From the Department of Cellular and Molecular Physiology and the Cell and Molecular Biology Program, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cAMP response element-binding protein (CREB)
mediates both basal and PKA-inducible transcription through two
separate and independently active domains, the constitutive activation domain (CAD) and the kinase-inducible domain, respectively. The CREB
CAD interacts with the general transcription factor TFIID through one
or more of the TATA-binding protein-associated factors (TAFs), one of
which is TAF110. The CAD is composed of three subdomains, rich in
either serine, hydrophobic amino acids, or glutamine. In the present
study, analysis of deletion mutants of the CAD showed that all three
CAD subdomains were required for effective interaction with TAF110 in a
yeast two-hybrid assay. Therefore, a library of random point mutations
within the CAD was analyzed in a reverse two-hybrid screen to identify
amino acids that are essential for interaction with the TAF.
Interaction defects resulted solely from mutations of hydrophobic amino
acid residues within the hydrophobic cluster to charged amino
acid residues. Together, the deletion and mutation analyses suggest
that the entire CAD provides an environment for a specific hydrophobic
interaction with TAF110 that is crucial for interaction. Our results
provide further evidence for a model of basal activation by CREB
involving interaction with TAF110 that promotes recruitment or
stabilization of TFIID binding to the promoter, which facilitates
pre-initiation complex assembly.
The cAMP response element is an enhancer that mediates both basal
and cAMP-induced transcriptional activation of a variety of genes in
many cell types (1-6). cAMP response element-binding protein
(CREB)1 binds constitutively
as a dimer to CREs in the promoter of the target gene and activates
basal transcription even in the absence of hormonal stimuli (3).
Extracellular stimuli that activate protein kinases lead to
phosphorylation of CREB on serine 133 by activated PKA, resulting in
further enhancement of transcriptional activation (7, 8). Mutation of
the serine 133 phosphorylation site in CREB abolishes kinase inducible
activation (7, 9) but leaves basal activation unaltered (3, 10). In
addition, previous work showed that CREB contains two separate and
independently acting transcriptional activation domains: a constitutive
activation domain (CAD) responsible for basal activation and a
kinase-inducible domain that mediates activation in response to
cAMP-activated PKA (3, 11). When the CREB CAD is fused to the
DNA-binding domain (DBD) of the yeast Gal4 protein, it activates basal
transcription from a Gal4 promoter nearly as well as the complete
unphosphorylated CREB (3, 11). Activation by CREB kinase-inducible
domain (KID) is proposed to be mediated by interaction with
CREB-binding protein, which, in turn, interacts with polymerase complex
components (12, 13). The CAD is composed of three subdomains with
distinct amino acid compositions; a serine/threonine rich region (ST3), a hydrophobic cluster (HC), and a glutamine-rich region (Q3). Deletion
of any of these regions results in a substantial loss of basal
activation by the CAD (14). It is not clear whether these subdomains
target a single factor or interact with different targets in the
preinitiation complex (PIC). We also showed that the CREB CAD binds
TFIID through interaction with one or more TAFs, rather than directly
with the TATA-binding protein (TBP) (15). Subsequently, Ferreri
et al. (17) showed that dTAF110, the interaction of which
with the glutamine-rich/hydrophobic domain of Sp1 had been extensively
characterized (16), also interacts with CREB.
Transcription of a protein-coding gene by RNA polymerase II requires
the assembly of a large complex of factors around the transcriptional
start site in the promoter of the gene. The characterization of these
general transcription factors (GTFs) has been an ongoing, ambitious
project in many laboratories and has lead to two basic models for basal
transcriptional activation (18, 19). In the first model, factors
assemble in a stepwise fashion (TFIID, TFIIB, TFIIA, TFIIF-polII,
TFIIE, and TFIIH) into a functional PIC with the ultimate goal of
positioning the polymerase complex at the proper start site and
allowing synthesis of an mRNA transcript to begin (20). The
specific order of addition in this model is based on in
vitro transcription assays and template binding assays with
purified GTFs (18, 21-23). A second model is based on recent in
vivo work that suggests that the majority of the GTFs, except
TFIID (and perhaps TFIIB), exist in the cell as large preformed
complexes termed holoenzymes (19) and that holoenzymes may be recruited
en bloc to the promoter (24, 25). Many activators have been
shown to interact directly with one or more of the GTFs, and this
interaction is currently thought to be a major mechanism for gene
activation. Importantly, for either the stepwise addition or holoenzyme
models, TFIID is proposed to be an obligatory first step in PIC formation.
