Department of Biochemistry and Molecular
Biology, Pennsylvania State University, University Park,
Pennsylvania 16802-4500
Received 15 June 2001/Returned for modification 19 July
2001/Accepted 30 July 2001
Yeast TAF90p is a component of at least two transcription
regulatory complexes, the general transcription factor TFIID and the
Spt-Ada-Gcn5 histone acetyltransferase complex (SAGA). Broad transcription defects have been observed in mutants of other
TAFIIs shared by TFIID and SAGA but not in the only two
TAF90 mutants isolated to date. Given that the numbers
of mutants analyzed thus far are small, we isolated and characterized
11 temperature-sensitive mutants of TAF90 and analyzed
their effects on transcription and integrity of the TFIID and SAGA
complexes. We found that the mutants displayed a variety of
allele-specific defects in their ability to support transcription and
maintain the structure of the TFIID and SAGA complexes. Sequencing of
the alleles revealed that all have mutations corresponding to
the C terminus of the protein, with most clustering within the
conserved WD40 repeats; thus, the C terminus of TAF90p is required for
its incorporation into TFIID and function in SAGA. Significantly,
inactivation of one allele, taf90-20, caused the
dramatic reduction in the levels of total mRNA and most specific
transcripts analyzed. Analysis of the structure and/or activity of both
TAF90p-containing complexes revealed that this allele is the most
disruptive of all. Our analysis defines the requirement for the WD40
repeats in preserving TFIID and SAGA function, demonstrates that the
defects associated with distinct mutations in TAF90 vary
considerably, and indicates that TAF90 can be classified
as a gene required for the transcription of a large number of genes.
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INTRODUCTION |
TFIID is one of several general
initiation factors required for the reconstitution of RNA polymerase II
(Pol II) transcription in vitro. In yeast, it is composed of the
TATA-binding protein (TBP) and at least 14 associated factors
collectively referred to as TAFIIs (23, 30,
34, 41, 42, 43, 46, 53). Individual TAFII
polypeptides contain distinct structural motifs and functional domains
that are likely to be responsible for their specialized functions. For
example, several functional domains have been identified in
hTAFII250, which include histone
acetyltransferase (HAT), kinase, and ubiquitin-conjugating domains and
bromodomains (8, 21, 32, 40). Many
TAFIIs contain another specialized motif, the
histone-like fold (11, 12, 13, 19, 36, 59). These include
TAF60p, TAF17p, TAF68/61p, TAF40p, TAF19p, TAF47p, TAF25p, and
TAF48p; for simplicity, only the yeast
TAFIIs are mentioned, although their eukaryotic
homologues also contain this motif (for reviews, see references
13, 19, and 36).
Perhaps the least understood motif found in
TAFIIs is the WD40 repeats of
TAF90/hTAFII100/dTAFII80
(9, 10, 42, 51, 52). The function of this domain is not
known, but it is speculated to be involved in protein-protein
interactions, as has been proposed for other WD40-containing proteins
(49). Despite the highly conserved nature of this region
of the protein, it has been reported that the WD40 repeat domain is not
necessary for incorporation of hTAFII100 into
TFIID (9). Moreover, the Schizosaccharomyces pombe homologue of TAF90,
taf72+, cannot complement the function of
Saccharomyces cerevisiae TAF90, but a chimera encoding the N
terminus of TAF90p and the C terminus of Taf72p can do so
(60), indicating that the N terminus of TAF90p provides
the species specificity. Therefore, all of the available evidence
suggests that the N terminus of TAF90p, and those of its homologues, is
most important for its incorporation into TFIID. That is not to say
that the C terminus is dispensable, as the only temperature-sensitive
mutants characterized to date have mutations located within the C
terminus (2).
There is an ongoing debate on what fraction of the genome is dependent
upon certain TAFII genes for its expression and
on what accounts for the differences observed between laboratories and
species (1, 17, 18). Mutation or depletion of certain TAFII subunits results in restricted
transcription defects (2, 20, 26, 33, 53), whereas
mutation of other subunits reduces the transcription of many genes
(3, 25, 31, 35, 37, 43, 45). The identification of certain
TAFIIs besides TFIID in nuclear complexes
(15, 22, 29, 39, 55) provided a possible explanation for
the differences observed among TAFII genes. For
example, genome-wide studies demonstrated that TAF145 and
GCN5 perform some redundant functions in transcription, as a
double taf145 gcn5 mutant shows defects in the transcription of a larger fraction of the genome than for the sum of the individual mutations (26). These observations suggest that certain
TAFIIs are responsible for the transcription of a
large number of genes because they are shared by TFIID and the
Spt-Ada-Gcn5 HAT complex (SAGA). Nonetheless, an inconsistency in this
theory is that mutation of TAF90, encoding a subunit of
TFIID and SAGA, affects only a small number of genes (2,
26). Of the TAFIIs shared between TFIID
and SAGA, TAF90p is the only one whose loss of function does not
broadly affect transcription. However, the number of mutants
characterized thus far has been small, and different mutant alleles
might be expected to have diverse consequences for the structures of
TFIID and SAGA. Another factor that complicates the interpretation of
the results obtained using various TAFII mutants
is that the effects of these mutations on TFIID and SAGA integrity have
not been thoroughly examined.
A comprehensive analysis of TAF90 was performed in an
attempt to understand its role in the function of TFIID and SAGA.
Eleven temperature-sensitive mutants were isolated and were
characterized in regard to their effects on transcription and on the
structures of TFIID and SAGA. We found that all of the mutants
contained amino acid substitutions within the conserved C terminus of
the protein, particularly within the WD40 repeats. Mutations within this region caused defects in the ability of TAF90p to interact with
TFIID and SAGA. While 10 of the 11 alleles displayed a variety of weak
and selective transcription phenotypes, a single allele of
TAF90 was identified that caused a rapid reduction in the
mRNA levels of a large number of genes. Our data indicate that the breadth and severity of the transcription defects of each mutant correlated strongly with their effects on TFIID structure but less so
with their effects on SAGA activity. The allele-specific defects we
report here illustrate the point that multiple alleles need to be
examined to fully understand the role of TAFIIs
in transcription.
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MATERIALS AND METHODS |
Yeast strains and genetic manipulations.
The strains used in
this study were as follows: YJR224 (mat
taf90::TRP1
[pRS316-TAF90] ade2-101
his3200 leu2-3,112
ura3-52 trp1-901 lys2-801
suc2-9), YJR226 (same as YJR224 except
containing pRS313-HA-TAF90 or
pRS313-HA-taf90-x in place of
pRS316-TAF90, where x is the allele number), YJR240
(mata
taf90::HIS3 [pRS316-TAF90] ade2-101
his3200 leu2-1
ura3-99 lys2-801), YJR241 (same as
YJR240 except containing pRS415-HA-TAF90 or
pRS415-HA-taf90-x), YJR500
(mata/
taf90::TRP1/TRP1
[pRS316-TAF90] his4-917
/ his4-917 lys2-17R2/lys2-17R2
leu2/leu2 ura3-52/ura3-52), YJR501 (mata taf90::TRP1
[pRS316-TAF90] his4-917 lys2-17R2
leu2 ura3-52), and YJR530 (same as YJR501 except
containing pRS415-HA-TAF90-x).
Mutants were created by hydroxylamine mutagenesis of
pRS313-HA-TAF90 in vitro for 60, 90, and 120 min at 70°C.
After purification of the plasmid, each pool was independently
transformed into YJR224 and plated onto histidine dropout plates. The
transformants were subsequently replica plated onto 5-fluoro-orotic
acid (48) and placed at 24, 30, and 37°C.
