(Received for publication, March 11, 1997, and in revised form, April 28, 1997)
From the Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Rtg3p and Rtg1p are basic
helix-loop-helix/leucine zipper protein transcription factors in yeast
that interact and bind to sites in an upstream activation sequence
element in the 5-flanking region of CIT2, a gene encoding
a peroxisomal isoform of citrate synthase. These factors are required
both for basal expression of CIT2 and its elevated
expression in cells with dysfunctional mitochondria, such as in
respiratory-deficient petite cells lacking mitochondrial DNA (
°).
This elevated expression of CIT2 is called the retrograde
response. Here we show that fusion constructs between the Gal4p DNA
binding domain and Rtg3p transactivate the expression of a
LacZ reporter gene under the control of a GAL1
promoter element. We have identified two activation domains in Rtg3p: a
strong carboxyl-terminal domain from amino acids 375-486, and a weaker
amino-terminal domain from amino acids 1-175; neither of these
activation domains contain the bHLH/Zip motif. We have also identified
a serine/threonine-rich domain of Rtg3p within amino acids 176-282
that is inhibitory to transactivation. In addition, the transcriptional
activity of the Gal4-Rtg3p fusion proteins does not require either
Rtg1p or Rtg2p; the latter is a protein containing an hsp70-like ATP binding domain that is also necessary for CIT2 expression.
In contrast, transcriptional activation by Gal4-Rtg1p fusion proteins requires the Rtg1p basic helix-loop-helix/leucine zipper protein domain, as well as Rtg3p and Rtg2p. These data suggest that
transcriptional activation by the Rtg1p-Rtg3p complex is largely the
function of Rtg3p. Experiments are also presented suggesting that Rtg3p is limiting for gene expression in respiratory-competent
(
+) cells.
Eukaryotic cells are able to monitor and respond to changes in
mitochondrial function through changes in nuclear gene expression (1-5). We have termed this pathway retrograde regulation, and have
suggested that it operates as a homeostatic mechanism for accommodating
various cellular activities to alterations in mitochondrial function
(6). One example of retrograde regulation in the yeast Saccharomyces cerevisiae is the elevated expression of the
CIT2 gene in cells with dysfunctional mitochondria (7).
CIT2 encodes a peroxisomal isoform of citrate synthase (8,
9), which functions in the glyoxylate cycle in one of two steps that
utilize acetyl coenzyme A. The glyoxylate cycle enables cells to
carry out a net conversion of acetate, available either as a sole
carbon source or produced via -oxidation, to carbohydrate by the
shuttling of glyoxylate cycle intermediates (e.g. succinate)
from the peroxisomes to mitochondria (10). Under conditions where the
activity of the tricarboxylic acid
(TCA)1 cycle is limiting, an
increased activity of the glyoxylate cycle would allow for a more
efficient use of two carbon compounds through anaplerotic pathways. We
have found that CIT2 transcription is activated as much as
30-fold in cells that have blocks in the TCA cycle or are
respiratory-deficient, for example, because of a complete loss of
mitochondrial DNA (
o petites) (7, 11, 12).
We have identified three genes, RTG1, RTG2, and
RTG3, that are required both for basal and
retrograde-regulated expression of CIT2 (11, 13).
Surprisingly, these genes are also required for oleic acid-induced
peroxisomal proliferation and the induction of enzymes of the
-oxidation pathway (12, 14), both of which occur when oleic acid is
present in the growth medium (15-17). Thus, the RTG genes
appear to play a pivotal role in controlling metabolic interactions
between mitochondria and peroxisomes.