TFIID consists of the TBP and a large group of TBP-associated factors
or TAFs, ranging in size from 15 to 250 kDa, which are generally well
conserved from yeast to mammals (26-28). In vitro transcription experiments have demonstrated that many activators will
function only if the complete TFIID complex is added and cannot
activate transcription if TBP alone is included (29, 30). Cellular TBP
is required for transcription by all three RNA polymerases, and it is
partitioned in vivo among multiprotein complexes for each
polymerase, including TFIID (28, 31). Many diverse transcriptional
activators have been shown to interact with the TAF subunits of TFIID,
including Sp1 (32) and bicoid (33) with TAF110, estrogen receptor with
TAF30 (34), CAAT transcription factor with TAF55 (35), NF- In the case of CREB, the CAD alone bound to an enhancer upstream of the
transcriptional start site activates transcription and the CAD
interacts with TFIID (3, 15). Because binding of TFIID must precede the
association of other general transcription factors with the promoter,
activators such as the CAD in CREB are thought to enhance transcription
initiation by facilitating the binding of the TFIID complex. This
general mechanism for gene activation has been suggested for many
activators (41).
The present study was designed to map the region(s) of the CAD involved
in protein-protein interaction with TAF110 and ultimately determine the
amino acid residues of the CAD that make crucial contacts with the TAF.
A yeast two-hybrid assay was used to determine which subdomains of the
CAD are required to support interaction with TAF110. In addition, the
CAD was subjected to random point mutagenesis and the mutated CAD
library was screened in a reverse two-hybrid system to identify amino
acids of the CREB CAD that are crucial for interaction with this
TAF.
Plasmids and Yeast Strains--
The same yeast expression
vectors were used in the two-hybrid and reverse two-hybrid experiments.
One, pGAD-110, contains the coding region for the N-terminal 308 amino
acids of the TAF110 protein fused to the activation domain of the yeast
Gal4 protein (amino acids 768-881) (42). This plasmid carries the
LEU2 selectable marker gene and the ampr
gene. A second yeast expression plasmid, pGN-1 (43), was modified to
contain the coding regions for CREB, the CREB CAD, or the various deletion mutations and point mutations of the CAD fused to the Gal4
(DBD amino acids 4-94). This plasmid carries the TRP1
selectable marker, as well as the ampr gene. The
yeast strain Y-153 (gal4 Yeast Transformation--
A LiAc protocol was used to introduce
plasmids into yeast (46). The appropriate yeast strain was grown in 50 ml of YEPD (0.5% yeast extract, 1.0% peptone, 25 µg/ml adenine, 2%
glucose) at 30 °C to an A600 of 0.2. Yeast
were centrifuged at 5000 rpm for 5 min at 4 °C, and the pellet was
washed with TE and re-centrifuged. The pellet was resuspended in 10 ml
of LiAc solution (10 mM Tris, pH 7.5, 1 mM
EDTA, 100 mM LiAc) and incubated for 1 h at 30 °C with gentle shaking. Cells were centrifuged as above and resuspended in
2 ml of LiAc solution. 200 µl of these competent yeast cells was
combined with 10 µg of each appropriate plasmid DNA and incubated at
room temperature for 10 min. One ml of 50% PEG 3350 was added, followed by mixing and incubation at 30 °C for 1 h. Cells were heat-shocked at 42 °C for 5 min, pelleted, and washed with
dH2O. Yeast were resuspended in 200 µl of
dH2O and plated on the appropriate selective medium. Plates
were incubated at 30 °C for 2-3 days to allow colony formation.
