From the Department of Plant Biology, Swedish
University of Agricultural Sciences, Uppsala Genetic Center, Box 7080, S-75007 Uppsala, Sweden, § Department of Medical
Biochemistry and Microbiology, Uppsala University, Box 582, S-75123
Uppsala, Sweden, and the
Department of Medical Biochemistry and
Biophysics, Umeå University, S-90187 Umeå, Sweden
Received for publication, August 11, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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It is possible to recruit RNA polymerase II to a
target promoter and, thus, activate transcription by fusing Mediator
subunits to a DNA binding domain. To investigate functional
interactions within Mediator, we have tested such fusions of the lexA
DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type,
med1, med2, gal11,
sin4, srb8, srb10, and
srb11 strains. We found that lexA-Med2 and lexA-Gal11 are
strong activators that are independent of all Mediator subunits tested.
lexA-Srb10 is a weak activator that depends on Srb8 and Srb11.
lexA-Med1 and lexA-Srb7 are both cryptic activators that become active
in the absence of Srb8, Srb10, Srb11, or Sin4. An unexpected finding
was that lexA-VP16 differs from Gal4-VP16 in that it is independent of
the activator binding Mediator module. Both lexA-Med1 and lexA-Srb7 are
stably associated with Med4 and Med8, which suggests that they are
incorporated into Mediator. Med4 and Med8 exist in two mobility forms
that differ in their association with lexA-Med1 and lexA-Srb7. Within purified Mediator, Med4 is present as a phosphorylated lower mobility form. Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8
to their low mobility forms.
RNA polymerase II (pol
II)1 transcribes all
protein-encoding genes and some small nuclear RNA genes in eukaryotes.
The yeast pol II holoenzyme (1) consists of a catalytic core enzyme of 12 subunits (2, 3) whose crystal structure has been solved (4, 5) and a
regulatory Mediator complex comprising 20 subunits. All subunits of the
Mediator have now been identified, and 13 of them are encoded by
previously known genes. The remaining seven subunits are novel proteins
named Med1, Med2, Med4, Med6, Med7, Med8, and Med11 (6-12).
Transcription can be reconstituted in vitro from highly
purified pol II core enzyme and five general transcription factors,
which are the TATA binding protein, TFIIB, TFIIE, TFIIF, and TFIIH.
These proteins are necessary and sufficient for basal transcription
in vitro, but they are unable to respond to transcriptional
activators (13). Mediator was originally defined as an activity in
yeast nuclear extracts that is required for such regulated
transcription (14, 15). In addition, Mediator enhances basal
transcription, stimulates phosphorylation of the C-terminal domain of
the largest pol II subunit by TFIIH, and binds to the C-terminal
domain. The latter interaction is important for the integrity of the
holoenzyme (1).
A partial truncation of the C-terminal domain causes a
temperature-sensitive phenotype (16) that is suppressed by mutations in
the SRB genes. Several of the Srb proteins are present both in Mediator and in another complex that contains the pol II core enzyme
and which also has been referred to as the pol II holoenzyme (17). It
differs from the holoenzyme described above in that it also contains
the Srb8-11 proteins, the SWI/SNF complex, some general transcription
factors, and other yet unidentified proteins (18, 19). It is likely
that this reflects the different purification procedures used and that
additional proteins found only in the second holoenzyme are more
loosely associated with pol II in vivo. In any case, their
absence from the first holoenzyme shows that they are not strictly
required for regulated transcription in vitro. An attempt to
further clarify the in vivo composition of the
holopolymerase was recently made by Hahn and co-workers (20). They
purified Mediator from yeast strains where Srb5, Srb6, or Rgr1 were
epitope-tagged. This allows purification of the holopolymerase without
the use of high ionic strength buffers and should, thus, better
preserve its in vivo composition. Mediator was found to be
present in two major forms of 1.9 and 0.55 MDa, respectively. The
larger complex is identical to the holopolymerase described by Kornberg
and coworkers (1, 14, 15), except that it also contains the
Srb8-11 module. The smaller complex is similar to free Mediator (9) in
that it lacks the Srb8-11 module, but it also lacks Nut1, Gal11, Rgr1,
Sin4, Med2, Pgd1, and Rox3. Notably, several of these proteins have
been proposed to form a distinct activator binding tail module
(21-23), which may be more loosely associated with the rest of the holoenzyme.
The Srb8-11 module, which contains the cyclin C-dependent
kinase, has been implicated in negative control of gene expression. Thus, SRB8-11 appeared in several genetic screens for genes
involved in repression (24). Because TUP1 and
CYC8 also appeared in the same genetic screens, it has been
suggested that Srb8-11 may be involved in transmitting repressive
signals from the Cyc8-Tup1 co-repressor complex to pol II. However,
other Mediator subunits such as Pgd1 and Srb7 (25, 26) have also been
proposed to be targets of Cyc8-Tup1. It should be emphasized that
mutations in the Srb8-11 module have a complex phenotype that also
includes reduced transcription of some highly expressed genes (6,
10).
Fusions between general transcription factors and DNA binding domains
can activate transcription from promoters that bind such fusion
proteins. An activation-by-recruitment model, which suggests that
physical recruitment of the basal RNA polymerase II machinery to a
promoter may be sufficient to activate transcription, has therefore
been proposed (27-29). Hybrid activators that contain parts of general
transcription factors or holoenzyme subunits have been called
non-classical activators, as opposed to classical activators with
conventional activation domains (30). Several Mediator subunits,
i.e. Med6, Gal11, and Sin4, have been shown to function as
non-classical activators (21, 30, 31). Srb2, Srb4, Srb5, and Srb6 can
activate transcription as lexA fusions but depend on the promoter
context for their ability to do so (30). A lexA-Srb11 fusion could
activate transcription (32), whereas a lexA-Srb10 fusion required
either overexpression of Srb11 or a mutation in the kinase active site
(33). Finally, we have found that lexA-Med1 is a cryptic activator that
becomes active only in the absence of Srb11 or if it is overexpressed (10).