RTG2 encodes a cytoplasmic protein containing an hsp70 type of ATP binding domain (18). Experiments to be presented elsewhere show that Rtg2p has properties of a heat shock protein that may function as a molecular chaperone in the control of gene expression.2 RTG1 and RTG3 encode basic helix-loop-helix leucine zipper (bHLH/Zip) transcription factors (11, 13). Rtg1p has some unusual features for a bHLH protein; in addition to an unusually long loop domain of some 39 amino acid residues, it has a very short basic DNA binding domain that lacks certain conserved amino acids found in the basic region of most other bHLH proteins. Those residues are known to make specific base contacts in the major groove of target site DNA (19-21). Rtg3p, on the other hand, has the features of a typical bHLH/Zip protein, including the conserved amino acids in the basic region of the bHLH motif. Recently, we showed that Rtg1p and Rtg3p bind, most likely as a heterodimer, to sequences in a 76-bp UASr element in the CIT2 promoter (13). The minimal DNA target site for binding of the Rtg1p-Rtg3p complex is not, however, an E box (22, 23), CANNTG, which is the core binding site for the great majority of bHLH transcription factors, but a novel sequence, GGTCAC, which we have called an R box (13). The CIT2 UASr element contains two such R boxes arranged as an inverted repeat separated by 28 bp of an AT-rich region. Both R box sites are required for full CIT2 expression and act synergistically in vivo (13).
In previous experiments, Rtg1p was tested for its ability to activate
transcription by fusing to it the DNA binding domain of Gal4p (24). In
wild-type cells, the Gal4-Rtg1p chimera was able to activate
transcription of an integrated LacZ reporter gene under the
control of a GAL1 promoter element (UASG).
Moreover, the activity showed a typical retrograde response,
i.e. expression was greater in ° petites than in
+ cells. Transcriptional activation by Gal4-Rtg1p was
absolutely dependent upon the presence of Rtg2p and at least one other
protein, which we suggested was a bHLH protein that interacted with
Rtg1p. That protein was subsequently identified as Rtg3p (13). To
characterize further the role of Rtg1p and Rtg3p in the control of
CIT2 expression, we have made a variety of Gal4p fusions to
Rtg3p. Here we show that Rtg3p contains at least two transcriptional
activation domains that can function independently of Rtg1p and Rtg2p.
We have also identified an apparent inhibitory domain of Rtg3p
transactivation rich in serine and threonine residues that could be
potential phosphorylation sites of the protein. We suggest that Rtg3p
may be limiting for CIT2 expression in respiratory-competent
(
+) cells and that Rtg2p could function to recruit Rtg3p
for interaction with Rtg1p.
The S. cerevisiae strains used in this study are derivatives of either
COP161 U7 (MATa ade1 lys1 ura3) (11) or SFY526 (MATa ade2 lys1 leu2 his3 trp1 gal4
gal80
Gal1-lacZ::URA3). SFY526
rtg1 was described
previously (24). SFY256
rtg2 and SFY526
rtg3 are derivatives of SFY526, in which the reading frame encoding amino
acids 23-573 of Rtg2p and the reading frame encoding amino acids
175-340 of Rtg3p were replaced, respectively, with the HIS3 gene. All strains exist both as a respiratory-competent
+ cells and respiratory-incompetent
° derivatives
lacking mtDNA. The
° strains were obtained by growth on rich
dextrose medium containing 20 µg/ml ethidium bromide. Each
°
derivative was stained with 4
,6
-diamino-2-phenylindole to ensure that
no mitochondrial DNA was present. Cells were grown on rich YP medium
(1% yeast extract, 2% Bacto-peptone) containing 2% glucose (YPD),
2% raffinose (YPR), or 3% glycerol (YPG) at 30 °C. Plasmids were
selected for by growth on minimal YNB medium (0.67% yeast nitrogen
base without amino acids) and 2% dextrose (YNDB), 2% raffinose
(YNBR), or 3% glycerol (YNBG) supplemented with 1% casamino acids or
individual amino acids as required.