Yeast Protein Lysate Preparation--
A glass bead lysis method
was used to make yeast protein lysates (47). The yeast clone of
interest was grown in 50 ml of double-selective liquid medium to an
A600 of approx. 0.8. Yeast were centrifuged at
5000 rpm at 4 °C for 10 min in a JA17 rotor, and the pellet was
resuspended in breaking buffer (50 mM
Na2HPO4, 50 mM
NaH2PO4, 10 mM EDTA, pH 7.2) with
protease inhibitors added (175 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM
dithiothreitol, 1 mM e-amino-n-caproic acid). The pellet was transferred to a 15-ml Corex tube on ice. Glass beads
(0.5 mm) were added up to the fluid meniscus, and the slurry was
vortexed eight times for 30 s with 2 min on ice between each round. The resulting crude yeast lysate was transferred to a
microcentrifuge tube and centrifuged at 13,000 rpm for 5 min at 4 °C
to pellet insoluble cellular debris and residual glass beads. A
standard Bradford assay was used to estimate total protein
concentration to ensure that comparable amounts of lysates were used in
quantitative PCR Mutagenesis--
A relaxed stringency PCR protocol that
promotes polymerase errors was adjusted to introduce Yeast Plasmid DNA Isolation--
The method described by Nasmyth
and Reed (50) was used for plasmid isolation from yeast. The yeast
clone of interest was grown in 5 ml of single selective medium (without
Trp, to select for only the CAD-G4 containing plasmid) to an
A600 of approximately 0.6. An aliquot of 1.5 ml
of the culture was centrifuged at 13,000 rpm at 4 °C for 5 min, and
the pellet was resuspended in 0.5 ml of S buffer (10 mM
KPO4, 10 mM EDTA, 50 mM
The CREB CAD Interacts with the N Terminus of TAF110 in a Yeast
Two-hybrid System--
We first established a functional yeast
two-hybrid assay to test mutants for interaction (51). CREB contains
two independent activation domains, kinase-inducible domain (KID) and
CAD, that were defined by functional analysis of CREB-Gal4 (CRG) fusion proteins (Fig. 1A) (3). The
CAD can be further subdivided into three subdomains, based on sequences
required for activation and prevalent amino acid composition: ST3, HC,
and Q3 (14). Based on previous reports of interaction between TAF110
and SP-1 or CREB (16, 17, 32), we used two yeast expression plasmids, one in which the N-terminal 308 amino acids of TAF110 are fused to the
Gal4 activation domain and that carries a selectable marker (Trp), and
a second expression plasmid carrying a different selectable marker
(Leu) and expressing the CREB activation domain-Gal4 DNA-binding domain
fusion proteins (CRG, CAD-G4, etc.). These plasmids were co-transformed
into the yeast strain Y-153 (leu The Three Subdomains of CAD Are Required for Full Interaction with
TAF110--
The results in Fig. 1B show that the
interaction of TAF110 with CREB maps to the CREB CAD. Previous work in
our laboratory showed that constitutive activity in vivo
required all three subdomains of the CAD, which may interact with one
or more different targets in the polymerase complex (14). In addition,
several different TAFs in TFIID have been shown to interact with
transcriptional activators. Thus, we were interested in determining
whether a specific subdomain in the CAD would mediate the interaction
with TAF110 or whether all three subdomains were required. We first tested CADs deleted of each of the three subdomains, ST3, HC, and Q3,
for interaction with TAF110 in an attempt to identify a smaller region
of interaction, with the ultimate goal of mapping the amino acid
residues involved. Site-directed mutagenesis was used to introduce
restriction sites flanking each region in question so that it could be
deleted. Each mutant was tested for interaction with TAF110 and
compared with the activity obtained with the full-length CAD (Fig.
2). Deletion of any of the three CAD
subdomains reduced the interaction with TAF110, as compared with
full-length CAD. In fact, Synthesis and Screening of a CAD Random Point Mutation
Library--
To determine the amino acid residues of CAD involved in
the interaction with TAF110, the CAD was subjected to random point mutagenesis, and the resulting library was screened in a reverse two-hybrid assay for interaction-deficient mutants. The lack of a
specific region within CAD responsible for TAF110 interaction led to
the decision to use the entire 87-amino acid CAD for point mutagenesis.
We employed a PCR-based mutagenesis protocol to introduce random point
mutations into the CAD coding region. This approach exploits the high
error rate of the native Taq polymerase and can be
fine-tuned by altering the Mg2+ concentration and cycle
number to introduce mutations into a given template at a desired rate
(49). PCR primers that flank the CAD coding sequence were chosen (Fig.
3A), and the region was
amplified under conditions designed to introduce, on average, one base
change error for each 300 base pairs synthesized (see under
"Experimental Procedures" for exact conditions used). DNA sequencing of clones from the mutagenized pool confirmed the expected error rate. The resulting mutagenized amplicon pool was then digested with restriction enzymes with recognition sites flanking the CAD coding
sequence and the DNA fragments were subcloned into the yeast expression
vector as a fusion with a wild type Gal4 DBD (Fig. 3A).
To screen the library for interaction-deficient CAD mutants, we
employed a reverse two-hybrid method (45) (Fig. 3A). This method uses a yeast strain (MaV203, a derivative of the Y-153 used
above) that contains a URA3 gene under control of a Gal4 promoter in addition to the GAL4-lacZ reporter gene.
