(Received for publication, October 12, 1995)
From the
The maize VP1 protein is a seed-specific regulator of gene
expression that effects the expression of a subset of abscisic acid
(ABA)-regulated genes that are expressed during the maturation program
of the seed. In addition, VP1 has pleiotropic effects on seed
development that are not related to ABA. In transient expression
assays, VP1 has been shown to transactivate gene expression through at
least two distinct promoter elements: the G boxes from the
ABA-inducible wheat Em gene and the SphI box from the
maize C1 gene. We have investigated how VP1 can transactivate
gene expression through diverse promoter elements by analyzing its
association in vitro with EmBP-1, a factor that binds the Em promoter. We demonstrate that VP1 can greatly enhance the
DNA binding activity of EmBP-1 in a gel retardation assay. This
enhancing activity has also been observed on transcription factors as
diverse as Opaque-2, Max, Sp1, and NF-B. Deletion of a small but
highly conserved region (BR2) in VP1 eliminates the enhancement in
vitro as well as the ability of VP1 to transactivate Em gene expression in a transient expression assay. A 40-amino acid
fragment from VP1 sandwiched between the maltose-binding protein and
LacZ can confer the enhancement function to this fusion protein in
vitro. A weak and relatively nonspecific interaction between BR2
and DNA is demonstrated by UV cross-linking. The in vitro properties we observe for VP1 might explain the regulatory effects
of VP1 on a diverse set of genes and why mutations in the vp1 locus have pleiotropic effects.
Phytohormones such as abscisic acid (ABA) ()are
characterized by their ability to mediate a wide range of physiological
responses, one of which is the expression of specific genes that are
organ-, tissue-, or cell-specific (1) . Such response pathways
are superimposed upon the developmental regulation of gene expression.
Genetic and biochemical approaches are being used to understand the
mechanism by which a wide variety of hormone-responsive genes are
regulated by a single hormone in a tissue-specific
manner(2, 3) . For example, a set of genes that are
expressed in the embryo of maturing seeds has been shown (by embryo
culture and mutational studies) to require
ABA(1, 2, 3) . However, it is becoming clear
that ABA-regulated genes are not only differentially expressed during
seed development, but are differentially controlled in response to ABA
in non-embryonic tissue. For example, a gene in maize (emb5)
with strong homology to the highly conserved seed protein Em gene(4) , as well as the globulin genes in maize (glb1 and glb2) are expressed exclusively in mid-maturation
embryos(5, 6, 7, 8) . Transcripts of
a catalase gene (cat1) and members of the Rab family (rab17 and rab28), however, accumulate not only in
response to ABA in developing embryos, but in response to ABA in
vegetative tissues as well(5, 9, 10) .
Although all of these genes have the common requirement for ABA for
their expression in embryos, they appear to be members of different
gene sets controlled by different developmental programs.
Mutations in the viviparous-1 (vp1) regulatory locus in maize and its equivalent in Arabidopsis, ABA-insensitive 3 (abi3), have pleiotropic effects during seed maturation, one of which is to control the sensitivity of cells to ABA. The vp1 mutant fails to accumulate transcripts of the emb5, glb1, glb2, and other members of the class I and II gene sets (5) in the presence of ABA, whereas the cat1 and rab28 genes are expressed in response to ABA in vp1 mutant embryos(10, 11) . Similar to the regulation of the emb5 gene, the homologous Em gene in rice (Emp1) and Em1 in Arabidopsis require both ABA and VP1/ABI3 for expression in the embryo(12, 13, 14) . Hence, Em and glb are examples of genes that require ABA and VP1 for expression in the embryo, while cat1 and rab28 are examples that require ABA but not VP1. The VP1/ABI3 protein, which is expressed only during seed development(6, 14) , may be the factor most directly responsible for the strict regulation of genes that are expressed exclusively in seeds, e.g. Em. Support for this comes from the work of Parcy et al.(14) , who demonstrated that in transgenic Arabidopsis plants, overexpression of ABI3 results in the ectopic expression of the endogenous Em gene (AtEm1) in vegetative tissue when exposed to ABA.