Standard molecular biology techniques
were used for the construction, amplification, and manipulation of
plasmids (25). The vector pGBT9 (2µ, TRP3, GenBank
accession no. U07646) was used to construct fusions to the Ga14p DNA
binding domain. The vector pGAD424 (2µ, LEU2, GenBank
accession no. U07647) was used to construct fusions to the Ga14p
activation domain. The RTG1 fusion plasmid p2H26-177 was
described previously (24). pGAD-RTG1 encodes the Gal4p activation
domain fused to amino acids 26-177 of Rtg1p. PCR primers, containing a
5 EcoRI and 3
PstI site, were used to construct
the series of GAL4-RTG3 fusion plasmids displayed in Fig. 1
and pGAD-RTG3 (encoding the full-length Rtg3p fused to the Ga14p
activation domain). pGBT-RTG2 encodes the Gal4p DNA binding domain
fused to full-length Rtg2p. The sequence of all PCR generated
constructs was verified using the Sequenase kit (U. S. Biochemical
Corp.).
A 1.5-kb KpnI-BamHI genomic RTG1 fragment was cloned into pRS416 (CEN, URA3) and the multicopy vector yplac195 (2µ, URA3) to give pRTG1-416 and p195-RTG1. A 3.1-kb genomic XbaI-BamHI RTG3 fragment was cloned into pRS416, yplac195, and yplac181 (2µ, LEU2) to give pRTG3-416, p195-RTG3, and p181-RTG3.
Yeast Transformation, Cell Extracts, andYeast were transformed using lithium acetate and heat
shock (26). Transformants carrying the desired plasmid or plasmid pairs
were selected for on YNBD supplemented with the necessary amino acids.
Liquid precultures were inoculated with a pool of 15-20 independent
transformants and grown in selective YNBG (+) or YNBR
(
°). Selective YNBR cultures were inoculated at a low density
(0.01 A600), grown at 30 °C and harvested at
mid-log phase (~0.9 A600). Cell extracts and
assays were carried out as described by Rose and Botstein (27). For
each plasmid-strain combination, these extracts was assayed in
triplicate.
COP161 U7 + and
° strains were transformed with an empty control plasmid, or
plasmid carrying RTG1 or RTG3 (either 2µ or CEN). Selective YNBG (
+) or YNBR (
°)
precultures were used to inoculate selective YNBR cultures at a low
density (0.01 A600). Cells were harvested at mid-log phase and total RNA was isolated (28). RNA was fractionated on
an agarose gel and probed for actin and CIT2 mRNA
abundance as described previously (7). Signals were quantified using a
Molecular Dynamics PhosphorImager.
In previous experiments, we showed that a chimeric protein
consisting of the DNA binding domain of Gal4p fused to Rtg1p was able
to activate expression of an integrated LacZ reporter gene under the control of a Gal4p-responsive UASG element (24).
However, those studies revealed that Rtg1p by itself could not
transactivate, but appeared to require, in addition to Rtg2p, one or
more proteins for transcriptional activity. That conclusion was
strengthened by the identification of a new bHLH/Zip protein encoded by
the RTG3 gene, which is also essential for CIT2
expression, and the finding that Rtg3p binds with Rtg1p at two sites in
the CIT2 UASr (13). To determine whether Rtg3p
is also required for transactivation mediated by the Gal4-Rtg1p
chimeric protein, where Rtg1p is directed to the USAG
element in the reporter gene solely by the Gal4p DNA binding domain,
SFY526rtg3 cells were transformed with p2H26-177 encoding the Gal4p DNA binding domain fused directly to the HLH domain
of Rtg1p (Fig. 1A). The
-galactosidase activity of this transformant was then compared with
p2H26-177 transformants of wild-type SFY526 cells and its
rtg1 and
rtg2 derivatives.
Fig. 2 shows that there is a retrograde
response in the transactivation by p2H26-17 in SFY526 transformants,
i.e. activity was greater in respiratory-deficient °
petites than in
+ cells of that strain. In addition,
activity both in
+ and in
° cells was comparably
greater in SFY256
rtg1 cells, presumably because
endogenous Rtg1p was not present to compete for limited interacting
proteins required for transactivation of the Gal4-Rtg1p fusion protein.
In contrast to these results, no transcriptional activation was
observed in either SFY526
rtg2 or SFY256
rtg3
transformants. Altogether, regulation of expression of the
UASG-LacZ reporter gene by Gal4-Rtg1p resembles
transcriptional control of the CIT2 gene, whereby expression
is elevated in
° cells, and is dependent on RTG1 and
RTG3 in both
+ and
° cells (11, 13).