Addition of the drug 5-fluoroorotic acid (5-FOA) is toxic to yeast when the URA3 gene product is expressed. Therefore, in a
two-hybrid assay in the presence of 5-FOA, cells containing CAD-G4 that
interacts with TAF-AD will activate Gal4-URA3, resulting in
cell death. Those clones with an interaction-defective CAD-G4 will
survive 5-FOA treatment. Double selective medium ensures the presence of both expression plasmids.
Preliminary experiments showed that the cytotoxicity of 5-FOA was
specific, because yeast containing the wild type CAD-DBD, and TAF110-AD
expression vectors or an expression plasmid for full-length Gal4 showed
normal growth without 5-FOA but no growth with 0.025% 5-FOA added
(Fig. 3B). In contrast, clones containing DBD plus
TAF110-AD, or CAD-DBD plus empty-AD, or both empty vectors are able to
grow normally in the absence or presence of 5-FOA. These preliminary
results established the utility of the reverse two-hybrid system for
screening our mutated CAD library to identify interaction-defective CADs.
The MaV203 yeast strain was cotransformed with the mutagenized CAD
library and TAF110-AD and plated on selective medium containing increasing concentrations of 5-FOA ranging from 0.005 to 0.05%. As
shown in Fig. 3C, the number of colonies formed by the
library in a representative experiment decreased as 5-FOA
concentrations increased. The CAD-G4 plasmids were recovered from
colonies isolated from plates with different drug concentrations, and
the CAD sequence was determined. The sharp drop in colony number
between 0 and 0.015% 5-FOA reflects the elimination of wild type CAD
clones or phenotypically wild type mutations. The majority of colonies found on the plate with the lowest concentration of 5-FOA (0.010%) were wild type. Importantly, many were found to be phenotypically wild
type despite having acquired amino acid mutations (Fig.
4A). Colonies from plates
selected at intermediate drug concentrations (0.015-0.025% 5-FOA)
contained CADs that were impaired for interaction with TAF110, as
discussed in detail below. Intermediate concentrations of 5-FOA
(0.015-0.025%) resulted in a significant number of clones that
produced full-length CAD-DBD fusion proteins with varying degrees of
reduction in their ability to interact with TAF110 (Fig. 4). The small
number of colonies surviving the highest dose of 0.050% 5-FOA was
found to contain CAD-G4s with stop codon insertions or, in a few cases,
frameshifts. These clones would not produce a protein capable of
binding DNA because the Gal4 DBD is fused downstream of the CAD.
To further characterize these mutations, the yeast colonies from which
plasmid DNA had been isolated and sequenced were grown and assayed for
To rule out the possibility that mutations that show up as
interaction-deficient in this screen are actually mutations that destabilize the protein, the mutant CAD-DBD fusion proteins were assayed for their stability in yeast. This was especially important for
those clones that appeared to be completely impaired for binding to
TAF110. Western blotting with antibody directed against the Gal4 DBD
was used to determine the stability of CAD-G4 proteins. Yeast
containing the putative interaction-defective clones were grown in
liquid selective medium, and whole-cell yeast protein lysates were made
and analyzed. The results in Fig. 4C show that the seven CAD
mutants identified in Fig. 4A that are most severely impaired for interaction with TAF110 produce proteins of the proper size in yeast. In addition, the amounts of the mutant fusion proteins were approximately equal to that of wild type CAD-DBD, as indicated by
the CAD control lanes on Western blots (Fig. 4C). However, this was not the case with all mutants isolated. One mutant that decreased binding to TAF110 did not produce a detectable, appropriately sized fusion protein (data not shown) and was therefore excluded from
the mutant set. This mutation (G188R) presumably destabilized the
CAD-DBD fusion protein in yeast and led to a false positive in the
screen for interaction defective mutants.
The TAFs of TFIID have been shown to interact with several
different transcription factors and are required for function of the
activator both in vitro and in vivo (32-36, 39).
In vitro experiments using the Drosophila
activators bicoid and hunchback, performed by Tjian et al.
(33), showed that the function of these activators is dependent on the
presence of TAF110 and TAF60 in the reaction, respectively. This same
study also showed that truncation of either TAF could inhibit
activation by the factor binding to it, both in vitro and
in vivo. More recent studies have demonstrated that TAFs
interact with and serve as coactivators for a wide range of
transcription factors in mammalian cells (32-36, 39), where they are
proposed to facilitate recruitment of the TFIID complex to the promoter.
