MOLECULAR AND CELLULAR BIOLOGY, June 2011, p. 2253–2261
0270-7306/11/$12.00 doi:10.1128/MCB.01464-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 11
Genome-Wide Transcriptional Dependence on Conserved Regions of Mot1䌤
Bryan J. Venters, Jordan D. Irvin,† Paul Gramlich,‡ and B. Franklin Pugh*
Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology,
Pennsylvania State University, University Park, Pennsylvania 16802
Received 22 December 2010/Returned for modification 25 January 2011/Accepted 21 March 2011
TATA binding protein (TBP) plays a central role in transcription complex assembly and is regulated by a
variety of transcription factors, including Mot1. Mot1 is an essential protein in Saccharomyces cerevisiae that
exerts both negative and positive effects on transcription via interactions with TBP. It contains two conserved
regions important for TBP interactions, another conserved region that hydrolyzes ATP to remove TBP from
DNA, and a fourth conserved region with unknown function. Whether these regions contribute equally to
transcriptional regulation genome-wide is unknown. Here, we employ a transient-replacement assay using
deletion derivatives in the conserved regions of Mot1 to investigate their contributions to gene regulation
throughout the S. cerevisiae genome. These four regions of Mot1 are essential for growth and are generally
required for all Mot1-regulated genes. Loss of the ATPase region, but not other conserved regions, caused TBP
to redistribute away from a subset of Mot1-inhibited genes, leading to decreased expression of those genes. A
corresponding increase in TBP occupancy and expression occurred at another set of genes that are normally
Mot1 independent. The data suggest that Mot1 uses ATP hydrolysis to redistribute accessible TBP away from
intrinsically preferred sites to other sites of intrinsically low preference.
ulated removal of TBP, which Mot1 is well suited to do. In this
context, Mot1 is a negative regulator.
Since TBP binds to the minor groove of DNA, which has
limited sequence specificity, TBP has relative high affinity for
nonspecific DNA (14). If bound inappropriately, this might
lead to aberrant or nonproductive assembly of the transcription machinery. Biochemical experiments have demonstrated
that Mot1 can remove nonspecifically bound TBP (41), perhaps acting as a chaperone allowing TBP to rebind in a productive mode. For example, at the URA1 gene, Mot1 can
promote transcription by removing a nonproductive TBP
bound in the reverse orientation (46). In this context, Mot1
operates as a positive regulator.
The mechanism by which Mot1 acts on TBP is well defined
biochemically, and this provides a basis for interpreting less
defined in vivo experiments. Because Mot1 is essential for
growth in S. cerevisiae (19), in vivo functional analysis of important regions of Mot1 is not straightforward in that loss of
function is lethal. Thus, an investigation into the genome-wide
functions of essential proteins like Mot1 is hampered by the
practical limitation that mutations that eliminate function
cause cell death. Temperature-sensitive mutations might alleviate this problem to some extent, but they are difficult to
target to specific regions of the protein and often vary in
severity. To circumvent this limitation, we utilized a transientreplacement strategy (34) to investigate the contributions of
conserved Mot1 domains to transcription and TBP recruitment
genome-wide. Our study revealed that Mot1-regulated transcription is dependent on nearly all conserved regions of Mot1.
Strikingly, transcriptional dependence for a subset of genes is
specifically altered when the Mot1 ATPase domain is deleted.
Genome-wide location analysis of TBP in a strain that lacks the
Mot1 ATPase region corroborates the expression-profiling experiments, suggesting a direct effect on TBP. Furthermore,
coimmunoprecipitation of TBP and the Mot1 ATPase deletion
mutant demonstrates that the two directly interact. These find-
From Saccharomyces cerevisiae to human, the TATA binding
protein (TBP) provides an indispensable role in nearly all
RNA polymerase I, II, and III transcription events (29). TBP
is the central component of a complex regulatory network
governing transcription complex assembly (33). Consequently,
TBP is subjected to an extraordinary level of regulation by
numerous transcription factors, one of which is Mot1 (6). Mot1
is a conserved Snf2/Swi2-related ATPase (21) that regulates
the dynamics of TBP-promoter interactions by removing TBP
from DNA using the energy of ATP hydrolysis (6, 45, 47). The
first 800 amino-terminal residues of Mot1 are both necessary
and sufficient for TBP binding (2, 7). The Snf2-related ATPase
domain resides within the last 600 carboxy-terminal residues
(7). Genome-wide expression studies using temperature-sensitive mot1 alleles indicate that Mot1 regulates between 3 and
15% of the yeast genome, some negatively and others positively (4, 17, 24).