These results are consistent with the notion that transactivation
occurs via an Rtg1p-Rtg3p heterodimeric complex, and that Rtg3p
contains one or more transactivation domains.
Although Rtg2p is required for Gal4-Rtg1p-mediated transactivation of the UASG-LacZ reporter gene, it is unlikely to be involved directly in transcriptional activation. First, we have fused full-length Rtg2p to the Gal4p DNA binding domain and found that this construct (pGBT-RTG2) by itself did not transactive UASG-LacZ reporter gene expression in wild-type cells (Table I, part A). Second, in a standard two-hybrid experiment testing protein-protein interactions (29), when pGBT-RTG2, encoding the Gal4p DNA binding domain fused to Rtg2p, was co-transformed with either pGAD-RTG1 or pGAD-RTG3 encoding, respectively, Rtg1p or Rtg3p fused to the Gal4p activation domain, no increase in reporter gene expression was detected. This result indicates that there is no direct interaction between Rtg2p and either Rtg1p or Rtg3p.
|
The results of the previous
experiments suggest that, for transactivation of the
UASG-LacZ reporter gene, either there is a
necessary interaction between Rtg1p and Rtg3p, or Rtg3p by itself is
sufficient for transcriptional activation. To distinguish between these
possibilities, we constructed a series of chimeric proteins consisting
of the Gal4p DNA binding domain fused to various domains of Rtg3p,
including the full-length protein (Fig. 1B). These
constructs were then tested for their ability to transactivate
expression of the UASG -LacZ reporter gene in
wild-type and mutant SFY526 cells. Fig. 3
shows that the full-length Gal4-Rtg3p chimera, p3GB1-486 (Fig.
1B), could transactivate in wild-type cells. Unlike
Gal4-Rtg1p, however, transactivation did not show a typical retrograde
response of higher activity in ° cells. Moreover, Gal4-Rtg3p could
transactivate in all three
rtg mutant backgrounds as well
as, or better than, in wild-type cells. Except for expression in
rtg1 cells, activity was lower in the
° petite than
in
+ cells. These results are in sharp contrast to
Gal4-Rtg1p transactivation, which requires both Rtg2p and Rtg3p and
which consistently shows a greater activity in
° cells. From these
experiments, we conclude that Rtg3p itself contains one or more domains
responsible for transcriptional activation, but these are not by
themselves responsive to retrograde control.
Rtg3p Contains Independent Amino- and Carboxyl-terminal Activation Domains
To define the activation domain(s) of Rtg3p, we
constructed a series of Gal4-Rtg3p fusions linking different portions
of the protein to the DNA binding domain of Gal4p (Fig. 1B).
These constructs were then tested for activity in + and
° derivatives of strain SFY526. The results of these experiments (Fig. 4) show, first, that there are at
least two independent activation domains in Rtg3p, a strong
COOH-terminal domain (amino acids 375-486) and a weaker
NH2-terminal domain (amino acids 1-175). Second, neither
of these domains contain the HLH/Zip protein dimerization motif,
indicating that transactivation does not require an HLH/Zip-mediated protein-protein interaction. Although there was somewhat greater activity in construct pGB300-486 containing the HLH/Zip domain, these
results, nevertheless, contrast sharply with those obtained with the
Gal4-Rtg1p fusions, where the HLH/Zip domain was found to be essential
for activity in UASG-LacZ reporter gene
expression (24).
A third significant outcome of these experiments is that the region of
Rtg3p from amino acids 176 to 282 was not only inactive in supporting
reporter gene expression but also appeared to be inhibitory to the
activity of the NH2- and COOH-terminal activation domains.