The complexity and interaction of regulatory circuits operative during seed maturation is demonstrated further by the fact that vp1 has very pleiotropic effects during grain development, suggesting an even broader regulatory function than the ABA and embryo responses. vp1 kernels exhibit a lack of anthocyanins in the non-embryonic aleurone tissue(15, 16) , as well as reduced activities of several seed enzymes in diverse metabolic pathways(17) . These data suggest that the VP1 protein may be involved in the regulation of a number of diverse genes and response pathways, only one of which is ABA perception and/or embryo-specific ABA-regulated gene expression. Although ABA is an important component of seed maturation and is essential for the expression of a subset of maturation genes, various processes and responses associated with seed maturation require factors other than ABA(18) , as well as regulatory loci whose control extends beyond ABA response pathways(19) . Since the Em gene appears to be exclusively expressed in embryos and requires both ABA and the seed-specific regulatory protein VP1, we have focused our studies toward understanding how VP1 interacts with the transcriptional controlling components of the Em gene.
ABA-responsive cis-sequences in the wheat Em promoter that have been identified using transient expression
assays in protoplasts of rice (20, 21, 22) and maize (6) ()are shown in Fig. 1A. A 76-bp
segment of the Em promoter (Region I) has been shown to confer
ABA responsiveness to the non-responsive 35S viral promoter (23) . Region I includes two copies of a CACGTG element (Em1a
and Em1b), which is a conserved element found in many plant (24) and other eukaryotic promoters(21) , which flank
an AGCAG element (Em2a) that is conserved in certain genes expressed in
seeds(25) . Deletion of Region I eliminates the ABA response,
while mutations in either copy of the Em1 (a or b) or Em2a sequences
dramatically reduces the ABA responsiveness of the Em promoter (23) . (
)
Figure 1:
Map of the Em promoter and
the EmBP-1 and VP1 proteins. A, position and sequence of
regulatory elements in the Em gene promoter. Boxes represent the Em1 (Em1a, Em1b, Em1c, and Em1d) and Em2 (Em2a and
Em2b) elements and SphI box identified by functional assays or
by their sequence homologies to known cis elements. Position
of these elements is given relative to the translation start site (the
A of the ATG codon is coordinate 0). The arrows indicate the
positions of the two putative transcriptional start
sites(20, 21) . Region I and Region II brackets
represent the extent of the probes used in the gel shift assays. B, schematic representation of the EmBP-1 and VP1
polypeptides. In the EmBP-1 map, the shaded boxes indicate the
position of the proline-rich, basic DNA binding, and leucine zipper
dimerization domains. The bar below the EmBP-1 protein
demonstrates that truncated bZIP retains only the basic DNA binding and
leucine zipper dimerization domains. In the VP1 protein, the
amino-terminal acidic domain is shown as the transcriptional activator.
The position of the two basic regions BR1 (amino acids 222-237)
and BR2 (amino acids 386-406) deleted from various VP1 constructs
are indicated by the shaded boxes. Bars above VP1
represent regions of high homology with ABI3, an Arabidopsis homolog of VP1(26) . Bars under
VP1 represent the extent of the deletion mutants expressed as maltose
binding fusion proteins. 190VP1 is a truncated version of VP1 that
is missing the first 190 amino acids.
190
BR1 and
190
BR2 are derived from
190VP1. The BR2-LacZ is a
translational fusion of amino acids 370-409 with the
COOH-terminal portion of the LacZ protein.
Previous mobility shift assays and
footprinting results with Region I demonstrated that the Em1a sequence
represents a high affinity binding site for nuclear factors such as
EmBP-1, a basic leucine zipper protein (bZIP) isolated from a wheat
embryo cDNA library(21) . Competition experiments with various
Em1 elements from both regions I and II using rice nuclear extracts or
bacterially expressed EmBP-1 indicates that the order of binding
preference for nuclear factors is the same as for EmBP-1. ()A mutation in the CACGTG core of the Em1a element
eliminates binding by nuclear extracts and EmBP-1 and abolishes
ABA-induced expression in a rice transient assay(21) .
Does
VP1 interact with the Em promoter in the transient assay, and
if so, does VP1 act through the same or different cis elements
and trans factors required for the ABA response? Transient
expression of the vp1 cDNA in maize protoplasts cotransfected
with the wheat or rice Em promoter linked to the reporter gene GUS, results in GUS expression in the absence of exogenous
ABA(6, 16) . In the presence of exogenous ABA, VP1
transactivation of the Em promoter shows a striking synergy in
its transcriptional response, demonstrating that VP1 can augment the
ABA signal in a transient expression system. Tetramers of either the
Em1a or Em1b sequences from the wheat Em promoter are
sufficient not only for the ABA response, but also for the VP1
transactivation and synergy between ABA and VP1. A mutation
in the CACGTG core of the Em1a element, which eliminates the ABA
response and EmBP-1 binding(21) , dramatically reduces the VP1
response in maize and rice protoplasts (22) .