In vitro biochemical experiments have largely defined mechanisms by which Mot1 regulates TBP-DNA interactions. Mot1
can bind and stabilize TBP-DNA interactions, but in the presence of ATP, it dissociates TBP from DNA and, to some
extent, Mot1 from TBP, thereby recycling both (2, 7, 12, 26).
This reaction is important for two reasons. First, dynamic assembly and disassembly of the transcription machinery impart
precise control over gene expression. Therefore, regulated recruitment of TBP to promoters must be accompanied by reg-
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Penn State University, 456 N. Frear Laboratory, University Park, PA 16802. Phone: (814) 863-8252. Fax: (814)
863-8595. E-mail: bfp2@psu.edu.
† Present address: Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD 21702-1201.
‡ Present address: Schering-Plough Research Institute, Union, NJ
07083.
䌤
Published ahead of print on 28 March 2011.
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TABLE 1. MOT1 yeast strains used in this study
Strain
Promoter
MOT1 allele
BY4743
yLAC1
yjdi408
yjdi410
yjdi412
yjdi414
yjdi416
yjdi418
yjdi420
MOT1
GAL1
GAL1
GAL1
GAL1
GAL1
GAL1
GAL1
GAL1
WT
WT
FHT-WT1
FHT-TBP1
FHT-TBP2
FHT-UK1
FHT-ATP1
Null
WT
a
Deletion (aa)a
05–80
307–432
1090–1259
1403–1867
MOT1::kanMX
Plasmid
pSH47
pMOT221
pMOT221
pMOT221
pMOT221
pMOT221
pMOT221
pMOT221
(mot1-42)
(mot1-42)
(mot1-42)
(mot1-42)
(mot1-42)
(mot1-42)
(mot1-42)
MAT
LEU2
LEU2
LEU2
LEU2
LEU2
LEU2
LEU2
Diploid
Diploid
Alpha
Alpha
Alpha
Alpha
Alpha
Alpha
Alpha
Reference
9
34
This
This
This
This
This
This
This
study
study
study
study
study
study
study
aa, amino acids.
ings reveal that Mot1-regulated genes are generally regulated
by all parts of Mot1 and that a TBP-binding portion of Mot1
can alter the selectivity of TBP for promoters.
MATERIALS AND METHODS
Plasmids. pCALF-T(PGK) (36) was converted to pCALF-FHT-T(PGK) 2.2
by inserting a 66-bp HIS-TEV oligonucleotide into the NdeI site. pUG6-FHT-P
(4,170 bp) was made by PCR amplifying 259 bp containing the FHT (Flu-HisTEV) sequence from the pCALFHT-T(PGK) 2.2 plasmid. The PCR product was
digested with SalI, and 161 bp was ligated into the SalI-digested pUG6 plasmid
(4,009 bp) so that the orientation was FHT-loxP-kanMX-loxP.
FHT-Mot1 mutant strains. A list of the yeast strains used in this study is
provided in Table 1. S. cerevisiae strain BY4743 (9) (Invitrogen) was used as the
parental strain. Initially, the strain was transformed with pSH47 (URA3) (25)
encoding galactose-inducible Cre recombinase. Oligonucleotides (70-mer) were
used to PCR amplify 1,991 bp of pFA6a-His3MX6-PGAL1 containing the HIS3
gene and GAL1 promoter (38). The PCR product was transformed into BY4743
using a high-efficiency lithium acetate method (25) to replace 550 bp of the
endogenous MOT1 promoter with the GAL1 promoter, creating strain yLAC1.
HIS⫹ homologous-recombination transformants were selected on complete synthetic medium lacking histidine and uracil (CSM-HIS-URA medium) and verified by colony PCR.