For example, construct p3GB1-282 was less active than construct
p3GB1-175, and construct p3GB176-286 was considerably less active
than p3GB300-486 or p3GB375-486 (Figs. 1B and 4). It is
noteworthy that the inactive, and apparently inhibitory, domain of
Rtg3p from amino acids 176-282 is very rich in serine and threonine
residues, and thus may be potentially important as phosphorylation
sites in the protein. Finally, as is the case with the full-length
fusion construct, all of the active, truncated Gal4-Rtg3p fusions were
also active in rtg1,
rtg2, and
rtg3 cells (data not shown). From these data, we conclude
that Rtg3p contains NH2- and COOH-terminal transactivation
domains, which, when directed to the Gal4p DNA binding site, do not
require the bHLH/Zip dimerization domain or the products of the two
other known RTG genes for activity.
The
findings presented thus far suggest that Rtg3p is the functional
transactivator of the Rtg1p-Rtg3p complex. Hence, reporter gene
expression mediated by Gal4-Rtg1p would require recruitment of Rtg3p
and interaction of these proteins via their bHLH/Zip motifs.
Gal4-Rtg3p, however, which contains independent transactivation domains, would not require Rtg1p for transactivation of reporter gene
expression from a UASG element. What then is the function of Rtg2p in transactivation? Rtg2p has no known DNA binding motifs, but
it does contain an hsp70-like ATP binding domain (18) and has some
properties of a heat shock protein and molecular
chaperone,2 such as a peptide-stimulated ATPase activity
(30). Thus, Rtg2p might function to promote a direct interaction
between Rtg1p and Rtg3p. To examine whether Rtg1p and Rtg3p can
interact in the absence of Rtg2p when both proteins are targeted to the
nucleus via plasmid-encoded heterologous nuclear localization signals, we used the construct, p2H26-177, which fuses the Gal4p DNA binding domain to Rtg1p, in a two-hybrid assay together with a second plasmid,
pGAD-RTG3, encoding full-length Rtg3p linked to the Gal4p activation
domain. The two constructs, p2H26-177 and pGAD-RTG3, were examined
singly (along with the control plasmids) and in combination for their
ability to transactivate the UASG-LacZ reporter gene in + and
° derivatives of wild-type,
rtg3, and
rtg2 SFY526 cells.
Table I, part B, shows that, as observed
previously (24), the construct encoding the Gal4p DNA binding domain
fused to Rtg1p (p2H26-177) was able to transactivate the reporter gene in wild-type SFY526 cells in a retrograde-responsive manner. By contrast, no transactivation was observed in cells transformed with
pGAD-RTG3, which encodes the Gal4p activation domain fused to Rtg3p.
However, in wild-type cells transformed with both p2H26-177 and
pGAD-RTG3, a high level of transactivation was observed that was
2-3.6-fold greater than with p2H26-177 alone. In rtg3
cells, neither p2H26-177 or pGAD-RTG3 alone showed any activity, but activity was observed in cells transformed with both plasmids, indicating an interaction between Rtg1p and Rtg3p. In
rtg2 cells, some transactivation by p2H-177 was observed
but the activity was only a fraction (10% or less) of that seen in
wild-type cells. Importantly, strong transactivation was observed in
rtg2 cells containing both p2H26-177 and pGAD-RTG3,
indicating that Rtg2p is not required for the direct interaction
between Rtg1p and Rtg3p. These findings are consistent with previous
observations that an Rtg1p-Rtg3p dependent complex is formed in
electrophoretic mobility shift assays of the 76-bp CIT2
UASr using whole cell extracts from a
rtg2
strain (11) and that recombinant Rtg1p and Rtg3p can interact and bind
to their target DNA sites in vitro (13). Finally, it is
interesting to note that the response of the p2H26-177/pGAD-RTG3 pair
in all cases showed an elevated expression in
° cells, suggesting
that some component of the retrograde response is post-transcriptional,
since both proteins were over expressed from constitutive
promoters.
Previous experiments showed that the
level of Rtg1p detected by Western blot analysis does not differ
significantly between + and
° cells (24),
suggesting that the CIT2 retrograde response is unlikely to
be controlled by a change in the level of Rtg1p. We have not been able
to determine whether the CIT2 retrograde response might be
controlled by changes in the level of Rtg3p because it is a very low
abundance protein both in
+ and
° cells (data not
shown). We have been able to detect the Rtg3p mRNA by RNase
protection experiments, however, and observed that
° cells contain
about twice as much Rtg3p mRNA as
+ cells (13).