In the present investigation, we have used activation by recruitment as
a tool to study functional interactions between different Mediator
subunits. We find that the non-classical activators differ in their
dependencies on other proteins. Thus, lexA-Med2 and lexA-Gal11 are
strong activators that function in all mutants tested. lexA-Srb10 is a
weak activator that is dependent on Srb11 and Srb8. lexA-Med1 and
lexA-Srb7, finally, are cryptic activators that function only in the
absence of either the Srb8-11 module or Sin4. The association of
lexA-Med1 and lexA-Srb7 with Mediator was further investigated by
co-immunoprecipitation. We found that both hybrid activators co-precipitate with Med4 and Med8, indicating that they are stably integrated into Mediator. Finally, we found that both Med4 and Med8
exist in two forms with different mobility. Only the low mobility form
of Med4 associates with lexA-Med1 and lexA-Srb7. Both forms of Med8
associate with lexA-Srb7, whereas the low mobility form preferentially
associates with lexA-Med1.
Yeast Strains--
All yeast strains used in this study (Table
I) were W303 congenic (34) and,
therefore, carry the following markers: MATa ade2-1 can1-101 his3-11,15 leu2-3,112 trp1-1 ura3-1. To
generate gal11- Plasmids--
The pDB185, pDB181, pDB198, and pDB223 plasmids
have been described (10, 36). To create pDB326, the lexA
HindIII-ApaI fragment of pDB198 was replaced by
the HindIII-ApaI fragment of pGWwt (37)
containing the Gal4 DNA binding domain. To create the lexA-Med2 fusion,
the MED2 open reading frame from a baculovirus expression
cassette (kindly provided by Claes Gustafsson) was cloned as an
EcoRI-XhoI fragment into
EcoRI-XhoI-digested pEG202, thus producing
pDB212. The whole lexA-Med2 expression cassette was then subcloned as
an SphI fragment into pFL39 (38), thus producing pDB213. The
lexA-Srb7, lexA-Srb10, and lexA-Gal11 fusion plasmids were all made by
cloning BamHI-XhoI-digested PCR products into
BamHI-XhoI-digested pDB223. SRB7 was
amplified by using AGGGATCCCAATGACAGATAGATTAACAC and
ACGTCTGAGTTATGTGCTCTTTTTTGAGTT as primers, SRB10 was
amplified by using the primers CGGGATCCAAATGTATAATGGCAAGGATA and
ACGCTCGAGCTATCTTCTGTTTTTCTTTCG, and GAL11 was amplified by
using the primers AGGGATCCCTATGTCTGCTGCTCCTGTCC and
CGGAATTCTCAAGATGCACTTGTCCAATT. To rule out PCR artifacts in the
complementation experiments, at least four different PCR clones were
tested for each fusion. Plasmids pGVwt and pGVfa, expressing wild type
Gal4-VP16 (39) and the Gal4-VP16-F442A mutant (22), respectively, were
kindly provided by Lawrence Myers. As 2-hybrid lacZ reporter
plasmids, we used pSH18-34 (40) for the lexA fusions, pLGSD5 (22) for
the Gal4 fusions, and pJK101 (41, 42) for both.
Immunoprecipitations and Western Blots--
Yeast cells were
grown to an A600 of 1.2 in 100 ml of yeast
nitrogen base containing 8% glucose and lacking uracil and
tryptophan, harvested by centrifugation at 230 × g for
5 min at 4 °C, washed in water, and resuspended in 0.5 pellet
volumes of 3× yeast lysis buffer (43). The cells were disrupted by
bead-beating at 4 °C (10 × 30 s) using 0.5-mm glass beads
and a Mini-BeadbeaterTM (Biospec products, Bartlesville,
OK). Whole cell extracts were isolated from the supernatant after
centrifugation for 60 min at 21,000 × g and 4 °C.
Total protein (40 µg) from the whole cell extracts of each strain was
trichloroacetic acid-precipitated, separated by SDS-PAGE, and analyzed
by Western blotting using antibodies specific for Med1, Med4, or Med8.
For immunoprecipitation, cell extracts corresponding to 1 mg of total
protein were diluted to 500 µl in yeast lysis buffer and precleared
with 30 µl of protein A-agarose (Roche Diagnostics) by rotation for
2 h at 4 °C. One additional tube for each lysate containing 500 µl of yeast lysis buffer and 5 µl of mouse monoclonal antibodies
specific for the DNA binding domain of lexA (Santa Cruz Biotechnology,
Santa Cruz, CA) was rotated for 2 h at 4 °C. The antibody-bound
beads were then washed with 1 ml of yeast lysis buffer. The precleared
lysates were centrifuged for 2 min at 6000 × g, and
the supernatants were added to the washed, antibody-bound beads. After
a 2-h rotation at 4 °C, the tubes were centrifuged as above, and the
beads were washed 3 times with 500 µl of phosphate-buffered saline
containing 0.8 M potassium acetate and 0.5% Nonidet P-40
and once with 500 µl of phosphate-buffered saline. The beads were
dissolved in an equal volume of 2× SDS-loading buffer and boiled for
10 min. Aliquots corresponding to 200 µg of total protein (before
immunoprecipitation) were separated on SDS-PAGE, transferred to
polyvinylidene difluoride membranes, and analyzed by Western blotting
as described above. The polyclonal rabbit antisera raised against Med1,
Med4, and Med8 have been previously described (9, 10).