In addition, there is evidence that TAFs play diverse roles at
different promoters during transcription initiation. Recent studies in
yeast indicate that TAFs are dispensable for activated transcription of
many genes in vivo (53, 54). TAFs appear to play a more
general role in promoter selectivity in that organism. In mammalian
cells, also, some TAFs bind to DNA and serve as promoter selectivity
factors at TATA-less promoters (28, 57). Other TAFs possess enzymatic
activities, such as protein kinase or histone acetyltransferase
activity (55). It is therefore likely that some TAFs are more important
than others for serving as coactivators for specific transcription
factors. In particular, TAF110 seems to be playing a major coactivator
role. Many reports have shown that TAF110, or its human homolog
hTAF130, interacts with various transcriptional activators, including
Sp1 (32), vitamin D receptor (37), thyroid hormone receptor (38),
adenovirus E1A (58), Drosophila bicoid (33), and the
CCAAT-binding factor (59). Sp1 was the first transcription factor shown
to interact with TAF110 (32). The strength of the interaction between
Sp1 and TAF110 correlates with the strength of the activator in
vivo (16). In our model (Fig. 5), we
propose that TAF110 serves as a coactivator by providing a target for
the CAD to recruit TFIID to the promoter and/or stabilize its
association with the TATA box. In either case, the interaction would
increase the association of TFIID with the promoter, a proposed
rate-limiting step in PIC assembly, thereby increasing the overall rate
of PIC formation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B with
TAF105 (36), vitamin D receptor with hTAF130 (37), thyroid receptor
with TAF110 (38), and p53 with TAF40 and TAF60 (39).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, leu
,
trp
, Gal:LacZ, etc.) is that described by the S. Elledge laboratory (44). The MaV203 yeast strain is a derivative of
Y-153 and was described by Vidal et al. (45). This strain is
ura3
and contains the URA3 gene under control
of a promoter that is kept silent by a sporulation repressor element
unless induced by binding of activator to the Gal4 containing upstream
activation sequence.
-galactosidase assays and for SDS-polyacrylamide gel
electrophoresis and Western blotting experiments.
-Galactosidase Assays--
To lift colonies for the
qualitative filter assay, a Whatman filter paper circle was placed on
the plate until wet. Yeast were lysed by placing the filter into liquid
nitrogen for 1 min. The filter was placed in a Petri dish and incubated
with 2 ml of Z buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4, 40 mM
-mercaptoethanol, 0.005% SDS) containing 20 mg/ml of
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside at
37 °C until a blue color was evident. For the quantitative liquid
assay (48), 2-5 mg of protein lysate was added to 1 ml of Z buffer and
incubated with 200 µl of 4 mg/ml O-nitrophenyl-
-D-galactopyranoside at
30 °C until a yellow color was evident. After color development, 0.5 ml of 1 M Na2CO3 was added to stop
the reaction, and the absorbance at 420 nm was determined spectrophotometrically. Values were adjusted for incubation time and
protein concentration to give the specific activity of the sample.
1 error per CAD
sequence of 300 base pairs (49). The plasmid pGEM3Z-CAD94, containing
the CREB CAD coding region, was used as a template for PCR with two
primers that annealed to opposite strands on either side of
NcoI and ClaI restriction sites that remove the
CAD from the plasmid. The PCR mixture contained 10 ng of template DNA,
5pmole of each primer, 10 mM of each dNTP, 4 mM
MgSO4, 50 mM KCl, 10 mM Tris, pH
8.4, and 5 units of Taq polymerase. Amplification of the CAD
was carried out for 33 cycles under the following conditions:
denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min,
extension at 72 °C for 2 min, followed by a final extension at
72 °C for 10 min. The resulting amplicon pool was digested with
NcoI/ClaI, and the DNA fragments were subcloned
into the pGN-CAD-G4 yeast expression vector in place of the wild type
CAD NcoI/ClaI fragment. The resulting library was
transformed into Escherichia coli and grown, and plasmid DNA
for transformation of yeast was prepared by a standard CsCl method.
-mercaptoethanol, pH 7.2). Yeast cell walls were digested by adding
5 µl of 20 mg/ml zymolase and incubating at 37 °C for 60 min.