These
data suggest that the Em1 elements in Region I of the Em promoter, and the bZIP factors which recognize them, are the sites
through which the ABA and VP1 signals can elicit enhanced
transcription(22) .
VP1 can also transactivate
through the SphI element in Region II of the Em promoter (Fig. 1A), but this is independent of the
ABA response, since Region II alone cannot support an ABA
response.
The NH
-terminal region of VP1 is
required for transactivation of the Em promoter and can serve
as an acidic transcriptional activator as evidenced by its ability to
be functionally replaced by the acidic domain of the Herpes simplex virus VP16 transcription factor(6) . Sequence comparison
of the maize VP1 protein (6, 26) with the product of
the genetic equivalent locus in Arabidopsis (abi3) as
well as with the rice (13) and bean (
)homologs shows
three distinct regions that have extremely high amino acid sequence
conservation.
In this paper, we investigate the in vitro interaction between recombinant VP1 and EmBP-1 proteins and the
wheat Em promoter. We demonstrate in vitro that the
VP1 protein enhances the binding activity of the bZIP transcription
factor EmBP-1 to Em1 sequences in the Em promoter. The
addition of VP1 to a DNA binding reaction with EmBP-1 appears to
increase the effective concentration of EmBP-1 at the Em1 sites and
additionally causes the formation of higher order complexes in a gel
shift assay when the target sites are of of low affinity. VP1 has
similar enhancement effects in vitro on a variety of
transcription factors with diverse DNA binding domains and different
DNA targets. A 21-amino acid region of the VP1 protein (BR2),
characterized by a basic, -helical forming sequence with a high
degree of conservation between maize, rice, bean, and Arabidopsis, is shown to be required for both transactivation
of the Em promoter by VP1 in a transient protoplast gene
expression assay, as well as for the in vitro DNA
binding/enhancing activity. A 40-amino acid fragment from VP1, which
includes BR2, sandwiched between the maltose-binding protein and LacZ
can confer the enhancement function to this fusion protein in
vitro. We show also a weak, relatively nonspecific DNA binding
activity of the BR2 region, which we speculate may alter the
conformation of DNA in vitro, and thus may be responsible for
these more general enhancement effects.
For expression of the
MBP-VP1 fusions, it was necessary to add EcoRI and HindIII sites next to the start and stop codons of the maize vp1 cDNA (obtained from D. R. McCarty, University of Florida)
by PCR amplification with the primers ANO-1 (GCGGAATTCATGGAAGCCTCCTC)
and ANO-3 (GCGAAGCTTTCAGATGCTCAC C). The pAN13 plasmid was then
constructed by inserting this amplified product in the EcoRI/HindIII sites of pPR997. Unfortunately, the
MBP-VP1 product of this plasmid turned out to be extremely unstable.
Plasmid pAN15 (MBP-VP1190), which encodes a VP1 protein that is
missing the first 190 amino acids, was obtained by the removal of the EcoRI/BamHI fragment of pAN13 and blunt ligating. We
also received from Don McCarty plasmids containing the Vp1 deletions
Vp1-85/87 (
BR1) and Vp1-103/104 (
BR2), where
amino acids 222-237 and 386-406, respectively, had been
removed. BamHI/HindIII inserts from both these
plasmids were subcloned in the EcoRI/HindIII sites of
pPR997. An expression plasmid encoding amino acids 372-405 of VP1
between the MBP and lacZ genes was constructed by inserting an MscI/PstI fragment of the vp1 cDNA in the EcoRI/PstI sites of pPR997.
Finally, an ApaI/HindIII fragment, containing the maize Opaque-2 cDNA (a gift from R. J. Schmidt, University of California, San Diego), was inserted in the EcoRI/HindIII sites of pPR997 and the EcoRI/BamHI insert of pPExSD, encoding a rat Max cDNA (obtained from E. M. Blackwood, University of Washington), was inserted in the same sites of the expression vector. For expression of MBP-GF-14, the BstUI/SmaI insert from a cDNA encoding a rice homolog of the maize GF-14 ((27) ; received from H. Uchimiya, University of Tokyo) was ligated in the XmnI site of pPR997.