Other Methods--
Treatment with calf intestine phosphatase was
performed as previously described (44). Briefly, 5 µl of purified
holo-RNA polymerase II was incubated at 37 °C for 1 h in the
presence or absence of calf intestine phosphatase (40 units). Holo-RNA
polymerase II for dephosphorylation assays was purified from a yeast
strain where the RGR1 gene encoding a Mediator subunit was
fused to the tandem affinity purification tag (45). The purification
was performed exactly as described previously (46). lexA-Med1, lexA-Med2, and lexA-Srb7 Can Functionally Replace the
Corresponding Wild Type Proteins--
We have previously shown that
the Med1 protein becomes a cryptic activator when it is fused to the
lexA DNA binding domain (10). To test how general a phenomenon this is
and to study functional interactions between different Mediator
subunits, we have now made lexA fusions to Med2, Gal11, Srb7, and
Srb10. As a first step in characterizing these new fusion proteins, we
attempted to determine if they could complement deletions of the
corresponding wild type genes. This is an important question because
non-classical activators are thought to function by being incorporated
into the holoenzyme in place of the wild type protein, thereby
recruiting pol II to the target promoter. However, this has to the best
of our knowledge never been proven experimentally. Complementation of a
deletion mutation would provide strong evidence that a fusion protein
can replace the wild type protein within the holoenzyme. We found that
lexA-Med1, lexA-Med2, and lexA-Srb7 are all able to complement
deletions of the corresponding genes (Fig.
1). In contrast, lexA-Gal11 and
lexA-Srb10 failed to do so. It should be noted that whereas
complementation provides evidence of functional replacement, lack of
complementation does not necessarily mean that a fusion protein cannot
be incorporated into the holoenzyme. It may also reflect steric
hindrances that prevent the incorporated protein from functioning
properly. We conclude that complementation supports the notion that
lexA-Med1, lexA-Med2, and lexA-Srb7 are incorporated into the pol II
holoenzyme, whereas the presence of lexA-Gal11 and lexA-Srb10 within
the holoenzyme must be ascertained by other means.
lexA-Med2 and lexA-Gal11 Are Strong Activators That Are Independent
of All Mediator Subunits Tested--
We proceeded to test the ability
of lexA-Med1, lexA-Med2, lexA-Gal11, lexA-Srb7, and lexA-Srb10 to
activate transcription both in wild type cells and in med1,
med2, gal11, sin4, srb8, srb10, and srb11 deletion strains (Table
II). We found that the lexA fusions fall
into three distinct groups with respect to how they respond to
mutations in other Mediator subunits. lexA-Med2 and lexA-Gal11 comprise
the first group and behave similarly to the classical activator
lexA-VP16, which we included as a control in these experiments. Thus,
lexA-Med2 and lexA-Gal11 are strong activators comparable in strength
to lexA-VP16 both in the wild type and in all mutant strains tested.
One difference between the two activators is seen in the
gal11 strain, where the activity of lexA-Gal11 is enhanced,
whereas the activity of lexA-Med2 is unaffected. Conceivably, this
could be due to increased incorporation of lexA-Gal11 into the Mediator
in the absence of wild type Gal11 protein. In conclusion, our results
show that both lexA-Med2 and lexA-Gal11 can function as activators in
the absence of all other Mediator subunits tested. It should be noted
that our finding that lexA-VP16 is independent of Med2, Gal11, and Sin4
was unexpected and is further discussed below.
lexA-Srb10 Is a Weak Activator That Requires Srb11 and Srb8--
A
second kind of result was obtained with the lexA-Srb10 fusion. We found
that it is a weak activator in all strains except in the
srb11 mutant, where it is completely inactive, and in the srb8 mutant, where its activity is reduced (Table II). These
findings suggest that the integrity of the Srb8-11 module is important for the ability of lexA-Srb10 to recruit the holopolymerase. This may
reflect the fact that cyclin-dependent kinases such as
Srb10 usually depend on their cyclins (in this case Srb11) both for activity and for interaction with the correct target protein (47, 48).
Accordingly, the ability of Srb10 to activate by recruitment would
suggest that it has at least one target within the pol II holoenzyme.
The rather low level of activity for lexA-Srb10 compared with other
fusions may result from a more loose association between the Srb8-11
module and Mediator. Alternatively, the inherent repressing activity of
the Srb8-11 module interferes with the ability of lexA-Srb10 to
activate transcription.
lexA-Med1 and lexA-Srb7 Are Cryptic Activators That Become Active
in srb8, srb10, srb11, and sin4 Mutants--
A third type of result
was obtained with lexA-Med1 and lexA-Srb7. We have previously shown
that lexA-Med1 is a cryptic activator that has little or no activity in
wild type cells but becomes active in srb11 cells (10). Here
we show that deletions of SRB8, SRB10, or
SIN4 have a similar effect (Table II). In contrast, deletions of MED1, GAL11, or MED2 have
no effect on lexA-Med1, which remains inactive in these strains. We
conclude that the cryptic activity of lexA-Med1 can be unmasked in at
least three ways, by mutations in the cyclin C-dependent
protein kinase complex (Srb8-11), by mutations in Sin4, and by
overexpression of lexA-Med1 itself (10). This suggests a possible
functional link between the cyclin C-dependent protein
kinase and Sin4.
Surprisingly, we found that lexA-Srb7 also is a cryptic activator that
behaves similarly to lexA-Med1. Thus, it has no activity in the wild
type, whereas deletions of SRB8, SRB10,
SRB11, and SIN4 all cause it to become active
(Table II). However, lexA-Srb7 differs from lexA-Med1 in three ways.
First, its activity when unmasked by the srb8,
srb10, or srb11 deletions is about 8-fold lower
than that of lexA-Med1. Second, the sin4 deletion has a much
stronger effect on lexA-Srb7 compared with the srb8,
srb10, and srb11 deletions (Table II). Third and
most important, we found that a deletion of MED1 activates
lexA-Srb7. This suggests that Med1 may be involved in transmitting the
negative effect that the Srb8-11 module exerts on lexA-Srb7 (see
"Discussion"). The fact that a similar effect was not observed for
lexA-Med1 is most likely due to the ability of lexA-Med1 itself to
complement the med1 deletion, thus making it phenotypically
wild type.