Temperature-sensitive mutants were identified based on growth at 24°C
but inviability at 37°C. The mutants were verified, and the coding
region was sequenced to identify the base substitutions. The mutants
were transferred into pRS415 by gap repair, and the resulting plasmids
were used to transfer the mutations into additional genetic backgrounds (YJR241 and YJR530). For the SPT analysis, strain FY632 was transformed with a taf90::TRP1 DNA fragment
generated by PCR (4), and the resulting transformants were
selected for on synthetic complete medium without tryptophan.
Individual colonies were screened by PCR and by Southern blotting to
confirm the disruption of TAF90. YJR500 cells were
transformed with pRS316-TAF90, and the transformants were
sporulated on solid medium for 4 days. Tetrads were dissected, and
genotypes of the resulting spores were identified by replica plating
onto selective media and mating type testing. The resulting strain,
YJR501, was transformed with pRS415 containing TAF90 or its
mutant derivatives. Next, transformants were selected for twice on
5-fluoro-orotic acid plates, and the temperature-sensitive mutants were
confirmed by monitoring growth at 24 and 37°C.
Analysis of RNA.
To measure poly(A)+
mRNA levels, total RNA was prepared by the acid-phenol extraction
method, and 10 µg was applied to charged nylon using a dot blot
manifold. The membrane was blocked and hybridized with a
32P-labeled dT20
oligonucleotide (Pharmacia) as described previously (54).
For Northern blotting (44) and S1 nuclease protection (7), total RNA was prepared by the glass bead disruption
procedure (2, 53). For the genes RPS5,
TRX1, CLB2, CLN2, SUC2,
HUG1, and scR1, gel-purified PCR-generated probes
were used. ADH2 RNA was detected using an oligonucleotide
probe specific for a region nonhomologous to ADH1.
Whole-cell extracts and immunoprecipitation (IP).
Yeast
cells were grown in 2% peptone-1% yeast extract-2% dextrose (YPD)
supplemented with 20 µg of adenine sulfate per ml (YAPD) (500 ml) to
an optical density at 600 nm (OD600) of
approximately 1.0 to 1.2. An equal volume of 24°C YPD (for
permissive-temperature extracts) or 50°C YPD (for
nonpermissive-temperature extracts) was added, and the cells were grown
for an additional 3 h at 24 or 37°C, respectively. Whole-cell
extracts were prepared as described previously (42, 58).
One milliliter of whole-cell extract was adjusted to a total protein
concentration of 1.5 mg/ml with 0.2 M buffer T (same as buffer B
described by Reese et al. [42] but with 0.2 M potassium
acetate), and the insoluble material was removed by centrifugation for
10 min at 14,000 rpm in a Microfuge. Two microliters of anti-TAF90p or
anti-TAF145p polyclonal antibody (53) was added, and the
mixture was incubated at 4°C for about 4 h. The tubes were
centrifuged for 10 min at 14,000 rpm, and the supernatant was
transferred to a new tube. Thirty microliters of a 50% slurry of
protein A beads (Repligen) in 0.2 M buffer T was added, and the tubes
were incubated overnight at 4°C with end-over-end mixing. The protein
A beads were collected by low-speed centrifugation (500 rpm, 4°C, 5 min), and the supernatant was carefully removed. The beads were washed
four times with 0.9 ml of ice-cold wash buffer (same as 0.2 M buffer T
but containing 0.1% NP-40). After the final wash, the supernatant was
carefully removed and the proteins were eluted by heating in 1.5×
sodium dodecyl sulfate (SDS) loading buffer. Samples were fractionated on 8% (14- by 10-cm) and 12.5% (8- by 10-cm) SDS-polyacrylamide gels
and transferred to nitrocellulose. For the inputs, 30 µg of extract
was loaded onto similar gels.
Purification of SAGA.
Six 500-ml cultures of yeast were
grown at room temperature in YAPD until an OD600
of 1 to 2 was reached and then were diluted with an equal volume of
prewarmed YAPD (50°C). SAGA was purified essentially as described by
Grant et al. (14). Extracts containing an equal quantity
of total protein (440 mg in about 40 to 43 ml) were mixed overnight at
4°C with 7.5 ml of Ni-nitrilotriacetic acid (Ni-NTA) agarose
(Qiagen). The slurry was poured into a 1.5- by 10-cm column (Kontes) at
4°C, washed with wash buffer (20 mM imidazole [pH 7.0], 100 mM
NaCl, 0.1% Tween 20, 10% glycerol, 2 µg of pepstatin A per ml, 2 µg of leupeptin per ml, 5 µg of aprotinin per ml, and 1 mM
phenylmethylsulfonyl fluoride [PMSF]), and eluted in same buffer
containing 300 mM imidazole (pH 7.0). The Ni-NTA column eluate was
loaded onto a Mono Q HR 5/5 column (Pharmacia), and the column was
washed with 1 column volume of 100 mM Q-buffer (50 mM Tris-HCl [pH
8.0], 100 mM NaCl, 0.1% Tween 20, 10% glycerol, 2 µg of pepstatin
A per ml, 2 µg of leupeptin per ml, 5 µg of aprotinin per ml, and 1 mM PMSF) and eluted using a linear gradient from 100 to 500 mM NaCl
through a volume of 25 ml. The SAGA-containing fractions were pooled
(about 3 ml) and concentrated to a final volume of about 400 µl using
a Centricon YM-30 filtration unit (Millipore). A 250-µl portion was
loaded onto a Superose 6 HR 10/30 column equilibrated with 40 mM HEPES (pH 7.5)-0.5 M NaCl-0.1% Tween 20-10% glycerol-1 mM PMSF and
eluted at a flow rate of 0.2 ml/min using a Biologic HR system
(Bio-Rad). Fractions (0.5 ml) were collected and frozen at 
80°C
after the addition of 50 µg of insulin.
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RESULTS |
Isolation and characterization of TAF90
mutants.
To study the function of TAF90, a screen for
temperature-sensitive mutants was performed. Eleven mutants that failed
to support growth at 37°C were isolated, and their mutations were
identified by DNA sequencing (Fig. 1).
All but two mutants (taf90-8 and -19) have
multiple amino acid substitutions. Strikingly, every mutant has
at least one amino acid change within the C terminus, and 9 of the 11 mutants have substitutions within a WD40 repeat. Interestingly, 8 out
of the 11 mutants have an amino acid substitution within the second
WD40 repeat. Several of the mutants have mutations that lie close to
the locations of those in two previously identified TAF90
mutants, referred to as taf90ts2-1 (S702D,
G793R) and taf90ts3-1 (G711E, G712S) by
Apone et al. (2). In fact, taf90-8 shares the
exact amino acid substitution (G793R) as one of the two mutations contained in taf90ts2-1. In no case did we
isolate a mutant that contained changes exclusively within the N
terminus of the protein. While we have not delineated the contributions
of each of the substitutions in the mutants with multiple mutations,
clearly the data as a whole suggest that mutations in the C terminus
largely are responsible for the loss of function of these mutants.
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FIG. 1.
Isolation of TAF90 mutants. A table of
amino acid substitutions and the locations of those in the C terminus
relative to the putative WD40 repeats are shown.
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The growth phenotypes of each allele were examined at 23, 30, and
37°C (Fig. 2A). As expected, none of
the mutants grew at 37°C, consistent with their isolation as
temperature-sensitive mutants. Differences in growth rates were
observed among the mutants at 23, 30, and 34°C (not shown),
suggesting a range of severity among the alleles. The
taf90-1, taf90-8, and taf90-10 mutants displayed a slight reduction in growth rate at 23°C, which was more
prevalent at 30°C (Fig. 2A). In addition, a number of mutants grew
significantly slower than wild-type cells at 23°C in liquid medium,
such as the taf90-1, taf90-3, and
taf90-19 mutants, despite showing comparatively milder
defects on solid medium at the same temperature (not shown).