Purified NF-B was a
gift from A. S. Baldwin (University of North Carolina). SP1 was
obtained from Promega.
The following DNA oligonucleotides used in the competitive binding assay were annealed and filled in with cold nucleotide triphosphates.
Only Region I of the Em promoter can function as an
independent ABA response element and in the synergistic interaction of
ABA with VP1(23) . To account for the enhanced
transcription from the Em promoter caused by expression of vp1 in a transient assay(6) , we asked whether the VP1
protein could interact directly with the DNA in Region I (Fig. 1A) or indirectly via an interaction with EmBP-1 (Fig. 1B), the bZIP transcription factor that binds
specifically to the Em1 sequences required for the ABA and VP1 effects.
Fig. 2demonstrates that under the conditions of our gel
shift assay, a bacterially expressed truncated VP1 (VP1190; see
``Experimental Procedures'' and Fig. 1B) does
not directly interact with Region I. Furthermore, the use of an
oligonucleotide selection assay similar to the one described by
Blackwell and Weintraub (31) has failed to identify any DNA
sequences that would bind to VP1
190 in a gel shift assay (data not
shown). However, when recombinant VP1
190 was added to a binding
reaction with EmBP-1 and a Region I probe, a striking enhancement of
EmBP-1 binding activity was seen (Fig. 2). This enhancement with
VP1
190 is not dependent on the maltose-binding protein since it is
observed whether VP1 and EmBP-1 are present as fusions
(+MBP) or after Factor Xa digestion
(-MBP). The amount of added protein was kept constant in
the reactions without VP1 by the addition of MBP-LacZ protein, which
was purified in a manner identical to the other fusion proteins. No
enhancement was observed when VP1 is replaced either with BSA or with
the MBP-LacZ, MBP-GF14, and MBP-Max fusion proteins. GF14 is a protein
shown to be associated with G box complexes(32) , and Max is a
mammalian transcription factor(33) . Bannister and Kouzarides (34) have demonstrated that basic peptides can enhance the DNA
binding activity of some transcription factors. The addition of
positively charged poly-L-lysine to levels 50 times greater
than the VP1 concentration fails to enhance the protein-DNA complex
(data not shown), demonstrating that the effect of VP1 enhancement is
not due to a nonspecific effect by a basic protein.
Figure 2:
Enhancement of EmBP-1 DNA binding
activity by VP1. EmBP-1 and VP1 as fusion protein (+MBP)
or digested with Factor Xa (-MBP) were assayed for DNA
binding to radiolabeled Region I or Region II probes in a gel shift
assay. Binding reactions contained approximately 375 ng of EmBP-1
fusion protein or 60 ng of Factor Xa-digested EmBP-1 (-MBP). Assays were carried out in the presence of
either 2 µg of MBP-LacZ (- lanes) or 2 µg of
MBP-VP1190 (+ lanes).
In addition to
Region I, which contains high affinity binding sites for EmBP-1, Region
II of the Em promoter was tested (Fig. 1A) for binding
activity with VP1. Region II contains two ACGT elements, Em1c and Em1d,
which have previously been shown to be low affinity binding sites for
EmBP-1. ()Fig. 2demonstrates that, like the result
with the Region I probe, VP1 alone does not form a complex with the
Region II probe. The addition, however, of VP1
190 to the binding
reaction with EmBP-1 enhances the original complex of EmBP-1 with the
region II probe and also causes the formation of a series of slower
migrating complexes to form. Under these binding conditions, the higher
order complexes are only apparent with the Region II probe. These same
complexes can be observed on the Region II probe in the absence of VP1,
when high concentrations of EmBP-1 are used in the binding reaction.
These complexes may, therefore, be the consequence of additional
molecules of EmBP-1 associating with the DNA probe and binding to less
favorable or cryptic sites. VP1, in this case, would be increasing the
apparent concentration of EmBP-1. In addition, we have noted that the
enhancement of EmBP-1 by VP1 is consistently greater when the target
probe bears a low affinity binding site (e.g. Region II
compared to Region I; data not shown).