lexA-VP16 Is Independent of the Activator Binding Module--
Our
finding that the classical activator lexA-VP16 is independent of all
Mediator subunits tested, including Med2, Pgd1, Gal11, and Sin4 (Table
II), was unexpected for two reasons. First, in vitro
experiments suggest that Gal11 is required for activation by Gal4-VP16
(21). Second, Myers et al. (22) find that the in
vivo activity of Gal4-VP16 is reduced 8-fold in med2
cells. It has, therefore, been suggested that the VP16-activating
domain is dependent on the activator binding tail module of the
Mediator, which includes Med2, Gal11, and Sin4. Because different
strains and plasmids were used in the two experiments, we obtained the plasmids used by Myers et al. (22) and tested them in
our own strains. We could confirm that a significant difference exists between Gal4-VP16 and lexA-VP16, with the former but not the latter dependent on Med2. In fact, we see an even larger effect than Myers
et al. (22), with a more than 100-fold reduction of
Gal4-VP16 activity in the med2 strain using the Gal4
binding reporter pLGSD5 (Table
III). We also tested the effect of
sin4 and gal11. We found a 14-fold reduction in
Gal4-VP16 activity in the sin4 strain and a 3-fold reduction
in the gal11 strain. Neither deletion has a significant
effect on lexA-VP16 (Table II). To rule out that the observed
differences are due to different reporters being used with lexA-VP16
and Gal4-VP16, we proceeded to test the activators with a reporter
(pJK101) that has both Gal4 and lexA binding sites (41, 42). The
results were similar, with Gal4-VP16 but not lexA-VP16 dependent on
Med2, Sin4, and, to some extent, also on Gal11 (Table III). We conclude
that Gal4-VP16 is dependent on Med2, Sin4, and Gal11, whereas lexA-VP16
shows no such dependencies. This suggests that the dependence of
Gal4-VP16 on the activator binding module may be mediated by the Gal4
DNA binding domain rather than by the VP16 activation domain.
The Dependence of Gal4-VP16 on the Activator Binding Module Is
Strongly Affected by the Gal4-VP16 Copy Number--
One further
difference in our experiments was that lexA-VP16 is expressed from a
single copy plasmid, whereas Gal4-VP16 is expressed from a high copy
number plasmid. We therefore proceeded to express Gal4-VP16 from the
single copy plasmid pDB326 and test it with both the pLGDSD5 and pJK101
reporters (Table III). We found that a reduced copy number for
Gal4-VP16 strongly affects both its activity in the wild type strain
and its dependence on subunits within the activator binding module.
Furthermore, quite different effects were seen for different subunits.
Thus, the activity in the wild type strain and in the gal11
mutant both drop 20-40-fold as compared with Gal4-VP16 on a high copy
number plasmid (Table III). In contrast, the residual activity in the
med2 mutant (11 and 64 units, respectively, for the 2 reporters) is unaffected by the Gal4-VP16 copy number. A third kind of
result was seen in the sin4 mutant, where the activity
actually increases 2-3-fold as compared with Gal4-VP16 on a high copy
number plasmid (Table III). These effects are statistically
significant; moreover, the same pattern was seen with both reporters.
In a final experiment, we decided to determine if the copy number
effects could be reproduced by reducing the level of Gal4-VP16 activity
through other means. For this, we used the attenuated activation domain
mutant Gal4-VP16-F442A (22). This mutant, when expressed from a high
copy number plasmid, has a 2-4-fold reduced activity in wild type
cells (Table III). We saw a similar 2-4-fold drop in activity also in
the med2, gal11, and sin4 mutants.
This is consistent with the findings of Myers et al. (22)
regarding med2 and sin4, although the effects on both Gal4-VP16 and Gal4-VP16-F442A activity were smaller in that case.
We conclude that reducing the level of Gal4-VP16 activity through an
attenuating mutation does not affect its dependence on Med2, Gal11, or Sin4.
Effects on the ADH1 Promoter in Mediator Mutants--
We have
previously noted that the amount of lexA-Med1 protein that can be
detected in Western blots increases 3-fold in strains lacking Srb11
(10). This raised the question if deletions of other Mediator subunits
would have a similar effect. We therefore transformed the deletion
strains with a reporter where the lacZ gene is expressed
from the same promoter (ADH1) that was used to express the
lexA fusion proteins. We found that all Mediator deletions tested cause
an increase in lacZ expression ranging from 2-fold in the
gal11 strains to 9-fold in the sin4 strain (Table
II). A med2 srb11 double deletion did not produce a stronger effect than either deletion alone (see below). We conclude that expression from the ADH1 promoter is increased in strains
where the integrity of the Mediator is disturbed. This effect is too small to explain the 20-80-fold increase in lexA-Med1 or lexA-Srb7 activity that is seen in some of the mutants. Furthermore, the effects
on the ADH1 promoter is seen also in the med2
mutant that has no effect on either lexA-Med1 or lexA-Srb7 (Table II).
Nevertheless, this effect should be kept in mind when interpreting the
lexA-Med1 and lexA-Srb7 results.
Med2 Is Required for Activation by lexA-Med1 and lexA-Srb7 in srb11
Cells--
Although lexA-Med1 fails to activate transcription when
expressed from a single copy plasmid in wild type cells, overexpression from a high copy number plasmid causes it to become active (10). The
activity achieved in this case is comparable with that seen with a
single copy lexA-Med1 plasmid in srb11 mutant cells.
Interestingly, this activity is reduced more than 10-fold in a
med2 strain that, together with our previous results that
Med2 is absent from Mediator in cells lacking Med1, indicate an
interaction between these two proteins (10). To determine if this
requirement also exists when lexA-Med1 is activated by an
srb11 mutation, we assayed its activity in a med2
srb11 double mutant strain. We found that the lexA-Med1 fusion is
inactive (1.7 ± 0.3 units) in the double mutant strain. This is
probably not caused by a failure to express lexA-Med1, since we found
that expression of the Both lexA-Med1 and lexA-Srb7 Are Stably Associated with Med4 and
Med8 in Wild Type and Mutant Cells--
To find out more about the
mechanism(s) of action of the two cryptic activators, lexA-Med1 and
lexA-Srb7, we decided to investigate to what extent they are associated
with other subunits within the Mediator. Extracts were prepared from
wild type cells expressing either lexA-Med1 or lexA-Srb7 and
precipitated with monoclonal antibodies specific for the lexA DNA
binding domain. Precipitated proteins were separated by SDS-PAGE and
analyzed by Western blots using antisera against Med4 and Med8. As a
control, we also included extracts from strains expressing lexA-VP16.
