promoters activated by c-Myb will
fall below a critical level of affinity and not be activated by AMV
v-Myb while other subclasses are activated by both factors. This may
result in a different spectrum of genes activated by AMV-Myb relative
to c-Myb.
Our analysis has revealed that several intrinsic properties of the minimal DNA binding domain of Myb are modified as a result of the AMV-specific mutations in R2. Even if we cannot directly link these changes to the observed phenotypic effects of the same mutations in transformed cells, it is quite probable that quantitative alterations in DNA binding properties will have important phenotypic effects. The prevailing alternative hypothesis has been to assume alterations in protein-protein interactions with a hypothetical partner. We cannot exclude that AMV v-Myb later will prove also to have modified interactions with such partner proteins, but our results show that we do not need to postulate such alterations to explain phenotypic effects of the AMV point mutations. Alterations in other subdomains of the protein also mutated in AMV v-Myb probably add to the changes analyzed in this work. It was recently reported that the transactivation properties of the protein was weakened as a result of some of these mutations (24, 39). Although it is intriguing that a more potent oncogene encodes a transcription factor with weakened DNA binding and reduced transactivation properties, the finding that most of the mutations have a clear effect probably reflects the long time through which these mutations have been selected for.
We thank M. Crabeel and J. J. Bonner for generous gift of plasmids and Scott Ness for providing antibodies. We thank K. Sletten for performing the amino acid sequencing. Thanks also to Hilde Nilsen for excellent technical assistance and to Kristin Brevik Andersson for critically reading the manuscript.