Fig. 3demonstrates that the VP1-mediated enhancement of EmBP-1 is dependent upon the concentration of EmBP-1 in the binding reaction. The ability of VP1 to enhance was measured over a range of EmBP-1 concentrations from 0.5 to 25 nm. Maximal enhancement was seen when EmBP-1 was present at concentrations below 1 nm. This result suggests that VP1 may be helping to overcome a concentration-dependent step that limits the extent of DNA binding.
Figure 3:
The degree of VP1-dependent enhancement is
dependent upon EmBP-1 concentration. Binding reactions were carried out
with varying quantities of MBP-EmBP-1 in the presence of 1.5 µg
MBP-VP1190 and the
P-labeled Em1a oligonucleotide.
The amount of retarded probe was quantified using a phosphorimager and
expressed as -fold enhancement.
As seen in Fig. 4,
the DNA binding activity of these widely differing transcription
factors, to their own or variant target sites, can be greatly enhanced
by the addition of VP1190. It is especially interesting to note
that for O2, detection of binding to the ACGT elements in Region II
(Em1c and 1d), which are quite different from its preferred target, was
dependent upon and enhanced by VP1. The enhancement of binding of this
diverse set of transcription factors indicates that the effect of VP1
is not confined to a specific class or target site.
Figure 4:
VP1 enhances the DNA binding activity of
other classes of transcription factors. Except where noted otherwise,
the DNA binding activity of the following transcription factors was
assayed in the presence of 2 µg of factor Xa-treated MBP-LacZ
(- lanes) or MBP-VP1190 (+ lanes). A, binding of 600 ng of purified MBP-Opaque2 to the Region I
or Region II probes. B, binding of 100 ng of purified
recombinant MBP-Max with the Region I probe. C, binding of
recombinant NF-
B p50 or p65 incubated with the
B probe. D, purified Sp1 (2 ng) was added to a radiolabeled GC box
probe in the presence of either 0.9 µg of MBP-LacZ (- lane) or 0.9 µg of MBP-VP1
190 (+ lane).
Figure 5: Vp1 increases the rate of association of EmBP-1 with its binding site. Binding reactions with EmBP-1 fused to MBP were carried out as in Fig. 3(+/- VP1) except that they were incubated for 5 or 10 min at room temperature and loaded onto a gel simultaneously.
Figure 6:
The enhancing activity of VP1 is mediated
through its BR2 domain. A, gel shift assay on the Region I
probe using 20 ng of Factor Xa-treated MBP-bZIP incubated with 300 ng
of either Factor Xa-treated MBP-LacZ or various VP1 deletion mutants
(see Fig. 2). B, same experiment as in panel A except that 8 ng of MBP-EmBP-1 was substituted for the MBP-bZIP. C, alignment of BR2 amino acid sequence from maize with that
from rice(13) , Arabidopsis(26) , and bean
(see Footnote 5). The position of the BR2 sequences within the context
of the full-length proteins are indicated by the coordinates at either end of the sequence. Vertical lines between the
sequences indicate conserved residues, while dots indicate
conservative substitutions. The bracketed region above the
sequence indicates the residues used in BR2-LacZ, and the bracket below indicates the region removed in the BR2
constructs.
To
test if the BR2 region can confer the enhancement function to another
protein, a fusion protein was obtained that is composed of a 40-amino
acid peptide from VP1 (amino acid residues 370-409), which
includes the amino acids removed in the VP1BR2 deletion,
sandwiched between MBP and LacZ. When this fusion protein was tested,
the binding activity of bZIP was enhanced as much as with the 502-amino
acid VP1
190. Identical results were observed when the full-length
EmBP-1 was used with the same series of recombinant VP1 (Fig. 6B), i.e. VP1
190, the VP1
BR1
deletion, and the MBP-BR2-LacZ fusion protein enhanced the EmBP-1 DNA
binding activity, while the VP1
BR2 deletion did not. (Note that in Fig. 6B the observed enhancement effect is less than
that seen in Fig. 2. This can be explained by the fact that the
concentration of EmBP-1 in the binding reaction is high enough to form
a visible complex, in the absence of VP1. Consequently, the overall
VP1-dependent enhancement is less). These results clearly demonstrate
that the amino acid residues 386-406 in VP1 are necessary and
sufficient for its enhancing properties and that deletion of another
charged region (BR1) in another part of the VP1 protein has no effect
on in vitro binding enhancement. Fig. 6C is an
alignment demonstrating the high degree of conservation of the BR2
domain between VP1 and its homologs in rice, Arabidopsis, and
bean.