Our assumption was that lexA-VP16 would function as a classical
activator by forming transient interactions with the Mediator.
Consistent with this, we found that although lexA-VP16 itself was
easily detected in the precipitate, there was no evidence of Med4 or
Med8 being co-precipitated with lexA-VP16 in any of the strains (data
not shown). We conclude that any interactions that occur between
lexA-VP16 and Mediator are either too transient or too weak to be
detected in our co-immunoprecipitation experiments.
In contrast, we found that both Med4 and Med8 co-precipitate with
lexA-Med1 in wild type cells (Fig. 2).
Similarly, Med4 and Med8 also co-precipitate with lexA-Srb7 (Fig.
3). This suggests that both cryptic
activators are more stably associated with Mediator than lexA-VP16 and
is consistent with the notion that these non-classical activators
function by being incorporated into the holoenzyme, thereby taking the
place of the wild type protein. The association of lexA-Med1 and
lexA-Srb7 with Med4 and Med8 is independent of all the other Mediator
subunits tested, since it was seen also in the med1,
med2, gal11, sin4, srb8,
srb10, and srb11 strains (Figs. 2 and 3). This
suggests that none of these subunits is required for assembly of the
middle and head regions of the Mediator, where Med1, Srb7, Med4, and
Med8 are found.
Two Distinct Forms of Med4 and Med8 Differ in Their Associations
with lexA-Med1 and lexA-Srb7--
Interestingly, we found that both
Med4 and Med8 give rise to doublet bands in the Western blots. This is
most clearly seen in the blots with whole cell extracts (Figs. 2 and
3). It further seems that the upper (low mobility) band is slightly
more abundant for Med4, whereas the lower (high mobility) band is
somewhat more pronounced for Med8. In the case of Med4, others have
already noted the presence of two distinct bands in protein gels. In
fact, the two bands were named Med4 and Med5 before sequencing revealed that they are that same polypeptide (9). It is noteworthy that the two
forms of Med4 and Med8 differ in their degree of association with
lexA-Med1 and lexA-Srb7. Thus, the two fusion proteins co-precipitate mainly with the low mobility form of Med4 (Figs. 2 and 3). For Med8,
the picture is more complex. Thus, lexA-Med1 associates preferentially
with the low mobility form of Med8 (Fig. 2), whereas lexA-Srb7
associates with both forms of Med8 (Fig. 3). These findings suggest
that assembly of Mediator is affected by the modification status of
Med4 and Med8. Furthermore, the fact that the high mobility form of
Med8 associates with lexA-Srb7 but only weakly or not at all with
lexA-Med1 further suggests that assembly of the Mediator could be a
multistep process (see "Discussion").
Med4 Is Phosphorylated When Present in Purified
Mediator--
Multiple bands in protein gels are frequently caused by
differential phosphorylation. We therefore proceeded to determine if
Med4 and Med8 are phosphorylated in vivo. For this we used a
yeast strain in which the Mediator subunit Rgr1 has been tandem affinity purification-tagged (45), thus allowing rapid isolation of the
holopolymerase by tandem affinity purification (46). The purified
holopolymerase was treated with calf intestine phosphatase, after which
the subunits were separated by SDS-PAGE and probed in a Western blot
with antisera against Med4 and Med8. As shown in Fig.
4, we found that Med4 from purified
holopolymerase preferentially exists as a low mobility form that is
converted into a higher mobility form upon phosphatase treatment. We
conclude that Med4 is phosphorylated when present in Mediator and that
this is a possible reason for the two mobility forms seen in Figs. 2
and 3. In contrast, we saw no evidence that Med8 from purified
holopolymerase is phosphorylated (Fig. 4).
Synthetic Interactions between med2, gal11, and
sin4--
Deletions of MED2, GAL11, and
SIN4 have been reported to produce similar phenotypes,
although some differences were observed for growth on galactose (22).
The differences that we saw between the congenic med2,
gal11, and sin4 strains used in our lexA
experiments, therefore, prompted us to examine their growth phenotypes.
We found that all three strains have a general growth defect that is
most severe in the med2 strain and least severe in the
gal11 strain. All three strains also have a reduced ability
to grow on galactose and gluconeogenic carbon sources such as acetate. However, in this case the effect was most pronounced in the
med2 strain and least pronounced in the sin4
strain. Finally, all three strains are temperature-sensitive at
38 °C, although the gal11 strain can grow on synthetic
media at 38 °C. This may reflect the fact that the gal11
strain is generally healthier than the med2 and
sin4 strains (Fig. 5). We
proceeded to test the sin4 disruption for genetic
interactions with the med2 and gal11 disruptions using tetrad analysis. We found that gal11 sin4 spores grow
more slowly than spores lacking either sin4 or
gal11 alone (Fig. 6), indicating a synthetic interaction between the two disruptions. A
similar interaction was seen between sin4 and
med2 (data not shown).
Partial Suppression of med2, gal11, and sin4 by Overexpression of
PDC1--
The fact that med2 cells are
temperature-sensitive makes it possible to screen for high copy number
suppressors. To this end we transformed a med2 strain with a
high copy number yeast genomic library. The transformed cells were
plated at 35 °C, a temperature at which the med2 strain
still can grow but is unable to form colonies after transformation
using the lithium acetate procedure. Plasmids were rescued from
colonies formed and tested by re-transformation. We found 14 plasmids
that did not contain MED2 itself (Table
IV). Seven contained the PDC1
gene encoding pyruvate decarboxylase (49), and four contained
PDC2, which encodes a transcriptional activator of
PDC1 (50). The remaining three plasmids contained ZDS1 (Zillion Different Screens), a gene that frequently is
found in suppressor screens, regardless of the mutant phenotype (51, 52). We conclude that overexpression of PDC1 either directly or through its activator PDC2 can suppress the inability of
med2 cells to form colonies at 35 °C. Two other
med2 phenotypes, failure to grow on galactose and
temperature sensitivity at 38 °C, were not suppressed by these
plasmids.