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FIG. 2.
Characterization of growth phenotypes. (A) Growth
phenotypes on rich medium. Cultures were grown in rich medium to an
OD600 of 1.0, and 2 µl of 10-fold serial dilutions was
spotted onto agar plates. Plates were grown for 3 days at the
temperatures indicated. (B) Recovery of TAF90 mutants
from exposure to the restrictive temperature. Cultures were grown in
liquid rich medium to an OD600 of 1.0, and then 2 µl of
10-fold serial dilutions was spotted onto solid rich medium. Plates
were grown either for 3 days at 23°C or for 12 or 24 h at 37°C
and then for 3 days at 23°C. Since the cells were plated onto agar
plates at room temperature, the exact time that the cells were exposed
to a temperature of 37°C is not known.
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It has been proposed that certain weak temperature-sensitive (leaky)
alleles of TAFII mutants fail to display true
loss-of-function phenotypes because they have residual activity at
37°C, despite their apparent lack of growth in liquid culture
(25, 31). For example, mutants with certain
temperature-sensitive alleles of TAF40 that display
restricted transcription phenotypes do not lose viability even after 2 days at the nonpermissive temperature, whereas a mutant with another
allele that displays broader transcription defects loses viability
during that time (25). These observations necessitated
that we analyze the ability of each mutant to recover from a transient
exposure to the nonpermissive temperature, so as to judge the
efficacy of the mutations in destroying TAF90 function. In
addition to the TAF90 mutants, we analyzed three well-characterized temperature-sensitive mutants considered to be tight
based on the rapid disruption of transcription that results from their
transfer to 37°C. These mutants are those carrying the
rpb1-1 allele of the gene encoding the largest subunit of Pol II (20, 38), the tbpts-1
allele of the gene encoding TBP (7), and the
taf17-L68P,E148G allele of the TBP-associated factor 17 (31). Serial dilutions of each culture were spotted onto
rich medium and either were grown solely at the permissive temperature
or were exposed to the restrictive temperature for 12 or 24 h
before being returned to the permissive temperature to resume growth.
The results indicate that most of the TAF90 mutants showed a
loss of viability similar to that of the rpb1-1,
tbpts-1, and taf17-L68P,E148G
mutants (Fig. 1B). The noted exceptions are the taf90-20 and
taf90-17 alleles. The taf90-20 mutant failed to
recover from a 12-h exposure to 37°C and was by far the most severely
affected among all the general transcription mutants examined here.
This mutant was even more strongly affected than the rpb1-1
and tbpts-1 strains. On the other hand, the
taf90-17 strain recovered even after a 24-h incubation at
37°C. Therefore, with the exception of the taf90-17
mutant, all of the TAF90 mutants described here were at
least as defective in their recovery from an exposure to the
restrictive temperature as the rpb1-1,
tbpts-1, and taf17-L68P,E148G
mutants. This analysis uncovered differences in the growth phenotypes
of the taf90-17 and taf90-19 mutants: the
taf90-19 allele is more deleterious to cell viability.
Moreover, the taf90-17 mutant grows well at 34°C, whereas
the taf90-19 mutant grows very poorly at this temperature
(not shown). Therefore, based upon these two growth phenotypes, the
additional amino acid substitution, G691S, contained in the
taf90-17 mutant is somewhat compensatory for the defects
caused by the S542F mutation found in both mutants.
Mutations in certain SAGA subunits can suppress Ty
insertions (SPT phenotype) and cause inositol and galactose
auxotrophies (16, 56); therefore, we screened each mutant
for these phenotypes. The TAF90 mutants were reconstructed
in a strain containing a Ty insertion at the HIS4
locus, his4-914 (56, 57), and plated onto
medium lacking histidine. Whereas a strain containing a mutation in
spt3 grew on medium lacking histidine, indicating
suppression, none of the TAF90 mutants showed this phenotype
(not shown). In addition, none of the mutants displayed obvious
galactose or inositol auxotrophies under conditions that would support
growth (not shown).
Allele-specific defects in Pol II-mediated transcription.
To
determine the effects of TAF90 mutations on Pol II-mediated
transcription, changes in mRNA levels were detected by probing a blot
of total RNA with radiolabeled oligo(dT). This method has been used
repeatedly to analyze transcription defects in
TAFII mutants (3, 25, 31, 46, 54).
From the data presented in Fig. 3, it
appears that shifting each mutant to 37°C had diverse consequences on
the levels of total poly(A)+ RNA. Based upon the
magnitude and kinetics of RNA loss, the mutants can be roughly grouped
into classes. The first category of mutants exhibited a reduction in
mRNA at the permissive temperature, a small drop at the 1-h time point,
and then maintenance of that level to 4 h. Examples of this class
are the taf90-1, taf90-15, taf90-17,
and taf90-19 mutants. The next class showed slight, gradual
reductions for the first 2 h and then a sharp decline at 4 h,
such as the taf90-8 mutant. Most mutants represent the third
type, which exhibited a steady decrease immediately after the
temperature shift. Examples of this class are the taf90-3, taf90-10, taf90-14, and taf90-16
mutants and the previously described taf90ts2-1 mutant (2, 54).
Even within this group the kinetics and magnitude of RNA loss varied
considerably, from the weaker phenotypes exhibited by the
taf90-3 and taf90ts2-1 mutants
to the stronger phenotype of the taf90-14 mutant. Finally, there is one mutant, taf90-20, which is clearly unique.
Shifting this mutant to 37°C resulted in a rapid, and significant,
reduction in poly(A)+ RNA levels (Fig. 3).
Significantly, the kinetics and magnitude of mRNA loss observed in the
taf90-20 mutant were very similar to those of the Pol II
mutant, rpb1-1. Specifically, the mRNA levels in the
taf90-20 mutant were reduced to 32% of the wild-type level
by 1 h and to 7% by 4 h, while the quantities in the
rpb1-1 strain declined to 20 and 6%, respectively. In
summary, it appears that a variety of transcription defects were
observed, ranging from weakly affected, as in the case of
taf90-1 and taf90-3, to very severe, as seen in
the taf90-20 mutant.
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FIG. 3.
Poly(A)+ RNA levels. (A) Cultures were grown
in YPAD to an OD600 of approximately 0.3 and then shifted
to 37°C. Aliquots were removed prior to the shift (0 h) and after 1, 2, and 4 h at 37°C, and RNA was isolated by acid-phenol
extraction. Ten micrograms of total RNA was spotted onto
nitrocellulose, and poly(A)+ RNA was detected by probing
with a 32P-labeled dT20 probe. (B)
Quantification. Spots were analyzed using a phosphorimager. The
percentage of wild-type (WT) levels was determined by comparing the
counts in each spot with those in the wild-type spot from 0 h. The
taf90-2 strain (#) is a previously described noncogenic
temperature-sensitive strain used as a control (2). The
relative levels of mRNA in the mutants versus the wild type varied
between 5 and 15% between experiments, but most points varied by less
than 10%.
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Next, we wanted to determine if the changes in total
poly(A)+ RNA described above reflect the loss of
a few very abundant RNAs or a reduction in the expression of many
different RNAs. If the latter is true, we expect that the kinetics and
magnitude of the reductions of specific transcripts will match those
observed in the poly(A)+ blots. Genes that have
been routinely used to characterize other TAFII
mutants were selected (2, 3, 25, 35, 36, 37, 43, 46, 53).