To gain some insight into whether Rtg3p might be limiting in the
retrograde response, we determined the effect of overexpression of
Rtg3p on UASG-LacZ reporter gene expression in
cells containing the Gal4-Rtg1p fusion protein. Both +
and
° derivatives of wild-type and
rtg2 SFY526 cells
were transformed with p2H26-177, and either p181-RTG3, a 2µ-based
multicopy plasmid containing the full-length RTG3 gene under
control of its own promoter or, as a control, the empty vector,
yplac181. Table II shows that in the
presence of the control plasmid, pGBT9, 181-RTG3 does not transactivate
in either
+ or
° wild-type SFY526 cells or in the
SFY526
rtg2 derivative. As expected, the Gal4-Rtg1p fusion
protein encoded by p2H26-177 transactivated in the presence of the
control plasmid, yplac181, in wild-type SFY526 cells but not in the
rtg2 derivative. When Rtg3p was overexpressed in the
transformants containing 181-RTG3, the Gal4-Rtg1p-mediated
transactivation was increased 2.8-fold in
+ cells and
1.9-fold in
° cells of the wild-type SFY526 strain. These data
suggest that Rtg3p is limiting both in
+ and
°
cells for Gal4-Rtg1p-mediated transactivation of reporter gene
expression. Table II also shows that cells overexpressing Rtg3p
(p181-RTG3) can overcome the failure of Gal4-Rtg1p to mediate transactivation in
rtg2 cells, suggesting that Rtg2p is
not required per se for Rtg3p function. These findings raise
the possibility that Rtg2p is required to make Rtg3p available for
interaction with Rtg1p.
|
The preceding experiments demonstrate that
increasing expression of RTG3 can increase the activity of a
Gal4-Rtg1p/Rtg3p complex directed to the Gal4p DNA binding sites in the
UASG. They do not, however, address whether increasing
levels of Rtg3p can increase transcription from the CIT2
UASr containing the natural DNA target sites for the
Rtg1p-Rtg3p complex. To examine that point, as well as to determine
whether overexpression of RTG1 also affects transcription from the CIT2 promoter, we transformed wild-type COP161U7
cells with the RTG1 or RTG3 genes on either a
centromere-based (pRS426) or 2µ-based multicopy (yplac195) plasmids.
In all cases, expression of RTG1 and RTG3 was
under control of their own promoters. Total cellular RNA was isolated
from log-phase cultures grown on YNBR + casamino acids medium and
analyzed by Northern blotting to determine CIT2 expression
relative to an internal control for actin mRNA (Fig.
5). The data were quantified by
PhosphorImager analysis, and the relative amount of CIT2
mRNA, normalized to the level of actin mRNA, is present below
each lane.
In this series of experiments, the retrograde response for
CIT2 expression (°/
+) was 5.6 (Fig. 5,
lanes 1 and 2). In
+ cells,
overexpression of Rtg1p from either the centromere or 2µ-based
plasmid resulted in a less than 3-fold increase in CIT2 mRNA abundance and little or no increase in
° cells
(lanes 3-6). There was a somewhat larger increase in
CIT2 mRNA when RTG3 was overexpressed in
+ cells from the centromere plasmid (lane 7),
and nearly a 10-fold increase in when RTG3 was overexpressed
from the 2µ-based plasmid (lane 9). In
° cells,
overexpressing RTG1 or RTG3 from either vector
led only to a relatively small increase in CIT2 expression above the control cells (compare lane 2 with lanes
4, 6, 8, and 10). These data
suggest, first, that the retrograde response is effectively limited by
Rtg3p and less so by Rtg1p, and second, that CIT2
transcription in
° cells is close to the limit of expression that
is regulated by the Rtg1p-Rtg3p heterodimeric complex.