We proceeded to test if other mediator mutants also are unable to form
colonies at 35 °C. We found that gal11 and
sin4 deletion mutants display a similar phenotype, although
less severe. In contrast, there was little or no effect on the plating
efficiency in the med1 and srb11 strains. We
further found that all three suppressor genes recovered in the
med2 screen can suppress the reduced plating efficiency of
the gal11 strain, with PDC1 being most efficient
and ZDS1 the least efficient (Table IV). In the case of
sin4, only PDC1 could clearly suppress the
reduced plating efficiency, but the underlying phenotype is weaker in
this case, making detection of suppression more difficult. We conclude
that med2, gal11, and sin4 strains are
similar in that their reduced plating efficiency is suppressed by
overexpression of pyruvate decarboxylase, a finding that suggests a
similar underlying defect in all three strains.
We have used activation by recruitment as a tool to study
functional interactions between different Mediator subunits.
Full-length Med1, Med2, Gal11, Srb7, and Srb10 proteins were fused to
the lexA DNA binding domain and tested for their ability to activate transcription in wild type, med1, med2,
gal11, sin4, srb8, srb10, and srb11 strains. As a positive control, we also studied
the effect of these mutations on a lexA-VP16 hybrid activator. Several conclusions can be drawn from our results (Table II).
First, we found that the Mediator subunits included in our study fall
into three distinct groups with respect to their functional interactions. The first group comprises Med2 and Gal11, both of which
are part of the proposed activator binding module (21, 22). They
resemble the classical activator lexA-VP16 in that their lexA fusions
are strong activators that do not depend on any other Mediator subunits
tested. lexA-Gal11 has been proposed to recruit pol II by replacing the
Gal11 wild type protein within the holoenzyme (29). Our finding that
lexA-Med2 can complement a med2 deletion (Fig. 1) makes a
strong argument for this activation by recruitment model in the case of
lexA-Med2. Several observations suggest that the activator binding
module may function primarily to enhance the expression of strongly
expressed genes. Thus, a limited number of genes, all of which are
strongly expressed, depend on Med2 for full expression (22). Similarly,
Gal11 is required for full expression of several strongly expressed
genes, including the GAL genes, which also depend on Med2.
Interestingly, we found that med2, gal11, and
sin4 mutants are partly suppressed by overexpression of
PDC1. This is a highly expressed gene that is important for
rapid growth on glucose and that is known to be
Med2-dependent (22). Its ability to suppress mutations in the activator binding module when overexpressed reinforces the notion
that the latter is required for high level expression of a limited set
of genes.
Our finding that the classical activator lexA-VP16 is independent of
the activator binding module is surprising given the fact that
Gal4-VP16 does exhibit such a dependence (Ref. 22 and Table III). It
suggests that the dependence could be mediated by the Gal4 part of
Gal4-VP16 rather than by the VP16 part. However, we cannot rule out
that other factors such as different expression levels for the two
hybrid proteins contribute to the observed differences. When we
expressed Gal4-VP16 from a single copy plasmid, its dependencies on the
activator binding module subunits were reduced and in one case (Sin4)
even reversed to a negative effect (Table III). We were unable to do
the opposite experiment, i.e. express lexA-VP16 from a high
copy number plasmid, since this plasmid was deleterious. This may
simply reflect the fact that lexA-VP16 is a much stronger activator
than Gal4-VP16 (cf. pDB198 and pDB326 in Table III). High
level expression of strong activators is known to be deleterious,
probably due to squelching (53). An alternative interpretation of our
results would, therefore, be that lexA-VP16 is independent of the
activator binding module because it is a very strong activator, whereas
the weaker activator Gal4-VP16 needs the module for full activity.
However, this is not consistent with the fact that Gal4-VP16 is less
dependent on the activator binding module when expressed in single copy nor does it fit with the finding that the attenuated Gal4-VP16-F442A mutant is as dependent on the activator binding module as is wild type
Gal4-VP16 (Ref. 9 and Table III). We conclude that the most likely
explanation for the observed differences between lexA-VP16 and
Gal4-VP16 is that the dependence of the latter on the activator binding
module is mediated by its Gal4 part, which is absent in lexA-VP16.
The second kind of result obtained with our fusion proteins is
represented by the cyclin C-dependent protein kinase Srb10. We found that lexA-Srb10 is a weak activator that is dependent on Srb11
(cyclin C) and Srb8 for its activity. Deletions of the SRB8-11 genes have identical phenotypes and show no
synergism with each other (32, 35). This indicates that the four
proteins function closely together and that they all are needed for the in vivo function of the kinase. It has been shown for other
cyclin-dependent kinases that the kinase depends on its
cyclin both for induction of its catalytic activity and for targeting
to the correct substrate, which is mediated by direct interactions
between the substrate and the cyclin (47, 48). It is, therefore, likely
that this is the case also for Srb10. It is not clear what the in
vivo substrate(s) of Srb10 are, although it is one of several
kinases that can phosphorylate the C-terminal domain of RNA polymerase
II. Our finding that lexA-Srb10 is dependent on Srb11 may suggest that
at least one substrate is present within Mediator and that Srb11 is
required for the interaction of Srb10 with this substrate. This would
also be consistent with a previous observation that a lexA-Srb10 fusion
became a stronger activator when Srb11 was overexpressed (32).