The steady-state levels of TRP3, ACT1,
DED1, TCM1, and MET19 RNAs were
examined by S1 nuclease protection, and the transcripts of
CLB2, CLN2, RPS5, and TRX1
were detected by Northern blotting. Shifting cells to the restrictive
temperature significantly affected the transcription of
TRP3, MET19, TCM1, and ACT1
in nearly every mutant (Fig. 4). The RNA
levels of these genes showed a gradual decrease over time, and the
severity of the defects varied among the mutants, similar to what was
observed in the analysis of poly(A)+ RNA
quantities. For example, shifting the taf90-1 mutant to
37°C only weakly affected the levels of total
poly(A)+ RNA up to 4 h, and similarly,
nearly wild-type levels of mRNA for each of these individual genes were
observed over the same time period (Fig. 4). Moreover, the
taf90-17 mutant displayed reduced total mRNA levels at
23°C, which were not strongly reduced further by the temperature
shift, and likewise it displayed a reproducible reduction in
TRP3, MET19, DED1, and ACT1
RNA levels at 23°C that was not exacerbated by the temperature shift.
Shifting the taf90-20 mutant to 37°C caused rapid and
dramatic losses of TRP3, MET19, TCM1,
and ACT1 mRNAs by 1 h, similar to what was observed for
the levels of poly(A)+ RNA. Therefore, the losses
of poly(A)+ RNA observed in each mutant closely
correlated with the magnitude and kinetics of the losses of the
individual transcripts examined here as well. In contrast to the case
for these other genes, DED1 transcription was only weakly
affected in all mutants, including the taf90-20 strain.
Therefore, even with the strongest allele, some genes are not strongly
affected by the inactivation of TAF90.
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FIG. 4.
Transcription analysis of specific messages. Cultures
were grown, treated, and processed as described in the legend to Fig.
3. (A) S1 analysis of specific messages. Ten micrograms of total RNA
was analyzed by S1 nuclease protection (7).
tRNAIle, a Pol III transcript, was used as an RNA control.
(B) Northern blotting. Fifteen micrograms of total RNA was fractionated
on formaldehyde-containing gels and transferred to a membrane, and the
specific transcripts were detected by hybridization with
32P-labeled probes corresponding to the coding
regions of CLB2, CLN2,
TRX1, and RPS5. The Pol III-transcribed
scR1 was used as a loading control.
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The messages for TRX1, RPS5, CLN2, and
CLB2 were analyzed because they are particularly sensitive
to mutations in TAF145 and other
TAFIIs (3, 20, 25, 47, 54; J. C. Reese, unpublished data). The results presented in Fig. 4B indicate
that the inactivation of most alleles, with the exception of
taf90-17, strongly affected the transcription of
RPS5, and all mutants displayed significant reductions in
TRX1 mRNA (Fig. 4B). Once again, inactivation of taf90-20 had the most deleterious effect on the levels of
TRX1 and RPS5 RNAs; their transcription is nearly
abolished by 1 h at the restrictive temperature. This is the only
allele that exhibited a reduction in these transcripts similar to that
for the rpb1-1 mutant. Even mutants such as
taf90-1, taf90-3, and taf90-8, which displayed weaker reductions in total poly(A)+ RNA
(Fig. 3) and in the specific transcripts analyzed in Fig. 4A at up to
4 h, exhibited more rapid and larger reductions in the expression
of these two genes. Thus, it appears that the expression of
RPS5 and TRX1 is especially sensitive to
TAF90 mutations and is not as prone to allelic variations.
Cyclin expression is abolished in certain TAFII
mutants, and in some cases this may explain the cell cycle phenotypes
observed in those mutants (20, 26, 28, 54). Our
preliminary analysis revealed that only the taf90-9 and
taf90-20 mutants displayed a convincing arrest in
G2/M at the restrictive temperature,
whereas only very weak arrest phenotypes were observed in other
mutants (J. C. Reese, A. K. Fisher, and T. A. Albright-Frey, unpublished data). The expression of the cyclins
CLN2 and CLB2 was analyzed following a
temperature shift, and with the exception of taf90-20, it
appears that the alleles had a weak effect or no effect on CLB2 and CLN2 transcription (Fig. 4B). Most
mutants, such as taf90-10, showed a decrease in
transcription only at the 4-h time point, a time when the decrease in
transcription may result from decreased cell vitality.
Activated transcription in TAF90 mutants.
The
genes analyzed thus far were actively transcribed at the moment the
mutants were inactivated. We next examined the activation of genes when
the mutants were exposed to the restrictive temperature. We first
examined the derepression of catabolite-repressed genes, specifically,
ADH2 and SUC2. It has been reported that the
depletion of TAF90p leads to a decrease in ADH2 gene
expression and that TFIID interacts with Ard1p, the specific activator
of this gene. (24). SUC2 was chosen for
analysis because we have found that its derepression is severely
impaired in certain TAFII mutants (Reese,
unpublished data). The wild-type strain and each of the mutants were
grown to log phase in high-dextrose medium, shifted to 37°C, and then
switched to prewarmed low-dextrose medium for 1.5 and 3 h.
Although none of these mutants displayed abated ADH2 transcription, mRNA levels were reduced in the taf90-1,
taf90-14, and taf90-17 mutants (Fig.
5A). Several alleles exhibited wild-type levels of ADH2 RNA, reaffirming the allele-specific nature
of these mutations. In addition, we found that the expression of ADH2 is only weakly affected in the taf90-20
mutant, which displayed the strongest transcription phenotype. We
cannot account for the differences between our results and those of
Komarnitsky et al. (24) with certainty. The previous study
utilized a copper-inducible shutoff strategy that required
significantly longer incubation times under the restrictive conditions,
which may have resulted in secondary effects that adversely affected
ADH2 expression.
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FIG. 5.
Induced transcription. (A) Derepression of
SUC2 and ADH2. Cultures of YJR241-x were
grown initially in YPAD at 23°C. Following the removal of an aliquot
from each culture, cells were centrifuged, resuspended in YPA
containing a low concentration of dextrose (0.05%) prewarmed to
37°C, and grown at the restrictive temperature. RNA was isolated from
aliquots that were removed after 1.5 and 3 h at 37°C in
low-dextrose medium. Transcripts were detected by Northern blotting.
scR1 was used as a loading control. (B) Analysis of a DNA
damage-induced gene. Cultures of YJR241-x were grown in rich medium and
shifted to the nonpermissive temperature. Aliquots were removed
(permissive [lanes P]) prior to the temperature shift. After 1 h
at 37°C, MMS was added to 0.02%. Aliquots were taken at 1.5 and
3 h after treatment with MMS. A separate culture was maintained at
37°C but left untreated (nonpermissive [lanes N]) for 4 h.
HUG1 RNA was analyzed by Northern blotting. scR1 was
used as a loading control.
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Several TAF90 mutants showed large reductions in the levels
of SUC2 mRNA (Fig. 5A), including the taf90-1 and
taf90-3 mutants, which we classify as having weaker
transcription phenotypes. Interestingly, the taf90-14 and
taf90-17 mutations appear to have no effect of SUC2 transcription, whereas they were two that had a
stronger effect on ADH2 expression. Our analysis of these
two catabolite-repressed genes highlights the allele- and gene-specific
nature of the transcription defects in each mutant and clearly
demonstrates the compensatory nature of the G691S substitution in the
taf90-17 mutant. The activation of SUC2 is not
strongly affected by the taf90-17 allele, whereas it is
drastically reduced by the taf90-19 allele.