Regions of MOT1 were deleted by replacing coding sequences with an FHT
tag. The FHT tag encodes three hemagglutinin (HA) (Flu) repeats, a decahistidine (H), sequence, and the TEV protease sequence (T). The kanamycin
resistance region of pUG6-FHT-p was PCR amplified with 68-mer oligonucleotides with 50-bp homology to distinct regions of MOT1. The PCR products were
transformed into yLAC1 and selected on CSM-HIS-URA (dextrose) plates containing 500 mg/ml G418 (Invitrogen). The kanamycin resistance cassette flanked
by loxP sites was removed by induction of Cre recombinase with 2% galactose for
4 h, leaving the FHT tag coding sequence upstream of the mutation in MOT1.
Kanamycin-sensitive colonies were identified by replica plating them on media
containing and lacking G418. Additionally, mutations were verified by colony
PCR with primers specific to each mutation.
Haploids. Kanamycin-sensitive FHT-Mot1 strains (Table 1) were plated on
CSM-HIS plus 5-fluoroorotic acid (5-FOA) to select cells that had lost pSH47
and verified by replica plating on CSM-HIS and CSM-HIS-URA. The strains
were then transformed with pMR13 (MOT1 wild type [WT] and URA3), and
transformants were selected on CSM-HIS-URA medium. The strains were
plated on presporulation medium (1% yeast extract, 2% peptone, and 10%
dextrose) for 2 days at 30°C. Cells were cultured in sporulation medium (0.3%
potassium acetate, 0.02% raffinose) for 3 days at 30°C. Two hundred microliters
of the culture was pelleted; resuspended in 1.2 M sorbitol, 10 mM Tris, pH 7.4;
and treated with 20 units of (1 mg/ml) zymolyase (MP Biomedicals) at room
temperature for 20 min. Tetrads were dissected according to standard yeast
techniques on YPD (yeast-peptone-dextrose) plates. Spores were replica plated
onto CSM-HIS-URA medium to select for the HIS3 gene (and therefore the
GAL1 promoter). Mating types of the mot1 strains were confirmed with MATa
and MATa sex tester strains. MATa leu⫺ HIS⫹ LYS⫹ tetrads were selected. The
strains were then transformed with pMOT221 (mot1-42 LEU2) or pAV20
(MOT1 WT LEU2) and selected on CSM-LEU medium. Cells that lost pMR13
(MOT1 WT URA3) were selected by plating them on CSM-LEU plus 5-FOA.
Colony PCR. For colony PCR, 1⫻ 25 mM MgCl2 buffer (Gene Choice), 2.5 U
Taq polymerase (Gene Choice), 0.0002 U Pfu polymerase (Stratagene), 0.4 mM
deoxynucleoside triphosphates (dNTPs), and 0.2 mM each primer were used per
50-ml reaction for 32 cycles.
Cell growth assays. MATa haploid FHT-Mot1 mutant strains carrying
pMOT221 (mot1-42 LEU2) were grown at 25°C in YPR (yeast-peptone-3%
raffinose) to mid-log phase. Cells (A600 ⫽ 0.5) were removed, and 2.5 ␮l of
10-fold serial dilutions was spotted on three sets of YPD (2% dextrose) and
YPG (2% galactose) plates and incubated at 25°C, 30°C, or 37°C. Photographs were taken after 48 h.
Microarray analysis. Microarrays were performed essentially as described
previously (13, 32). Briefly, cultures were grown to an A600 of ⬃0.6, induced with
2% galactose in YPR medium for 60 min at 25°C, and shifted to 37°C for 45 min
to inactivate the temperature-sensitive copy of Mot1 encoded by the mot1-42
allele. Cells were harvested by centrifugation at room temperature, washed in
RNase-free DEPC (diethyl pyrocarbonate)-treated double-distilled H2O, and
frozen in liquid nitrogen.
Total RNA was isolated as described previously (32), and poly(A) tail mRNA
was purified using oligo(dT) cellulose (Ambion) according to the manufacturer’s
instructions. Reverse transcription, labeling with fluorescent dyes (Cy3 and Cy5;
Amersham), hybridization, and scanning were all performed as described previously (13, 32). Four micrograms of mRNA was used for hybridizations instead of
the conventional 2 ␮g. Slides were treated with Dye Saver2 (Genisphere) according to the manufacturer’s instructions to preserve signal intensity. Data sets
were mode normalized by using R software (Bioconductor) to mode-center
replicates (dye swaps) (data are available at http://atlas.bx.psu.edu/).