A third kind of result was obtained for lexA-Med1 and lexA-Srb7. We
have previously shown that lexA-Med1 is a cryptic activator that lacks
activity in the wild type but becomes active in the absence of Srb11
(10). We have now extended this finding by demonstrating that loss of
Srb8, Srb10, or Sin4 also activates lexA-Med1. This was an expected
result for Srb8 and Srb10 since we know that the Srb8-11 module is a
functional unit. The fact that loss of Sin4 activates lexA-Med1 is more
surprising, since Sin4 is thought to be part of the activator binding
module that also includes Med2, Gal11, and Pgd1 (21, 22). It should be noted, however, that Sin4 resembles Srb8-11 in that it also appears to
play a role in transcriptional repression. Thus, SIN4 and
ROX3 were the only bona fide Mediator subunit
genes that were recovered in the same genetic screens for relief of
repression as SRB8-11 (24).
Surprisingly, we found that lexA-Srb7 also is a cryptic activator.
Although its activity is lower than for lexA-Med1, the pattern of
activation is almost identical. Thus, lexA-Srb7 is inactive in wild
type cells but becomes active in the absence of Srb8, Srb10, Srb11, or
Sin4. It differs from lexA-Med1 in that it is activated also in the
absence of Med1. This difference is, however, most likely due to the
ability of lexA-Med1 itself to complement the med1 deletion.
We conclude that the two cryptic activators are activated under the
same conditions, which suggests a common underlying mechanism. This is
notable since Srb7 is an essential protein, whereas Med1 is not. Loss
of Med1 instead causes a mild phenotype very similar to that of
srb10 or srb11 (10). Similar to srb10
and srb11 mutations, med1 mutations can also partially suppress the constitutive glucose repression phenotype of
snf1 cells. These similarities suggest a possible link
between Med1 and the Srb8-11 complex (10). Interestingly, the latter has been proposed to be involved in repression mediated by the general
co-repressor Tup1, perhaps serving as one of its downstream targets
(54). Srb7 is known to interact with Tup1 (26), thus providing a
possible functional link between Srb8-11, Med1, and Srb7.
It is conceivable that the ability of lexA-Med1 and lexA-Srb7 to
activate transcription in the absence of Srb8-11 reflects the
mechanism by which the latter complex inhibits gene expression in wild
type cells. According to this model, Med1 and/or Srb7 would provide a
signal or activity that can stimulate transcription but which is
normally inhibited by the Srb8-11 complex. Signals that cause
activation of transcription would unveil this cryptic activator
function perhaps by modulating the kinase activity of Srb10. It has
been proposed that Tup1 inhibits gene expression by binding to Srb7,
thus preventing it from interacting with Med6. The latter interaction
is thought to be essential for activation of transcription (26). If
this notion is correct, lexA-Srb7 may well activate transcription
through the same mechanism.
In an attempt to learn more about the two cryptic activators, we
investigated to what extent they are associated with other Mediator
proteins in wild type and mutant cells. We found that lexA-Med1 and
lexA-Srb7 are stably associated with Med4 and Med8 both in wild type
cells and in the med1, med2, gal11,
sin4, srb8, srb10, and
srb11 strains (Figs. 1 and 2). In contrast, this was not the
case for the classical activator lexA-VP16. This suggests that
lexA-Med1 and lexA-Srb7 both activate transcription by recruitment, i.e. by stably integrating into the Mediator and, thus,
recruiting it to the target promoter. This is also consistent with our
finding that both fusion proteins can complement deletions of the
corresponding wild type genes. Our finding that lexA-Srb7, but not
lexA-Med1, associates with wild type Med1 provides further support for
the notion that lexA-Med1 replaces wild type Med1 within Mediator. The
fact that the association of lexA-Med1 and lexA-Srb7 with both Med4 and
Med8 remained unaffected in all the gene disruption strains tested may
seem surprising. However, none of these disruptions affect essential
core subunits of Mediator.
Of particular interest is our finding that both Med4 and Med8 exist in
two different mobility forms and that these forms differ in their
ability to associate with lexA-Med1 and lexA-Srb7 (Figs. 2 and 3).
Thus, only the low mobility form of Med4 associates with lexA-Med1 and
lexA-Srb7. Of the two Med8 species, the low mobility form associates
preferentially with lexA-Med1, whereas both forms associate with
lexA-Srb7. The latter finding is noteworthy since it suggests that
assembly of Mediator could be a multistep process. According to this
interpretation, a core complex containing Srb7, Med8, and Med4 (as well
as other subunits from the head and middle domains) would be formed
first. The fact that only the low mobility form of Med4 co-precipitates
with lexA-Srb7 suggests that Med4 has to be converted to this form to
be included in the complex or, alternatively, that conversion of Med4
to its high mobility form causes it to dissociate from Srb7 and Med8.
In either case, modification of Med4 would modulate assembly or
disassembly of Mediator. In a second step, the core complex would bind
Med1 and perhaps also other subunits such as the activator binding tail
domain. However, this would happen only after Med8 has been converted
to its low mobility form, since only the latter co-precipitates with
lexA-Med1. As with Med4, one may argue that disassembly is the
regulated step in which case conversion of Med8 to its high mobility
form would trigger dissociation of Med1. The nature of the
modifications remains to be determined, but our experiments with
purified holopolymerase show that Med4 is phosphorylated when
present in this complex and that the phosphorylation converts Med4 into
a lower mobility form (Fig. 4). Differential phosphorylation is,
therefore, a possible explanation for the two forms of Med4. In
contrast, we saw no evidence that Med8 is phosphorylated in the
purified holopolymerase. It is, therefore, possible that the two forms
of Med8 could reflect some other kind of modification. In this context,
it is interesting to note that a mouse Med8 homolog recently was shown
to be an Elongin BC-interacting protein that can assemble with Cul2 and
Rbx1 to reconstitute a ubiquitin ligase (55). In conclusion, our
results suggest that assembly of Mediator is a multistep process that
involves conversion of both Med4 and Med8 to their low mobility forms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1::HIS3,
the GAL11 coding sequence between two Bsu36I
sites was replaced by a HIS3 BamHI fragment. To generate
srb8-
1::LEU2, the
SRB8 coding sequence between the Bst1107I and
NcoI sites was replaced by a LEU2
HpaI-SalI fragment. The
med1-
2::HIS3,
med2-
1::HIS3, srb10-
1::LEU2, and
srb11-
1::HIS3 strains
have been described (10, 35). Strains DY1699 and DY1702 were kindly
provided by David Stillman.