Depletion of TAF90p or inactivation of a temperature-sensitive allele
strongly reduces the expression of DNA damage-induced genes
(27). We therefore examined the expression of a
representative of this class of genes, HUG1, in the mutants.
Cells were shifted to 37°C and treated for 1.5 and 3 h with the
DNA-damaging agent methyl methanesulfonate (MMS). A separate untreated
aliquot of cells was maintained at 37°C for 3 h to detect
changes in the uninduced level of transcription (Fig. 5B). The results
presented in Fig. 5B indicate that inactivation of every allele
affected the DNA damage-induced expression of HUG1.
Expression was significantly reduced even in the taf90-1,
taf90-3, and taf90-17 mutants (although to a
lesser extent), which display the most selective transcription defects
of all of the mutants described here. In agreement with our previous
analysis of the TAFII dependence of DNA
damage-inducible genes (27), these alleles appeared to
specifically affect the derepression of this gene, as the uninduced
level of RNA was not significantly reduced even after 3 h at
37°C (Fig. 5).
Some mutants of TAFIIs shared by TFIID and SAGA
affect Gcn4p-activated transcription (3, 35, 37);
therefore, we examined the expression of the Gcn4p-responsive gene
HIS3 in the mutants. HIS3 contains several
transcription start sites; a nonconsensus TATA element
(TC) is responsible for constitutive
transcription from position +1, while a consensus TATA element
(TR) is responsible for transcription from +13
(5). Gcn4p preferentially activates transcription from the
+13 start site (5, 6). The cells were grown for 3 h
at 37°C in complete medium (noninducing conditions) or in medium
lacking histidine for 1.5 h at 37°C, followed by another
1.5 h in the presence of 30 mM 3-aminotriazole (inducing conditions). Comparing the levels of HIS3 mRNA when the
cells are grown under noninduced conditions at 23°C (permissive)
versus 37°C (nonpermissive) gives an indication of the effect of each mutant on the basal transcription level. In all but one mutant, shifting the cells to 37°C for 3 h did not affect the uninduced levels of the +1 or +13 transcripts from HIS3 (Fig.
6). A reduction in the +1 transcript was
observed in the taf90-20 mutant in this and other
experiments. In contrast, the level of starvation-induced transcription
from the +13 transcript was strongly reduced for every allele. The
magnitudes of induction varied between 33 and 6% of the wild-type
level, but most were reduced to approximately 20%. Not surprisingly,
the taf90-20 mutant displayed the largest reduction in
induced transcription, to 6% of wild-type levels. Interestingly,
unlike what was observed in the numerous examples presented above, the
taf90-19 and taf90-17 alleles were equally defective for the activation of HIS3 by Gcn4p. Thus, the
compensatory nature of the G691S substitution is not universal and
displays some gene specificity. These results demonstrate that
TAF90 is essential for the activation of HIS3 by
Gcn4, similar to the case for other TAFIIs found
in both TFIID and SAGA.
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FIG. 6.
Effects of TAF90 mutations on
Gcn4p-mediated transcription. Cells were grown under noninducing
conditions in synthetic complete medium at 24°C (lanes N) and at
37°C for 3 h (lanes N, 37). A separate culture was grown in
synthetic complete medium without histidine, shifted to 37°C for
1.5 h, and incubated for another 1.5 h in the presence of 30 mM 3-aminotriazole (lanes I, 37). Analysis of HIS3 was
performed by S1 nuclease protection. The induction level (+13 I/N) is
the ratio of the +13 transcript from cells grown under inducing
conditions at 37°C to that from cells grown under noninducing
conditions at 37°C. The reduced tRNA signal in the
taf90-19 sample is a gel-loading artifact and is not
reproducible. WT, wild type.
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Inactivation of TAF90 mutants reduces TFIID and SAGA subunit
levels.
Inactivation of certain TAFII
temperature-sensitive alleles, or depletion of its protein, results in
the degradation of multiple TFIID and SAGA subunits (25, 31, 43,
46, 53). Codegradation of TAFIIs is a
reasonable indicator of the disruption of complex structure; therefore,
we monitored the levels of TAFIIs, TBP, and two
SAGA components after shifting the cells to 37°C. In many mutants,
the levels of TFIID subunits were not strongly reduced until 4 h
at 37°C (Fig. 7). The clearest
exception was the taf90-20 mutant, which showed a steady
decrease in TAF90p, TAF145p, TAF60p, and, to a lesser degree, TBP
immediately upon the temperature shift. Thus, by this criterion the
taf90-20 allele is the most detrimental to the structure of
the TFIID complex. Overall, the degree to which inactivation of these
mutants reduced TAFII levels correlated with
their growth and transcriptional defects. The taf90-20 and
taf90-17 mutants had the strongest and weakest effects on
the levels of TFIID subunits, respectively, and likewise they had the
most and least severe growth and transcriptional defects. Additionally,
the taf90-19 mutant displayed stronger growth and transcriptional defects than the taf90-17 mutant (Fig. 2, 4,
and 5) and likewise displayed more drastic reductions in
TAFIIs. Finally, the taf90-1 mutant,
which showed significant growth defects at the permissive temperature
(Fig. 2A and data not shown), had measurable reductions in TFIID and
SAGA subunits, even when grown at 23°C. This is most obvious for
TAF145p, TAF60p, TAF47p, TAF17p (see below), and Ada1p. The
steady-state levels of two SAGA-specific components, Ada1 and Spt7,
were also examined. Inactivation of each allele had very different
consequences for these two proteins. Whereas Spt7p remained relatively
stable in most mutants until the 4-h time point, the levels of Ada1p
were significantly reduced earlier in the temperature shift. This is
particularly apparent in the taf90-8, taf90-14,
taf90-17, and taf90-19 mutants, which showed
noticeable reductions in Ada1p by 1 h. Interestingly, the taf90-20 mutant, which had the largest reductions in TFIID
subunits, exhibited relatively minor reductions in Spt7p and Ada1p up
until the 4-h time point. Therefore, in regard to the degradation of these two SAGA subunits, this allele appears to be no more destructive than the other mutations.
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FIG. 7.
Steady-state levels of TFIID and SAGA subunits following
a temperature shift. Wild-type and mutant strains were grown at 24°C
in YPAD to an OD600 of approximately 0.4, and after removal
of an aliquot for the t = 0 time point, they were
transferred to a 37°C shaking water bath. Aliquots of culture were
removed after 1, 2, and 4 h at the nonpermissive temperature.
Extracts prepared from these cells (20 µg of protein) were
fractionated by SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose, and the specific proteins were detected by
immunoblotting.
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Mutations in TAF90 affect its ability to associate
with TFIID and SAGA.
Next, we examined the ability of each mutant
to bind to TFIID and SAGA in extracts prepared from cells grown at the
permissive temperature and after 3 h of growth at 37°C by co-IP
using anti-TAF90p antisera. The ability of each mutant to incorporate
into TFIID was judged by the IP efficiencies of Tsm1p, TAF145p, and
TAF47p, since they are not subunits of SAGA (15).
Likewise, immunoblotting for Spt7p, Ada3p, and Ada1p monitored their
association with the SAGA complex. Unfortunately, the antibodies to
Ada3p were not of sufficient quality to detect the levels of these
proteins in whole-cell extracts; nonetheless, they could be used to
analyze the immunopurified material, and these results are presented. The loads and IP material are presented in Fig.