Lysis buffer (0.1 ml) (0.25 M Tris, pH 7.5, 25 mM EDTA, 2.5% SDS) was added, and the sample was vortexed
and incubated at 65 °C for 30 min. After precipitation with 166 µl
of 3 M KOAc (pH 5.8) on ice for 10 min, samples were
centrifuged, and nucleic acid was precipitated from the supernatant
with 0.8 ml of EtOH. The pellet was washed, dried, and resuspended in
40 µl of dH2O. An aliquot of 2 µl of this preparation was used to transform E. coli by electroporation. Clones
were screened for the proper plasmid construct and sequenced.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
trp
, gal4
), which contains the
lacZ gene under control of a Gal4 responsive promoter (44).
Activation of this hybrid gene, as a result of interaction between the
two fusion proteins, leads to the production of
-galactosidase,
which can be measured by qualitative and quantitative assays. The
extent of this interaction can be qualitatively determined by using the
synthetic substrate 5-bromo-4-chloro-3-indolyl
b-D-galactopyranoside in a filter lift assay to give a blue
color to those colonies expressing
-galactosidase (data not shown).
In addition, a quantitative
-galactosidase assay of yeast extracts
was used to determine the relative strength of the interactions, which
is directly proportional to the amount of
-galactosidase
activity (
-galactosidase activity/mg of protein) (Fig.
1B). Neither of the test plasmids, CRG or TAF-AD, had
any activity alone, but significant
-galactosidase activity was
observed when full-length CAD-G4 was cotransformed with a TAF110-AD expression construct (Fig. 1B). This response
indicates that CAD-G4 recruited TAF110-AD to the upstream activation
sequence-lacZ gene promoter, resulting in activation of the
lacZ gene. The specificity of this interaction, together
with the lack of activity of CREB in yeast, provides a useful model for
determining interactions between various CAD mutants and
TAF110.
View larger version (27K):
[in a new window]
Fig. 1.
Domains of CRG and interaction with TAF110 in
a yeast two-hybrid system. A, schematic representation
of the CRG fusion protein indicating the various domains within the
amino acid sequence. The kinase inducible domain (KID) is
indicated, including its PKA phosphorylation site at serine 133, and
the three subdomains of the CAD are shown: ST3, HC, and Q3. CRG has the
CREB DNA-binding domain replaced with the DBD of the yeast GAL4
protein, amino acids 4-94. B, CAD and the N-terminal 308 amino acids of TAF110 were tested for interaction in a yeast two-hybrid
system. Yeast strain Y153 (see under "Experimental Procedures") was
transformed with the indicated plasmid DNA and plated on
double-selective medium. Individual clones were picked from each plate,
and whole-cell protein lysates were assayed for -galactosidase
activity, which is indicative of the strength of the interaction.
Values are adjusted for concentration of lysates and incubation time in
the assay. Data represent three independent experiments with three
clones assayed for each. Error bars depict S.E.
HC and
Q3 showed little, if any,
activity. However,
ST3 did retain approximately 35% of full-length
CAD activity, so we further truncated the remaining region to see
whether a discreet interaction target for TAF110 might reside within
the HC/Q3 region. We deleted the N-terminal half of HC (
NHC), the C-terminal half of Q3 (
CQ3), and both (
NHC/
CQ3). When these mutants were tested for interaction with TAF110, a gradual loss of
activity was observed for successive truncation of the HC/Q3 region of
the CAD (Fig. 2). The results of these yeast two-hybrid experiments
lead to the conclusion that elements throughout the entire CAD are
necessary for interaction with TAF110, either for a conducive
conformation or through direct amino acid contacts with the TAF.
View larger version (19K):
[in a new window]
Fig. 2.
Interaction of CAD deletion mutations with
TAF110. Deletion mutants of CAD were generated by inserting
restriction sites at the indicated boundaries using site-directed
mutagenesis. The indicated regions of CAD were then deleted, and the
resulting constructs cloned into the yeast expression vector. Each
mutation was then tested for interaction with TAF110 in the yeast
two-hybrid system, and -galactosidase activity was assayed as
indicated previously. Data are representative of three experiments with
three clones assayed in each. Error bars depict S.E.
View larger version (23K):
[in a new window]
Fig. 3.
Generation and screening of a CAD point
mutation library. A, the CREB CAD coding region was
amplified using polymerase chain reaction with two primers that bind
flanking the sequence as indicated. Conditions and amplification cycle
number were chosen to favor the introduction of 1 point mutation per
CAD sequence of 300 base pairs amplified (see under "Experimental
Procedures" for conditions). The resulting amplicon pool was then
subcloned into the yeast expression vector in fusion with a wild type
Gal4 DBD. The library of CAD point mutations was transformed along with
the TAF110 1-308-AD expression plasmid into the reverse two-hybrid
yeast strain MaV203 (see under "Experimental Procedures") and
plated on double selective medium containing 5-FOA at concentrations of
0.0, 0.005, 0.010, 0.015, 0.020, 0.025, and 0.050%. B,
yeast clones containing the indicated expression constructs were
streaked on double-selective medium with or without 0.025% 5-FOA.