Genes were filtered by several criteria to minimize false positives. (i) Genes
were eliminated if their signals on the array were greater than 25% saturated. (ii)
The mean foreground signal minus the median background signal had to be
greater than the standard deviation of background signal. (iii) Quality data were
needed from both replicates of the dye swap. (iv) The directional change of the
mutant’s signal (relative to the reference) had to be equivalent in the replicates.
False-discovery rates (FDRs) were determined using a modified version of a
method described previously (35). The false-discovery rate is reported as a
percentage of the number of expression changes above and below a given threshold (⫾0.59; log2 scale) in the homotypic control (yjdi420) compared to each
FHT-Mot1 mutant expression experiment (yjdi410 to -416). The FDRs are 4.3%,
2.0%, 2.4%, 3.0%, and 2.9% for TBP1, TBP2, UK1, ATP1, and Null, respectively.
K-means clustering was performed using Cluster (22) on 515 genes that contained data in 80% of the experiments and that had a change of at least 1.5-fold
(log2 ratio ⫽ 0.59) in one of the mutants. K was chosen to equal 6 clusters (K ⫽
6). Clusters 5 and 6 were merged because they were visually indistinguishable.
Clustering information was visualized using Treeview (22).
ChIP-chip. ChIP-chip was performed essentially as described previously (48),
with minor changes noted below. Briefly, cultures were grown to an A600 of ⬃0.6,
induced with 2% galactose in YPR medium for 60 min at 25°C, and shifted to
37°C for 45 min to inactivate the temperature-sensitive copy of Mot1 encoded by
the mot1-42 allele. The cells were then fixed by adding formaldehyde to a final
concentration of 1% for 2 h at 25°C (instead of a typical 15-min cross-link time)
and quenched for 5 min with glycine. The cultures were diluted 2-fold with the
same volume of temperature-adjusted distilled water just prior to addition of
formaldehyde to achieve a medium temperature of 25°C. The harvested cells
were lysed with glass beads, and the chromatin pellet was washed and sonicated.
Sheared chromatin was immunoprecipitated with IgG-Sepharose. This ChIPenriched DNA was amplified by ligation-mediated PCR (LMPCR) as described
elsewhere (27), and 100- to 250-bp LMPCR-amplified fragments were gel puri-
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IN VIVO STRUCTURE-FUNCTION STUDY OF Mot1
fied according to the manufacturer’s protocol (Qiagen) and subsequently hybridized to low-density tiled spotted microarrays containing ⬎21,000 oligonucleotide
probes as described previously (48). Briefly, each yeast gene is interrogated with
a set of at least 3 oligonucleotide probes, which survey the relative occupancy
levels for a given transcription factor at the ⫺250 and ⫺60 sites relative to the
translational start site and the downstream portion of the open reading frame
(ORF). Data were filtered and analyzed as previously described (49) (data are
available at http://atlas.bx.psu.edu/).
Coimmunoprecipitation and immunoblotting. Briefly, FHT-Mot1 mutant
strains were grown as described above for ChIP-chip, except the cells were not
cross-linked with formaldehyde. The harvested cells were then pelleted and flash
frozen in liquid nitrogen. The cells were lysed with glass beads in NP-S buffer (10
mM Tris-Cl, pH 7.5, 0.5 mM Spermidine, 0.075% Igepal (Sigma), 50 mM NaCl,
5 mM MgCl2, 1 mM CaCl2). Chromatin pellets were washed in NP-S buffer, the
chromatin was micrococcal nuclease digested (15 units) in a volume of 300 ␮l for
20 min, and then the chromatin was solubilized by washing the spun pellet with
FA lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1%
Na deoxycholate, 2 mM EDTA). This native nuclear extract was immunoprecipitated with anti-TBP rabbit polyclonal antibody serum, and the eluate was
assayed by Western blot analysis for interacting FHT-Mot1 mutants.