8A and B, respectively. The quantity of
TAF90p immunoprecipitated from each sample largely correlated with its
relative abundance in the extracts (input samples) and in many cases
was not significantly different for samples from cells grown at 23 and
37°C (Fig. 8B). Only the samples from the taf90-16 and
taf90-20 mutants showed significantly reduced amounts of
TAF90p in the IPs. Virtually every mutant displayed a
temperature-dependent defect in its ability to interact with most TFIID
subunits examined here. The two exceptions were the taf90-1
mutant, which showed a temperature-independent reduction in its ability
to interact with other TFIID subunits, and the taf90-17
mutant, which showed only minor reductions in extracts from cells grown
at 37°C (Fig. 8B). The taf90-20 mutant showed the
strongest defects. However, since the levels of
TAFIIs were severely reduced in the extracts of
this mutant, we can be certain only that the taf90-20 allele
is extremely disruptive to the TFIID complex. Small amounts of TAF90p,
TAF60p, TAF68p, and TAF25p was detected in the IP material from the
extracts of this mutant grown at 37°C, which may be attributed to
TAFIIs contained in the SAGA complex (see below).
This assay also highlights differences between the taf90-17
and taf90-19 alleles. In agreement with what was observed
for these two alleles in regard to their effects on the steady-state
levels of TAFIIs (Fig. 7), the binding of TFIID
subunits to the taf90-19p mutant protein was reduced to a greater
degree than that to the taf90-17p mutant protein.
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FIG. 8.
Co-IP studies with extracts of TAF90
mutants. Whole-cell extracts were prepared from cells grown at room
temperature (approximately 24°C) or at 37°C for 3 h. Complexes
containing TAF90p were immunoprecipitated from cell extracts using
antibodies raised against TAF90p or TAF145p. Cell extracts and IPs were
analyzed by Western blotting using the antibodies indicated. The input
material (A) and complexes immunoprecipitated using antibody raised
against TAF90p (B) or TAF145p (C) are shown.
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Interestingly, while the levels of Tsm1p in the 37°C extracts were
not significantly reduced in each of the mutants (Fig. 8A), very strong
reductions in its ability to co-IP with the TAF90p mutants were
observed (Fig. 8B). Moreover, the taf90-3,
taf90-16, and taf90-19 mutants appeared to be
more strongly affected in their ability to co-IP Tsm1p versus either
TAF145p or TAF47p, suggesting that these mutants somewhat selectively
affect the interaction of Tsm1p with TFIID. The ability of TBP to co-IP
with TAF90p was severely compromised in all but the taf90-1
mutant when the cells were grown at 37°C (Fig. 8B). Therefore, the
majority of the TBP in these extracts (Fig. 8A) likely represents free TBP or TBP distributed among other complexes. In a number of cases, the
reduction of TBP in the IP material was disproportionately lower
than that of TAF145p; therefore, the TAF145p-TBP interaction alone is not sufficient to maintain TBP in the TFIID complex. A similar
result was also found in the analysis of a TAF68/61 mutant
(43).
The presence of Spt7p, Ada1p, and Ada3p in the immunoprecipitates was
examined to ascertain each mutant's ability to incorporate into the
SAGA complex. Most mutants, such as taf90-3,
taf90-8, taf90-9, taf90-10,
taf90-14, taf90-16, taf90-17, and
taf90-19, displayed temperature-dependent interactions with
Spt7p, Ada1p, and Ada3p, while the taf90-1 mutant displayed
a temperature-independent reduction in the IP efficiency of these three
SAGA subunits. Thus, most of the mutants described here are defective
for interacting with SAGA in a manner similar to that for TFIID. An
interesting exception is the taf90-20 mutant. This allele is
the most disruptive to TFIID, but the quantities of these three SAGA
subunits detected in the IPs were not significantly reduced. Therefore,
the mutations contained in this allele do not affect its ability to
interact with at least some SAGA components, and it is significantly
more competent to bind to SAGA than most of the other mutants described here. Comparing the results for the taf90-17 and
taf90-19 alleles revealed differences in the ability of
these two mutants to interact with SAGA versus TFIID components.
Whereas the additional amino acid substitution contained in the
taf90-17 mutant protein stabilized its interaction with
multiple TFIID subunits (see above), it did not confer the same effect
on SAGA components: the amounts of Spt7p, Ada1p, and Ada3p contained in
the IPs from extracts of the taf90-17 and
taf90-19 mutants were similar (Fig. 8B). Thus, it appears
that the additional amino acid substitution is compensatory for TFIID
but not for SAGA interactions. This hypothesis is consistent with the
results indicating that both mutants are equally defective for
HIS3 activation (Fig. 6), a process heavily dependent upon SAGA function (for reviews, see references 16 and
56).
While the results of Fig. 7 suggest otherwise, it is a formal
possibility that these mutations leave TFIID denuded of TAF90p but
otherwise intact after a temperature shift. The assay shown in Fig. 8B
measures the ability of TAF90p to interact with TFIID rather than the
integrity of the complex; therefore, we repeated the IPs using
antiserum to TAF145p (Fig. 8C). Overall, the results obtained using
anti-TAF145p antibodies to co-IP TAFIIs confirmed those in Fig. 8B; that is, the quantities of
TAFIIs coimmunoprecipitated from extracts of
mutants grown at 37°C were significantly reduced. The noted
difference is that less Tsm1p, TAF68p, and TAF47p were detected in the
anti-TAF145 IPs from the extracts of the taf90-1 and
taf90-10 mutants grown at the permissive temperature than in
the anti-TAF90p IPs (compare Fig. 8B and C). In addition, no TFIID
subunits were detected in the anti-TAF145p IPs from the 37°C extracts
of the taf90-20 mutant, even in overexposed blots (not
shown); therefore, the small quantities of TAF90p, TAF68p, TAF60p, and
TAF25p detected in the TAF90p IPs (Fig. 8B) likely originated from the
SAGA complex. In conclusion, our results show that each mutation has
different effects on TFIID structure, with taf90-1 and
taf90-17 being the least severe and taf90-20
being the most. In addition, comparison of the abilities of the
taf90-17p, taf90-19p, and taf90-20p mutant proteins to copurify with
TFIID and SAGA components suggests that TAF90p interacts with these two
complexes differently.
TAF90 mutations reduce SAGA abundance and activity.
The
results described in Fig. 8 indicate that inactivation of most TAF90p
mutants adversely affects SAGA subunit levels and/or their ability to
interact with the complex. Nonetheless, these are indirect assays for
SAGA complex activity, and the ability to co-IP SAGA subunits, as is
observed in the taf90-20 mutant, does not necessarily
indicate that the mutant complex is functional. The preparation and
fractionation of extracts from each of these 11 TAF90
mutants to analyze SAGA, at least in duplicate, would be a significant
undertaking. Therefore, three mutants were selected to more carefully
determine their effect on the ability of the SAGA complex to acetylate
nucleosomes. The taf90-20 mutant was chosen since it
completely disrupts TFIID structure and yet appears not to strongly
affect its ability to copurify with some SAGA components (Fig. 8B).
Thus, it is important to determine if SAGA, or a subcomplex, containing
the mutant protein is active. The taf90-17 mutant was chosen
because it has the weakest effect on TFIID structure and transcription
yet displayed measurable defects in its ability to bind SAGA subunits.
Finally, taf90-10 was chosen as a representative of a mutant
that displayed defects in both complexes and exhibited a moderate
transcription phenotype.
Extracts were prepared from cells grown at 37°C for 3 h, and HAT
complexes were purified by sequential chromatography over Ni-NTA
affinity and Mono Q ion-exchange columns (14). Aliquots of
alternating Mono Q fractions were used in HAT assays and analyzed by
Western blotting. Each of the four distinct HAT complexes
(14) retained on Ni-NTA-agarose were resolved in the
sample from wild-type cells, and their locations in the elution profile
are indicated above Fig. 9A. Their
identities were assigned by Western blotting for unique subunits (see
below and data not shown) and by histone substrate specificity. In each
of the mutants, four HAT activities were also resolved, but the amount
contained in the fractions where SAGA elutes (fractions 38 to 42) was
significantly reduced (Fig. 9B to D). In contrast, the activities of
the TAF90p-independent NuA4, ADA, and NuA3 complexes were comparable to
those of the wild-type sample and were within the normal variation
observed between different preparations (Fig. 9 and data not shown).