McCarty et al.(6) and Vasil et
al. have demonstrated that VP1 expressed from a 35S
promoter (and the shrunken1 intron) transactivated different
Em-GUS constructs in maize protoplasts. We constructed plasmids in
which the same full-length VP1 and VP1
BR2 were fused to a 35S
promoter and tested for Em-GUS transactivation in our rice protoplast
transient assay(23) . Table 1shows the results of a
transient Em-GUS gene expression assay where full-length VP1
or VP1
BR2 are cotransfected with the reporter Em-GUS. Expression
of full-length VP1 resulted in a clear transactivation of Em,
whereas expression of the BR2 deletion failed to transactivate an
Em-GUS construct. Although the BR2 deletion construct has an intact
activation domain (amino acids 1-122), which was shown to be
required for transactivation of the Em-GUS fusion(6) , it still
fails to transactivate in the rice transient assay. A ribonuclease
protection assay on total RNA from replicate transfections demonstrates
that full-length transcripts of the transgenes are present in the
protoplasts (data not shown). These results clearly demonstrate that
the amino acid residues 386-406 in VP1 are not only necessary and
sufficient for the ability of VP1 to enhance the binding of EmBP-1 to
DNA in vitro, but are required for transactivation of Em in an in vivo transient expression assay.
Several recombinant proteins were
incubated with a Region I probe that had been labeled with
bromodeoxyuridine and [-
P]dCTP and
cross-linked with UV-light to capture a transient interaction. Unbound
DNA was then digested with DNase and the proteins separated on
SDS-PAGE. Fig. 7shows binding (i.e. labeling) of
MBP-BR2-LacZ and our positive control MBP-EmBP-1 to DNA. No labeling (i.e. binding) was observed using BSA or the MBP-fusion
proteins LacZ, VP1
190 or VP1
BR2. To determine if the
interaction of MBP-BR2-LacZ with DNA is specific, the cross-linking
reactions were repeated in the presence of a 250-fold excess of either
the Em1a element Em2 or pBluescript DNA that had been cleaved into
small fragments with the restriction endonucleases AluI, BstUI and HaeIII. As can be seen in Fig. 7B, binding of MBP-BR2-LacZ to region I was
reduced by 40-60% with all competitors. This is in contrast to
the results obtained with the specific DNA-binding factor EmBP-1, the
labeling of which was totally competed only by excess Em1a.
Figure 7:
DNA binding activity of the BR2 domain. A, autoradiogram of an SDS-PAGE gel after UV cross-linking of
approximately 2 µg of BSA, MBP-LacZ, MBP-VP1190,
MBP-VP1
BR2, MBP-BR2-LacZ, and MBP-EmBP-1 with a Region I probe. B, labeling of MBP-BR2-LacZ or MBP-EmBP-1 by UV cross-linking
with a Region I probe. Where indicated, we added a 250-fold excess of
the following competitors: Em1a, Em2a, or digested vector
DNA.
Our results show that the bacterially expressed VP1190
protein can enhance the in vitro DNA binding of the bZIP
transcription factor EmBP-1 to its CACGTG binding sites in Region I of
the Em promoter. The enhancing effect on EmBP-1 binding
appears to be highly selective for VP1
190 since a number of other
(nonspecific) recombinant proteins expressed and purified in a manner
similar to VP1
190 (e.g. GF14, LacZ, or Max) failed to
enhance the binding of EmBP-1 to its target.
However, the ability of
VP1190 to enhance the binding activity of transcription factors is
not limited to EmBP-1 or its preferred binding sites. The binding of
O2, another member of the plant bZIP class, to ACGT sites in both
Region I and Region II of the Em promoter is also enhanced by
VP1. In addition to these members of the bZIP class, we show that VP1
also enhances the binding of members of the Rel, zinc finger, and basic
helix-loop-helix class of DNA-binding proteins to their target
elements, some of which are different from the ACGT sites for EmBP-1
and O2. Although dimerization is required for the bZIPs, Max, and
NF-
B to bind DNA, SP1 does not require dimerization to bind
DNA(38) . Hence, there is no apparent specificity of the VP1
effect with respect to class of transcription factor, target site, or
requirement for dimerization.