For Western blotting, FHT-Mot1 mutant coimmunoprecipitation eluates were
electrophoresed in 6% SDS-PAGE and transferred to a polyvinylidene difluoride
(PVDF) membrane (Pall Gelman Laboratory) in Western transfer buffer for 60
min at 1.5 A. FHT-Mot1 mutants were detected with 1:3,300 anti-HA (HA.11;
Babco) and 1:5,000 anti-mouse–horseradish peroxidase (HRP) antibodies (Amersham) and exposed to Hyperfilm (Amersham) with enhanced chemiluminescence (ECL) (Amersham).
Comparisons with public microarray data. The relationships to the top and
bottom 10th percentiles of the expression and ChIP-chip data were calculated in
Excel with the data downloaded from the referenced laboratory or journal’s websites. The percent rank of the distribution was calculated with the PERCENTRANK
function. Next, the numbers of genes that appear in the top 10% (⬎0.9 in
PERCENTRANK) or the bottom 10% (⬍0.1 in PERCENTRANK) and also appear in each cluster were calculated. The CHITEST function of Excel was then used
to calculate P values from the observed and expected values.
RESULTS
Replacement strategy for essential Mot1 functions. Mot1 is
demarcated by several regions that are highly conserved from
yeast to human (12, 16). To test their in vivo importance, we
targeted four conserved regions for deletion, creating mutants
named TBP1, TBP2, UK1, and ATP1 (Fig. 1A to C). The
names reflect the associated functional regions (e.g., the TBP1
deletion removes one of two TBP interaction domains, UK1 is
unknown, and ATP1 removes the ATPase domain). Deletion
of each region of MOT1 was achieved by homologous recombination using a PCR-amplified cassette containing kanMX
flanked by loxP Cre recombination sites (34). The cassette also
contained coding sequences that allowed the deleted region to
be replaced by an FHT epitope tag that encodes a triple-HA
tag, a decahistidine tag, and a TEV protease cleavage site.
After selection for recombinants on G418 plates and subsequent excision of kanMX with the Cre recombinase, the deleted region was replaced with the FHT tag and a single loxP
site, both of which maintained an open reading frame through
the replaced region. Cell viability was maintained in a resident
mot1-42 temperature-sensitive allele, which allowed subsequent temperature inactivation of the mot1-42 allele at 37°C
(Fig. 2A). The location of each deletion mutation was verified
by PCR across the deletion borders, with the appropriate-size
products detected (not shown). Expression of the deletion
mutants was placed under the control of the GAL1 promoter.
Immunoblot analysis demonstrated the presence of an appropriate-size band that reacted with anti-HA antibodies and was
present only after the addition of galactose to the cells (Fig.
2255
2B). The untagged Mot1 (WT) and null controls were not
detected because both lack the FHT tag and thus are not
recognized by the HA antibody. All mutants except UK1 were
expressed at levels roughly equivalent to that of the wild-type
Mot1 containing an FHT tag at the N terminus (WT1). UK1
was expressed at about 50% of the WT1 level. Importantly, the
expression levels of the mutants were not diminished after
inactivation of the mot1-42 allele at 37°C for 45 min.
Four conserved regions of Mot1 contribute essential functions to Mot1. Haploid strains carrying a galactose-inducible
chromosomal copy of one of the mot1 deletion mutants and a
plasmid-borne temperature-sensitive mot1-42 (16) were tested
for the ability of the mot1 domain deletion mutants to support
viability. The mot1-42 supporting cell viability was inactivated
at 37°C and replaced with the domain deletion mutants by
adding galactose to the medium. At 25°C and 30°C, mot1-42
remained functional, allowing cell growth, as expected. In the
presence or absence of the galactose-induced domain deletion
mutants (Fig. 3, 25°C and 30°C), viability was unaffected,
indicating that the domain deletion mutants did not have a
dominant-negative effect on cell growth. At 37°C, wild-type
Mot1 (WT and WT1) and all mutants failed to support
viability in dextrose medium, where these proteins are not
expressed, verifying the temperature-sensitive nature of the
mot1-42 allele (16). As positive controls, both the untagged
(WT) and tagged (WT1) inducible Mot1 supported growth
in galactose medium at 37°C. However, none of the Mot1
domain deletion mutants support growth, indicating that
each of the four conserved domains is essential in S. cerevisiae. These findings are consistent with related studies presented elsewhere (1, 7, 16).