Next, we examined the elution profiles of individual SAGA components by
Western blotting. The Mono Q elution profile of the HAT complexes retained on Ni-agarose from wild-type extracts reveals that all of the
SAGA subunits examined coeluted predominantly with their HAT activity
in fractions 38 to 42, with the exception of those found in the ADA and
NuA4 complexes (Fig. 9A). Western blotting of the fractions from each
of the mutants revealed that significant amounts of most subunits were
also in earlier fractions (Fig. 9B to D), indicating a breakdown of the
mutant SAGA complexes. This is most obvious in the fractionation
profile for the taf90-20 mutant (Fig. 9B). Specifically,
Tra1p broadly eluted in fractions 18 to 40, and significant amounts of
Spt7p, Ada3p, Ada2p, Gcn5p, and TAF68p were detected in fractions 30 to
34 (Fig. 9B). It is not know if this represents a stable subcomplex of
SAGA or free subunits that coincidentally coelute in these fractions.
In addition to the appearance of breakdown products, the amounts of
SAGA components eluting in fractions 38 to 42 were somewhat reduced in
the taf90-17 (Fig. 9C) and taf90-20 (Fig. 9B)
samples and were drastically reduced in the taf90-8 (Fig.
9D) samples (for a more accurate estimation, see Fig. 10).
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FIG. 9.
Purification of HAT complexes from TAF90
mutants. Extracts prepared from mutants grown at 37°C for 3 h
were subjected to chromatographic analysis as described in Materials
and Methods. Aliquots of the Mono Q column fractions were analyzed by
Western blotting and HAT assays using nucleosomes as substrates.
Elution profiles for the wild type (A) and the taf90-20
(B), taf90-17 (C), and taf90-10 (D)
mutants are shown. Note that the blots presented in panel D were
deliberately exposed longer than those in panels A to C to detect the
small amounts of SAGA subunits eluting in fractions 38 to 42.
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The reduced HAT activity observed in each TAF90 mutant may
be due to a reduction in the amount of SAGA, an altered complex with
decreased catalytic activity, or both. Therefore, we directly compared
the SAGA levels and activity in the peak Mono Q column fractions. Equal
amounts (volumes) of the pooled fractions were used to measure the HAT
activity using oligonucleosomes as substrates (Fig.
10A, left panel), and an aliquot was
withdrawn and subjected to immunoblotting following the HAT assay (Fig.
10A, right panel). The results show that the levels of Gcn5p in the
Mono Q fractions were reduced about 2.5- and 5-fold in the
taf90-20 and taf90-17 mutants, respectively,
whereas the level was reduced approximately 10-fold in the
taf90-10 mutant. The level of HAT activity in the taf90-17 samples was roughly proportional to the levels of
Gcn5p, but the taf90-20 fractions contained an additional
reduction in activity that cannot be accounted for by reduced SAGA
levels (Fig. 10A, right panel). For example, this mutant has 43% of
wild-type Gcn5p levels but 25% of wild-type HAT activity (Fig. 10A).
Thus, even though the SAGA complex from the taf90-20 mutant
is largely intact, its mutations reduce the activity of the complex.
Higher HAT activity was detected in the taf90-10 fractions
compared to the level of Gcn5p. This is likely due to the contamination
of these fractions with quantities of the NuA3 complex, which elutes near the SAGA complex (14) (Fig. 9A).
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FIG. 10.
Analysis of mutant SAGA complexes. (A) SAGA has reduced
HAT activity in TAF90 mutants. Fractions containing SAGA
(fractions 38 to 42 from Fig. 9) were pooled and analyzed by Western
blotting and HAT assays using oligonucleosomes as substrates. The
relative levels of Gcn5p were determined from appropriately exposed
blots using NIH Image software and are expressed as the percentage of
the wild-type (WT) level. HAT activity was compared to that of SAGA
isolated from wild-type cells, which was set at 100%. (B) Size
exclusion chromatography. The pooled SAGA-containing fractions from the
Mono Q chromatography (fractions 38 to 42 from Fig. 9) were
concentrated by filtration and fractionated on a Superose 6 column.
Each fraction was analyzed by Western blotting and HAT assays using
nucleosomes as a substrate. The void volume corresponds to fraction 14. No HAT activity or SAGA components were detected in later-eluting
fractions, and thus only fractions 15 to 23 are shown.
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The integrity and activity of the mutant SAGA complexes from the
taf90-17 and taf90-20 strains were analyzed by
gel filtration chromatography. Unfortunately, the quantities of the
SAGA complex from the taf90-10 mutant were not sufficient
for this analysis. The results presented in Fig. 10B show that the
wild-type and mutant SAGA complexes eluted from the Superose 6 column
with the expected mass of approximately 2 MDa (14, 15).
While this analysis cannot rule out the possibility of a subtle change
in structure or composition, it does suggest that the size of the
mutant complexes is not significantly different from that of the wild
type. In addition, we did not detect the presence of additional
complexes eluting in later fractions by either HAT assays or Western
blotting for SAGA subunits (data not shown). It is important to note
that as observed in the Mono Q fractions, the nucleosomal HAT activity was significantly reduced (Fig. 10B). The HAT activities, measured in
liquid assays, for the taf90-20 and taf90-17
mutants were approximately 19 and 15% of that for the wild type,
respectively (not shown). We observed a slightly larger difference in
HAT activities between the mutants and the wild-type complexes eluting
from the sizing column versus that observed in the Mono Q fractions.
This may be attributed to a further reduction in activity in the
mutants during the processing of the samples for size exclusion
chromatography or their separation from contaminating NuA3 complex.
From these results, it appears that mutations in TAF90, like
for TAF68/61 (15), disturb the function of the
SAGA complex by reducing the total amount of the SAGA complex present
in the cell and impairing its ability to acetylate nucleosomes.
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DISCUSSION |
TAF90 mutants display allele-specific and broad
transcription defects.
The relatively thorough characterization of
a large number of mutants has allowed us to make a number of
observations regarding TAF90 function. The first significant
conclusion is that allele-specific defects in transcription can be
observed among different mutants of the same gene, despite the fact
that all alleles seem tight based upon their temperature-sensitive
growth phenotypes. There were a number of clear examples where the
expression of a gene was strongly reduced in one or more mutants but
not in others. This argues that results obtained with any single
temperature-sensitive allele may not reveal all of the functions of
this gene. While we have largely concentrated our discussion on cases
where the differences between alleles are very obvious, even the subtle differences in the magnitude and kinetics of RNA loss observed among
all of the alleles described here could have consequences for the
classification of certain genes as being TAF90 dependent when a cutoff is applied. By no means does our study diminish the
impact of the genome-wide studies performed to date. However, it
already has been recognized that analyzing the genome-wide expression
profile of any given mutant under a single growth condition does not
constitute a comprehensive analysis of its function (for a review, see
reference 61), and now it is clear that multiple alleles
of the same gene may be necessary when conditional mutants are used.