C, colony number was determined for yeast containing the CAD
point mutation library clones and TAF110-AD at the indicated
concentrations of 5-FOA. The results of a typical experiment are
depicted.
View larger version (34K):
[in a new window]
Fig. 4.
Analysis of the CAD point mutation library in
a yeast reverse two-hybrid system. A, yeast clones
containing the CAD point mutation library and TAF110-AD were chosen
from plates with various concentrations of 5-FOA. Plasmid DNA was
recovered from the yeast and sequenced to determine the presence and
identity of the mutation. B, the mutations depicted in the
graph without an asterisk were isolated at 0.015-0.025% 5-FOA. Those
mutations with an asterisk were isolated at 0.010% 5-FOA.
Yeast clones bearing point mutations were grown in liquid selective
medium and assayed for -galactosidase activity using an
O-nitrophenyl-
-D-galactopyranoside liquid
assay as described under "Experimental Procedures." C,
yeast clones with interaction-defective CAD mutants were grown in
selective liquid medium, and whole-cell protein lysates were made.
Proteins were separated by SDS-polyacrylamide gel electrophoresis, and
a Western blot was performed using a primary antibody directed against
the Gal4 DBD and visualized with chemiluminescence.
-galactosidase activity. Interestingly, all mutations that reduced
interaction with TAF110 lie within the HC domain and represent changes
in hydrophobic amino acid residues to charged amino acid residues.
These mutations decreased interaction with TAF110 to varying degrees,
ranging from near complete elimination of interaction with mutations
G189R, L193Q, and L207Q to less severe disruption of the interaction in
I191R, I191T, L204Q, and L207S. Mutations in the ST3 and Q3 domains of
CAD were isolated only under less stringent conditions (low 5-FOA
concentrations). These mutations, along with a few mutations of polar
amino acids within the HC domain (indicated in Fig. 4 by
asterisks), produced little or no effect on TAF110 binding.
This result was not surprising because it is likely that many clones
that are mutated, but phenotypically normal for binding to TAF110,
would survive a very low dose of 5-FOA. No mutations in ST3 or Q3 or of
hydrophilic amino acids in HC were isolated from plates with higher
doses of 5-FOA (
0.020%). Thus, it would appear that the central
hydrophobic residues of the CAD are most crucial to its interaction
with TAF110.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (52K):
[in a new window]
Fig. 5.
Model for basal activation by the CREB
CAD. The proposed model shows CREB interacting with the TAF110
subunit of TFIID through the CAD. This interaction would recruit TFIID
to the promoter DNA and/or stabilize its binding to the TATA-box. Once
this step in PIC formation is achieved, the remainder of the complex
can form, ultimately positioning RNA polymerase II properly at the
transcriptional start site.
Given that the CAD interacts with holo-TFIID in vitro (15) and that all three CAD subdomains are required for function in vivo, we were interested to know whether the CAD-TFIID interaction was mediated by multiple TAFs. We observed no interaction with TAF55 (data not shown), a TAF that has been shown to interact with multiple transcription factors, including Sp1 (60). Also, others observed that CREB did not interact with TAF250.2 In addition, we found that our previous report of an interaction between CREB and TFIIB (15) cannot be replicated in vitro or in a two-hybrid assay (data not shown). At this point, the finding that all three CAD subdomains are required for interaction with TAF110, together with the lack of evidence for association with other GTFs, suggests that the CAD interaction with a single GTF component, TAF110, may be its primary mode of action.
TAF110 (or dTAF110) is the Drosophila homolog of the human hTAF130, which was recently cloned (37). Our study with TAF110 was undertaken before the human clone had been obtained and was based on preliminary observations made by others (17). dTAF110 and hTAF130 share much sequence identity, especially at the N terminus, where CREB interacts with the TAF, and at the C terminus, where the TAF binds to TAF250, the scaffolding TAF that integrates it in the TFIID complex (37). It was recently shown by Saluja et al. (61) that hTAF130 and dTAF110 interact with transcriptional activators, such as Sp1 and CREB, in a similar manner, suggesting that our studies of CREB interactions with dTAF110 will be directly applicable to a mechanism that involves hTAF130 in vivo.