These results raise the interesting
possibility that the nonspecific enhancement effect displayed by VP1 in vitro could be reflected in vivo by VP1
transactivating a number of widely differing genes through diverse
promoter elements and transcription factors. This is demonstrated by
the ability of VP1 to transactivate C1, a regulatory gene
active in the control of anthocyanin biosynthesis in the non-embryonic
aleurone tissue, and to transactivate the non-ABA-responsive Region II
of the Em promoter (both require an SphI element
(CATGCATG) and not the CACGTG motif essential for its effect on Region
I of the Em promoter (16) ). These
activities of VP1 in vivo and in vitro are consistent
with the very pleiotropic effects of VP1 during seed
development(17) . Hence, the enhancement effect by VP1 that we
observed may be part of the mechanism by which VP1 modulates the
expression of a broad range of genes and their associated diverse
promoter elements and transcription factors.
However, the role of VP1 is not simply to facilitate a general increase in the expression levels of genes expressed during seed development. In maize, for example, at least two other ABA-regulated genes do not require VP1 interactions for expression in the embryo(10, 11) . In Arabidopsis, although the abi3-4 mutation inhibits the accumulation of numerous members of the different temporal classes of mRNA expressed during seed development, abi3-4 does not equally affect the accumulation of all mRNAs in any one temporal class. Hence, the regulatory networks modulated by VP1/ABI3 in vivo is neither confined exclusively to any one temporal stage or tissue within the seed nor to all ABA-regulated genes during seed development. Perhaps the characteristic in common among the genes in the set controlled by VP1 is that they are expressed exclusively during seed development (e.g. Em), in part regulated by the seed-specific expression of VP1/ABI3. This is supported by the recent demonstration that overexpression of ABI3 in transgenic Arabidopsis results in the expression of AtEm1 in vegetative tissue where AtEm1 is normally not expressed (14) . Whereas many other non-VP1/ABI3 regulated genes are expressed in a temporally and spatially specific pattern during seed development in response to various signals including ABA, they may also be expressed at other times during the life cycle. In any case, it is apparent that specific regulatory factors other than VP1 must be involved in the complex regulation of gene expression in seed maturation and that the nonspecific enhancing activity we see with VP1 in vitro must somehow be targeted to a specific subset of genes in the seed that are under VP1 regulation.
The pleiotropic
nature of vp1/abi3 in maize and Arabidopsis and the
absence of specificity associated with the in vitro enhancing
phenomenon is reminiscent of properties associated with several
transcriptional activators encoded by animal viruses. The Tax protein
encoded by human T-cell lymphotrophic virus type 1 is a transcriptional
activator that can transactivate not only its own viral promoter but
also a variety of cellular enhancers with diverse sequence elements.
Armstrong et al.(39) have demonstrated, using gel
shift assays, that the Tax protein has the ability to enhance the DNA
binding of a variety of transcription factors from different structural
classes including ATF, NF-B, SP1, and GAL4. This enhancement
phenomenon may explain the highly pleiotropic effects of Tax on animal
cells. Several in vitro characteristics of Tax appear to be
similar to the properties of VP1
190. The Tax enhancement is not
associated with a direct Tax-DNA interaction or a supershift
(indicative of protein-protein interaction) in gel retardation assays.
Like VP1
190, Tax has been shown to increase the on rate of bZIP
proteins as well as achieve maximal enhancement of DNA binding, when
the concentration of DNA-binding protein is low(40) . Wagner
and Green proposed that Tax works by enhancing the ability of bZIP
transcription factors to dimerize via a protein-protein interaction
with Tax. This mechanism does not account for the observation that Tax
has been shown to associate with multiple classes of cellular
enhancer-binding proteins that are not bZIP factors, including
NF-
B and SRF(41) . The mechanism by which Tax enhances the
DNA binding activity of such a wide range of proteins is not
understood.
A different viral transcriptional activator from adenovirus, E1a, also transactivates through a number of different promoter elements. A recent report (42) presents biochemical evidence that E1a can associate with diverse transcription factor DNA binding domains, including those from AP-1, SP1, and USF. E1a appears to associate with various promoters via protein-protein interactions with a variety of transcriptional activators. However, the protein-protein association is not stable enough to survive gel electrophoresis and therefore does not result in a ``supershift.'' Likewise, our results with VP1 and the various DNA-binding proteins in our gel shift analysis show an enhancement of the original protein-DNA complex, but no detection of a supershift that would indicate formation of a stable ternary complex. However, unlike E1a, we have been unable to detect protein-protein interaction between EmBP-1 and VP1. If protein-protein interaction plays a role, it is extremely transient and we have been unable to capture it using the various approaches mentioned above.