Transcription of the CIT2 gene requires the bHLH/Zip proteins, Rtg1p and Rtg3p, which bind as heterodimers to two identical, non-E box target sites (R boxes, GGTCAC) in the UASr element of the CIT2 promoter (13); neither protein alone is able to bind to the R box in vitro or to activate CIT2 transcription in vivo. CIT2 transcription also requires Rtg2p, a protein with no obvious DNA binding motifs, that is related to the hsp70 family of heat shock proteins (18).2 Based on two-hybrid analysis carried out in the current study, we conclude that Rtg2p neither binds to Rtg1p or Rtg3p nor is required for the direct interaction of these transcription factors with each other.
Transactivation by Rtg3pTo understand better how the Rtg
proteins function both in basal and retrograde regulated expression of
the CIT2 gene, we have extended our previous analysis of the
transcriptional activity of fusion proteins between the Gal4p DNA
binding domain and Rtg1p (24) to include a similar analysis of
Gal4-Rtg3p chimeras; these were analyzed in wild-type +
and
° cells and in different rtg mutant backgrounds.
Such fusions allow an assessment of the transcriptional activity of
Rtg3p and its various domains without requiring DNA binding to
endogenous target sites. Our experiments show that Gal4-Rtg1p fusion
proteins require Rtg3p and Rtg2p to activate transcription of a
LacZ reporter gene. By contrast, Gal4-Rtg3p fusion proteins
can transactivate reporter gene expression in the absence of either
Rtg1p or Rtg2p. These findings suggest that transcriptional activation
by the Rtg1p-Rtg3p complex is largely the responsibility of Rtg3p.
Although a number of bHLH proteins can interact with target DNA sites
as homo- or heterodimers, neither Rtg1p or Rtg3p alone binds to an R
box DNA target site (13); therefore, one function of the constitutively expressed Rtg1p may be to position the transactivator, Rtg3p, at the R
box site by formation of a heterodimer capable of binding DNA. This
strategy would conform to well documented examples of transcriptional
activation or repression determined by specific heterodimeric complexes
between bHLH/Zip proteins, such as the Myc-Max and Mad-Max heterodimers
(31-33).
From the analysis of a series of constructs of fusions of the Gal4p DNA binding domain to regions of Rtg3p, we identified two transactivation domains, one within the NH2-terminal region from amino acids 1 to 175 and the other within the COOH-terminal region from amino acids 375 to 486. This organization is similar to that of the skeletal muscle specific bHLH transcription factor, myogenin, which also has NH2- and COOH-terminal transactivation domains (34). It is important to note that neither of the Rtg3p activation domains contain the bHLH/Zip protein dimerization motif. This finding is consistent with the observation that Gal4-Rtg3p can activate transcription in the absence of Rtg1p. Conversely, transactivation by Gal4-Rtg1p requires both its bHLH/Zip domain (24) and Rtg3p, suggesting that in this one-hybrid assay the Rtg1p bHLH/Zip domain in the Gal4-Rtg1p fusions directs endogenous Rtg3p to the Gal4p DNA binding site for activation of reporter gene expression.
A Potential Regulatory Domain of Rtg3pA central domain of Rtg3p from amino acids 176 to 282 that was tested for transactivation was not only inactive but also inhibitory to activation by other domains; constructs lacking that central domain had significantly more activity than the constructs containing it (for example, p3GB1-282 versus p3GB1-175 shown in Fig. 4). This apparently inhibitory domain of Rtg3p is very rich in serine and threonine residues and thus could contain potential phosphorylation sites that might play a role in regulating the activity of the Rtg1p-Rtg3p complex. That phosphorylation can play a role in regulating the activity of bHLH proteins is suggested from studies showing that there is a dimerization-dependent phosphorylation of the muscle-specific bHLH transcription factors myogenin (35) and MyoD (36). The potential for a direct regulatory role of this phosphorylation is suggested by the finding that mutations of the dimerization-dependent phosphorylation sites enhance the transcriptional activity of myogenin heterodimeric complexes (35). It will be of interest to learn whether the serine/threonine-rich region of Rtg3p is a target for phosphorylation of the protein that might affect the transcriptional activity of the Rtg1p-Rtg3p complex.