The second important observation is that mutations in TAF90
that cause a broad transcription defect can be isolated, as seen in
mutants of other TAFIIs that are shared by TFIID
and SAGA. Mutants of TAF68/61, TAF60,
TAF25, and TAF17 that satisfy this criterion had
been described previously (3, 31, 37, 43, 46), but until
this study such a mutant of TAF90 had not been. The
thermoinactivation of this mutant results in the rapid reduction in
total poly(A)+ RNA and in most of the specific
messages analyzed here. Significantly, the magnitude and timing of
these reductions were strikingly similar to those observed in the
rpb1-1 mutant, which is considered the standard for judging
primary transcription defects (20, 26). This phenotype is
unlikely to result from a reduction in only a small number of abundant
messages, because we found that many specific messages, some of which
are of low to medium abundance, were reduced likewise. Therefore,
mutations in TAF90 that abolish transcription of many
yeast genes can be isolated. We recognize that the analysis performed
on this mutant thus far cannot accurately determine the exact fraction
of the genome that is affected. Nonetheless, it is clear that this
mutation does have much stronger effects on the expression of more
genes than any of the other TAF90 mutations described here
or elsewhere. Equally clear is that inactivation of even the strongest
mutant described here did not significantly affect the expression of
all genes examined here, thus reinforcing the notion that
TAFIIs are unlikely to be obligate transcription factors for the entire genome.
The cause of the broad transcription defects observed in certain
TAFII mutants is slowly being revealed. It has
been proposed that redundancy between TFIID and SAGA explains this
phenotype (26). While this explanation may account for an
increase in the total number of genes affected in these mutants, it
cannot solely account for the differences between these mutants and
mutants of other TAFIIs found only in TFIID. The
first evidence for this is that mutations in TAF40 or
depletion of TAF48p, two TAFIIs not in SAGA,
cause reduced expression of many genes (25, 43). The
second is the results presented here. Many of the TAF90
mutants described here show significant reductions in their ability to interact with the SAGA components Ada3p, Ada1p, and Spt7p in co-IP experiments, yet they do not display the broad transcription phenotypes exhibited by the taf90-20 mutant. Moreover, we found that
the levels and activities of SAGA complex isolated from the
taf90-17, taf90-10, and taf90-8 (not
shown) mutants were reduced at least as much as those of the
taf90-20 mutant. In light of these results, redundancy
between SAGA and TFIID is at best an important contributing factor to
the broad transcription defects observed in certain TAFII mutants.
A reoccurring phenotype exhibited by most TAFII
mutants that show reduced expression of a large number of genes is that
shifting them to the restrictive temperature results in dramatic
reductions in the steady-state levels of most TFIID subunits (25,
31, 43, 46). This also is true of the taf90-20 allele
characterized here. It is striking how well the magnitudes of the
transcription defects described here match the consequences of each
mutant for the structure of TFIID. The TAF90 mutants that
display the most dramatic reductions in TAFII
levels when shifted to 37°C, and are the least competent to bind to
TFIID in the co-IP studies, are also those that show the strongest
transcription phenotypes. This is particularly apparent when comparing
the weakest and strongest classes of mutants, taf90-1 and
taf90-17 versus taf90-20, respectively. Therefore, our study argues that it is the rapid loss of many TFIID
subunits that, when combined, results in broad transcription defects.
This hypothesis is supported by a recent genome-wide analysis of
transcription that showed that the combined effect of the loss of
function of each individual TFIID subunit can account for the
expression of greater than 70% of the genome (26). What now needs to be debated is what type of mutant, or depletion system, is
most appropriate to assign functions to TAF90. Depletion
strategies and mutants such as taf90-20 are prone to
secondary effects. It is likely that a comprehensive analysis of
multiple alleles will be necessary.
The C terminus of TAF90p is required for TFIID and SAGA
function.
This study has established the importance of the WD40
repeats and the C terminus in maintaining the integrity of the TFIID and SAGA complexes. This result would not have been predicted from
previous analysis of hTAF100, which suggested that the WD40 repeats are
dispensable for its incorporation into TFIID or other TAF-containing
complexes (9). Does this mean that human TFIID and yeast
TFIID are different? It is more likely that these results are explained
by the incorporation of the truncated hTAF100 into partial TFIID
complexes or alternative TAF-containing complexes that had not been
identified at the time of that study. We demonstrate that the C
terminus of TAF90p is important for its association with TFIID and
SAGA, but we cannot say that it is sufficient or that the N terminus
does not make important contributions to this function as well. It is
likely that regions across the entire length of the protein are
required for TFIID and SAGA function. Interestingly, inactivation of
nearly every mutant severely compromises its ability to co-IP with
Tsm1p, TAF17p, and TBP, even though it retained some ability to
copurify with other TAFIIs. This was even true
for some of the alleles that have weaker effects on the co-IP of other
TFIID subunits, such as TAF47p and TAF145p. This suggests that the
interaction of Tsm1, TAF17p, and TBP is strictly dependent upon the
integrity of the C terminus of TAF90p. This agrees very well with
another analysis of hTAFII100, which demonstrated
that it directly interacts with hTAFII31 (TAF17p homologue) and TBP (52). It is not known if Tsm1p, or its
homologues, binds to the WD40 repeats of TAF90p.
Our results also indicate that TAF90 is required for SAGA
abundance and activity, as the complexes isolated from the
taf90-8 (not shown), taf90-10,
taf90-17, and taf90-20 mutants are significantly reduced in abundance and/or activity. Even though we did not purify SAGA from every mutant, essentially every allele showed impaired interactions with some SAGA subunits in co-IP studies, in particular Ada1p. Since Ada1 is essential for the structure and function of the
SAGA complex (50), we can predict with a high degree of
certainty that the activity of SAGA is compromised to some degree in
each of the mutants described here. This prediction is supported by the
inability of all mutants to support wild-type levels of
starvation-induced transcription of HIS3, which is
particularly sensitive to mutations in SAGA subunits. The C terminus of
TAF90p is required for both SAGA and TFIID function, but it is likely that it binds to these two complexes differently. The first line of
evidence for this is that the mutations contained in the
taf90-20 allele are the most disruptive to the integrity of
TFIID and the least disruptive to that of SAGA. The second is the
phenotypes of the taf90-17 and taf90-19 alleles.
It is clear that the taf90-19 allele has stronger effects on
cell viability, on the integrity of the TFIID complex, and on
transcription. However, both alleles are equally disruptive to the SAGA
complex as judged by the co-IP studies (Fig. 8B) and are equally
defective for the activation of HIS3 (Fig. 6).
Interestingly, both alleles contain the same S542F mutation, but
taf90-17 has an additional amino acid substitution that is
compensatory in nature in regard to its interaction with TFIID but not
SAGA. Molecular modeling of the WD40 repeat region of TAF90p, based on
the known structure of another WD40 repeat protein, predicts that these
mutations are on the same surface of the protein (not shown). Whereas
we did not isolate either a TFIID- or a SAGA-specific mutant in this
screen, scoring for a temperature-sensitive phenotype may not be
selective enough for the identification of such a mutant. Our discovery
and analysis of the taf90-17, taf90-19, and
taf90-20 mutants suggest that isolating such a mutant is
possible. Alternative screens that are more selective, such as an SPT
phenotype for SAGA-specific mutants, may result in the identification
of these types of mutants, which will further our understanding of the
role of TAF90 in these two important transcription
regulatory complexes.
We are especially gratefully to Patrick Grant and Jerry Workman
for advice on SAGA purification and antibodies. We thank Michael Green,
Fred Winston, Tony Weil, Shelley Berger, Steve Buratowski, Rick Young,
and Kevin Struhl for providing strains used in our studies and members
of the Reese lab and the Penn State gene regulation group for advice
and comments on this work.
This research was supported by funds provided by the National
Institutes of Health (grant GM58672) to J.C.R.
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Abstract
Introduction