Yeast strains
-Galactosidase assays were performed as described in Balciunas et al. (10). Cells were grown in 8% synthetic glucose media with dropout selection for the appropriate markers in each case.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
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Fig. 1.
lexA-VP16 fusions that were used in this
work. The ability of each fusion to complement the corresponding
deletion mutant is shown to the right. The phenotypes that
were tested for complementation were the growth defect on galactose for
gal11 and med2, the temperature sensitivity and
generally poor growth on glucose for med2, the lethality of
an srb7 deletion, and growth on lactate in a snf1
mig1 mutant background for med1 and srb10
(10). Because of its size, the lexA-Gal11 fusion is not drawn to
scale.
Transcriptional activity of different lexA fusion proteins in wild type
and mutant strains
-galactosidase units ± S.E.
Transcriptional activity of different Gal4-VP 16 constructions in wild
type, med2, gal11, and sin4 strains
-galactosidase units ± S.E.
-galactosidase gene from the ADH1
promoter used to express lexA-Med1 produces a level of activity
(24 ± 4 units) in the med2 srb11 double mutant that is comparable with that seen in the srb11 and med2
single mutants (Table II). Instead, it suggests that Med2 is required
for transcriptional activation by the lexA-Med1 cryptic activator
irrespective of how it is activated. We next proceeded to determine the
ability of lexA-Srb7 to activate transcription in the med2
srb11 strain. Just as for lexA-Med1, we found that it is inactive
(1.5 ± 0.4 units) in the double mutant strain. It should be noted
that Med2 is unlikely to be required for incorporation of Srb7 into the Mediator since SRB7 is an essential gene, whereas
MED2 is not. Moreover, lexA-Srb7 co-immunoprecipitates with
other Mediator subunits in med2 cells (see below),
which reinforces the notion that its incorporation into the Mediator is
independent of Med2. Therefore, the epistasis of med2 over
srb11 in this case must reflect a partial functional
dependence of lexA-Srb7 on Med2.
View larger version (85K):
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Fig. 2.
Co-immunoprecipitation of Med4 and Med8 with
lexA-Med1. Whole cell extracts were prepared from different yeast
strains expressing lexA-Med1 from a plasmid and immunoprecipitated with
monoclonal antibodies specific for the lexA DNA binding domain. The
precipitated protein was analyzed by Western blots as described under
"Experimental Procedures" using the antisera indicated to the
left. It should be noted that in the whole cell extract, two
proteins are detected by the anti-Med1 antiserum; the lexA-Med1 fusion
(upper band) and wild type (wt) Med1 (lower
band). Only the lexA-Med1 fusion is present in the
immunoprecipitate. ld (load), whole cell extract;
ip, immunoprecipitated proteins.
View larger version (90K):
[in a new window]
Fig. 3.
Co-immunoprecipitation of Med1, Med4, and
Med8 with lexA-Srb7. Whole cell extracts were prepared from
different yeast strains expressing lexA-Srb7 from a plasmid and
immunoprecipitated with monoclonal antibodies specific for the lexA DNA
binding domain. The precipitated protein was analyzed by Western blots
as described under "Experimental Procedures" using the antisera
indicated to the left. ld (load), whole cell
extract; ip, immunoprecipitated proteins. wt,
wild type.
View larger version (64K):
[in a new window]
Fig. 4.
Med4 is phosphorylated in purified
holopolymerase. Holopolymerase was purified from cells in which
its Rgr1 subunit has been tandem affinity purification-tagged (45) as
previously described (46). The purified holopolymerase was incubated
for 1 h at 37 °C in the presence or absence of calf intestine
phosphatase (CIP) and then analyzed by Western blots as
described under "Experimental Procedures" using antisera against
Med4 and Med8 (9).
View larger version (86K):
[in a new window]
Fig. 5.
Phenotypic differences between
med2, gal11, and sin4
strains. Wild type and mutant strains (Table I) were patched
onto rich glucose medium (YPD) and then replicated to rich
glucose medium, synthetic glucose medium (SGlu), synthetic
acetate medium (SAc), or synthetic galactose medium
containing 20 µg/ml of ethidium bromide (SGal EB). The
plates were incubated at 30 °C unless otherwise stated.
wt, wild type.
View larger version (72K):
[in a new window]
Fig. 6.
Synthetic interaction between sin4
and gal11 disruptions. A diploid obtained
by crossing strains H966 and DY1699 (Table I) was sporulated, and
tetrads were dissected onto rich glucose medium (YPD). After
colonies had formed, the plates were replicated to synthetic media
lacking either leucine or histidine to identify cells carrying the
sin4 and gal11 disruptions, respectively.
Effects of med2 high copy number suppressors in different Mediator
mutants
(no colonies) to +++ (comparable to the wild type strain) in an
experiment where all strains were transformed with similar amounts of
plasmid DNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Roger Brent, Claes Gustafsson, Roger Kornberg, Lawrence Myers, and David Stillman for generous gifts of strains, antibodies, and plasmids.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Research Council and the Swedish Cancer Society (to H. R. and S. B.), the Kempe Foundation (to M. H.), and the Swedish Foundation for Strategic Research and the Human Frontier Science Program (to S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Genetics, Cell Biology, and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St. S. E., Minneapolis, MN 55455.
** To whom correspondence should be addressed: Dept. of Plant Biology, Swedish University of Agricultural Sciences, Box 7080, S-750 07 Uppsala, Sweden. Tel.: 46-18-673313; Fax: 46-18-673279; E-mail: Hans.Ronne@vbiol.slu.se.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M206946200
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ABBREVIATIONS |
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The abbreviation used is: pol II, RNA polymerase II.
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REFERENCES |
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