The nature of the mutants isolated in the reverse two-hybrid screen
(hydrophobic charged) suggests that a hydrophobic surface on the
CAD may contact a similar surface on TAF110. Gill et al. (16) showed that hydrophobic residues in the activation domain of Sp1,
which are arranged similarly to those in the CREB HC (Fig. 4A), mediate interaction with TAF110. This pattern of
hydrophobic residues is present in many activation domains and was
first recognized in the viral activator VP16 (62). In VP16, mutation of
key hydrophobic residues leads to a dramatic loss of activation.
Upon analysis of codon usage and the probabilities of obtaining hydrophobic versus charged residues for single base changes in the codons of the hydrophobic residues in HC, it was found that some, Gly-189, Ala-190, and Ile-191, had a 50% chance of being changed to a charged amino acid residue or of being changed to another hydrophobic residue. Other hydrophobic residues, however, had a much smaller probability of being changed to a charged residue with a single base change (Leu-193 = 33%, Leu-204 = 33%, and Leu-207 = 17%). These results show that the hydrophobic residues for which interaction-defective mutants were isolated were either random or were biased in favor of a conservative mutation to another hydrophobic residue. The fact that mutants were isolated in which each of these hydrophobic residues was changed to a charged residue (in the case of Ile-191 and Leu-207, to two different charged residues), even though the mutagenesis was either random or biased in favor of conservative mutations, makes it more likely that the interaction-defective phenotype between CAD and TAF110 is caused by the presence of charged residues on an interacting hydrophobic surface in CAD, which would have a repulsive effect.
Both the reverse two-hybrid screen and our deletion mutation analysis suggest that the remaining two regions of the CAD, ST3 and Q3, contribute to CAD conformation, rather than being directly involved in contacting TAF110. All point mutations isolated in the ST3 or Q3 subdomains were found only under a low concentration of 5-FOA (0.010%), and all were phenotypically wild type. However, deletion of either, or even part, of these two subdomains significantly reduced interaction with TAF110 (Fig. 2). In addition, all three subdomains of the CAD are required for full activation of transcription in mammalian cells (15). These results suggest that interaction with TAF110 is at least part of the mechanism of CAD activation.
The fact that our CAD-DBD fusion protein is incapable of activating transcription from a Gal4-lacZ reporter in yeast (Fig. 1B), whereas the same fusion construct activates transcription from a Gal4-CAT reporter in mammalian cells (3), is also very interesting. This same observation has been made for other activators that interact with TAF110, including Sp1 (32, 52, 56). Yeast contain homologs of most of the TAF subunits of human TFIID but does not contain a TAF110 homolog (40). Thus, if TAF110 is the target for Sp1 and CREB in the general transcription machinery, the lack of a TAF110 homolog in yeast could explain why these activators do not stimulate transcription in yeast.
We have presented here evidence that supports a mechanism for CREB
activation of basal transcription that utilizes TAF110 as a coactivator
to recruit TFIID to the promoter and facilitate PIC assembly (Fig. 5).
Although additional experimentation is required to further test our
current model for activation, there is mounting evidence in the
literature that the TAF subunits of TFIID mediate selective activator
function in higher eukaryotes. In particular, TAF110 (hTAF130) seems to
be a widely used coactivator for transcriptional activators, including
the CAD of CREB, that regulate transcription in metazoans but not in yeast.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Hopper and members of his laboratory for providing plasmids, yeast strains, technical assistance, and critical discussions of this work. We also thank Dr. A. Hopper and members of her laboratory for technical advice on yeast experiments. We thank the Tjian laboratory for providing TAF110 clones and David Spector for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: Dept. of Cellular and
Molecular Physiology, H166, The Pennsylvania State University College
of Medicine, 500 University Dr., Hershey, PA 17033. Tel.: 717-531-6182; Fax: 717-531-7667; E-mail: pquinn{at}psu.edu.
2 J. Goodrich, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CREB, cAMP response element-binding protein; DBD, DNA-binding domain; CRG, CREB-Gal4; CAD, constitutive activation domain; ST3, serine/threonine-rich region; HC, hydrophobic cluster; Q3, glutamine-rich region; GTF, general transcription factor; TBP, TATA-binding protein; TAF, TBP-associated factor; PIC, preinitiation complex; URA, uracil; 5-FOA, 5-fluoroorotic acid; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|