The dioxin receptor, also known as the aryl hydrocarbon receptor, is another example of a bHLH protein with a regulatory domain that may bear on the activity of the Rtg1p-Rtg3p complex. The dioxin receptor is a ligand-responsive member of the bHLH/PAS family, which forms a heterodimer with the bHLH/PAS protein, Arnt, and binds to specific xenobiotic-responsive elements regulating transcription of genes involved in drug metabolism (37-40). The dioxin receptor has been shown to have a strong COOH-terminal transactivation domain, which is attenuated by a central 82-amino acid ligand binding domain of the protein (41). It is interesting that this inhibitory domain of the dioxin receptor is a site where a regulator molecule, an hsp90, interacts to prevent dimerization with Arnt (42, 43).
We suggest that Rtg3p, which is a low abundance protein both in
+ and
° cells, is recruited to the R box through
its interaction with the more abundant and constitutively expressed
Rtg1p. Although the details of how the complex contacts the R box sites
remain to be established, it is likely that Rtg3p interacts with the CA
dinucleotide of the R box because the basic region of the Rtg3p bHLH/Zip motif contains the critical conserved amino acid residues, such as an Arg, His, and Glu, with characteristic spacing that have
been shown for other bHLH proteins to make specific contacts with the
CA dinucleotide of the CANNTG E box half-site in the major groove of
DNA (19, 21, 44). Those key amino acid residues are not present as such
in the truncated basic domain of Rtg1p. It is also significant that the
Rtg1p-Rtg3p complex does not bind in vitro to common E box
sites that were tested under conditions where robust binding was
observed to the R box (13). Thus, the unique structural characteristics
of Rtg1p may be responsible for determining the unusual DNA binding
specificity of this bHLH/Zip complex.
What is the role of Rtg2p in the transcriptional process mediated by
Rtg1p-Rtg3? Since overexpression of Rtg3p can elevate target gene
expression in + cells, it is conceivable that Rtg2p
could function to modulate the expression of RTG3. This is
unlikely, however, for a number of reasons. First, we have not detected
any effect of an rtg2 null allele on the level of expression
of RTG3 mRNA (data not shown). Second, the abundance of
RTG3 mRNA does not vary by more than 2-fold between
+ and
° cells (13). And finally, electrophoretic
mobility shift assays using whole cell extracts from a
rtg2 mutant strain, in which there is no CIT2
expression, yield the same Rtg1p-Rtg3p-dependent complex
with a CIT2 UASr probe as do extracts from
wild-type cells (11, 13). An alternative possibly suggested by our data
is that in
+ cells, Rtg3p is sequestered in an inactive
form by one or more proteins, and that Rtg2p functions to regulate the
availability of Rtg3p for transcriptional activation. This would be
analogous to the sequestration of transcription factors such as NF-
B
by I-
B (reviewed by Verma et al. (45)), the E protein
family of bHLH proteins by Id (46) and the bHLH/PAS dioxin-responsive and related Sim family of transcription factors by hsp90 (43, 47,
48).
Overexpressing Rtg3p in + cells, in which the majority
of the endogenous protein might be sequestered in an inactive form, could result in an increase in gene expression by effectively titrating
out the protein(s) responsible for the sequestration. The finding that
overexpression of Rtg1p also resulted in some increase in
CIT2 expression in
+ cells, but to a lesser
extent than was observed with Rtg3p, could be accounted for by a
competition for the free and sequestered forms of Rtg3p. The presence
of an hsp70-like ATP binding domain in Rtg2p (18) and observations that
Rtg2p has the properties of a heat shock protein2 raise the
possibility that Rtg2p could function as a molecular chaperone to
promote the formation of the Rtg1p-Rtg3p heterodimer for
transcriptional activation.
We thank Stan Fields and Paul Bartel for generous gift of plasmids. We also thank members of the Butow laboratory for many helpful discussions.