Our efforts
to identify the domain of VP1 responsible for enhancement activity, and
by so doing elucidate the possible mode of action of VP1, has been
guided by the work of McCarty et al.(15) . The vp1-Mc allele is a transposable element insertion in the vp1 locus causing the production of a truncated VP1 protein
missing the COOH-terminal 150 amino acids. This allele specifically
eliminates expression of the anthocyanin but not the maturation
pathway, i.e. the ABA-insensitive phenotype. Hence, our
efforts have been focused on the sequence of VP1 upstream of the
missing COOH terminus in the vp1-Mc allele: in particular, two
regions that are highly conserved between Arabidopsis and
maize (BR1 and BR2). Our in vitro results clearly show that a
21-amino acid deletion of the BR2 domain of VP1190 eliminates the
enhancing function of VP1. That this region is also sufficient for the
VP1 effect is further supported by the fact that a 40-amino acid
peptide containing the BR2 region can confer full enhancing activity to
MBP-LacZ.
The functional relevance of the in vitro effects
of the BR2 domain of VP1 is supported by transient expression studies
in rice protoplasts. When the deletion of BR2 from the full-length VP1
protein, is overexpressed behind a 35S promoter, a protein results that
fails to transactivate an Em-GUS construct (Table 1). Although
the BR2 deletion fails to activate the Em promoter, the
NH-terminal acidic activation domain (amino acids
1-122) that is required for transactivation of Em-GUS (6) is present and intact. Taken together, these results
demonstrate that both the NH
-terminal acidic region and the
BR2 segment are required for the VP1 function. Since BR2-LacZ lacks an
activation domain, we did not detect transactivation by BR2-LacZ alone
(data not shown).
Because VP1 enhances the binding of widely
different transcription factors and target sites, and the effect of VP1
on EmBP-1 binding is confined to the DNA binding region (bZIP), we
reasoned that some essential functional domain of VP1, e.g. BR2, could be interacting with DNA in a relatively
sequence-independent way. Such an interaction could alter the
conformation of DNA (e.g. promote bending) to enhance binding
of a number of different sequence-specific factors, such as EmBP-1, O2,
etc. The BR2 domain of VP1 is a positively charged region with
helix-forming potential that can form plausible DNA-binding structures.
This region is highly conserved between VP1 and its homologs in Arabidopsis, rice, and bean (Fig. 6C). It is
clear that the enhancement by VP1190 is not associated with a
stable VP1-DNA interaction, as evidenced by our inability to detect a
shifted complex in a gel retardation assay. However, it appears that
the BR2 domain may mediate the transient association of VP1 with DNA
because of our ability to cross-link MBP-BR2-LacZ to region I. The
observation that BR2 is sufficient both for the enhancing activity and
DNA association suggests that a transient association of VP1 with DNA
might account for its ability to enhance the binding activity of other
proteins.
We are unable to explain the lack of cross-linking on
MBP-VP1190. However, one possibility is that the conformation of
BR2 within the context of MBP-VP1
190 is such that its association
with DNA is too transient or unstable to capture with UV cross-linking.
Removing BR2 from MBP-VP1
190 and placing it within the context of
a heterologous protein may serve to unmask the BR2 domain resulting in
a more stable association with DNA. Another possibility is that within
the context of the whole protein, the DNA is not fully protected
against DNase digestion. In any case, the observation that BR2 is
sufficient both for the enhancing activity and DNA association suggests
that a transient association of VP1 with DNA might account for its
ability to enhance the binding activity of other proteins.
It is possible that VP1 enhances the DNA binding activity of EmBP-1 by transient stabilization of a local structural conformation in the CACGTG core and that binding of EmBP-1 and VP1 to the same DNA is mutually exclusive. bZIP factors have recently been shown to achieve sequence-specific recognition by binding to a preorganized distorted DNA structure that constitutes the bZIP recognition target(43) .
Recently it has been proposed that positively charged protein domains may contribute to protein-induced DNA bends(44) . In addition, it has been shown that proteins that are known to bend DNA, for example HMG-1, are associated with enhanced binding of other DNA-binding proteins in gel retardation assays(45, 46) . Future investigations will focus on whether the association of the positively charged BR2 domain with DNA results in altered DNA structure.