Mot1-regulated genes display strong transcriptional dependence on all regions of Mot1. Inasmuch as Mot1 regulates
genes both negatively and positively, we tested whether the
four conserved regions of Mot1 make distinct gene-specific
contributions to gene expression on a genome-wide scale. Expression of the Mot1 mutants was induced with galactose for
60 min, and then the resident functional mot1-42 allele was
shut down by an abrupt temperature shift from 25°C to 37°C
for 45 min (Fig. 2A). During this time, we expect heat shockregulated genes to change in expression within 15 min of the
temperature shift and then return to near-normal expression
levels by 45 min (11, 23).
To place any changes in gene expression into the appropriate context, each expression profile conducted on a mutant was
also conducted in parallel using a galactose-inducible untagged
wild-type MOT1 allele (WT; yjdi420) (Table 1). Thus, if a
mutant is as functional as wild-type Mot1, then no changes in
gene expression are expected. Changes in gene expression
were mode centered, meaning that the most frequent binned
ratio (mutant/WT) corresponded to no change in gene expression. This centering is valid, since most genes are not appreciably regulated by Mot1 (4, 17, 24). Log2-transformed changes
in gene expression are presented as a cluster plot (Fig. 4A),
where red and green denote increased and decreased expression, respectively. Black denotes no change. Each row corresponds to a protein-coding gene, and each column corresponds
to the expression profile for a Mot1 mutant. The 515 genes that
met a specified cutoff (Fig. 4A) for changes in expression in at
least one set of experiments are shown. The data were clus-
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VENTERS ET AL.
MOL. CELL. BIOL.
FIG. 1. Mutagenesis of conserved regions in the MOT1 gene. (A) Schematic of the MOT1 gene showing the approximate locations of four
conserved regions. (B) Schematic of the constructed Mot1 mutants. The null control represents WT1 in which the kanMX selection gene was not
removed. (C) Conservation of the four regions of Mot1 targeted for mutagenesis in this study. S. cerevisiae (Sc) and Homo sapiens (Hs) Mot1
protein sequences were aligned using the BLAST algorithm (3).
tered by K-means (22) into five clusters, representing the maximum number of visually nonredundant clusters. The homotypic control expression profile, reflecting comparisons
between two independent biological replicates of galactoseinduced wild-type Mot1, produced no appreciable changes in
expression (Fig. 4A, WT), as expected.
The homotypic control also provided a measure of intrinsic
variability in the data. The mean log2 ratio was 0.045 (1.03- ⫾
0.12-fold [standard deviation] change). To ensure that the
FHT tag was not perturbing expression, an FHT-tagged wildtype Mot1 control (Fig. 4A, WT1) was tested. Changes in gene
expression were very modest compared to the untagged reference (log2 average ⫽ 0.033, or 1.02-fold ⫾ 0.12-fold change),
indicating that the FHT tag had little or no effect on expression
genome-wide. In particular, the standard deviation in gene
expression was not significantly different than that observed
with the homotypic control.
Changes in gene expression of the galactose-induced null
mutant (empty cassette in the mot1-42 strain) at 37°C provided
a measure of the maximal level of expected change in expression and correlated well with changes in gene expression reported for the mot1-1 or mot1-14 allele (4, 17) (Fig. 4B). Thus,
VOL. 31, 2011
IN VIVO STRUCTURE-FUNCTION STUDY OF Mot1
2257
FIG. 2. Transient-replacement strategy. (A) The transient-replacement approach simultaneously expresses the mot1 test allele from an
inducible galactose-driven promoter while inactivating the temperature-sensitive (ts) version expressed from the mot1-42 allele supporting viability.
Consequently, at the time of harvest, the Mot1 mutants expressed from the galactose promoter have replaced the ts copy of Mot1. (B) Galactose
induction of Mot1 derivatives. An immunoblot probed with monoclonal anti-HA antibodies is shown, revealing the FHT epitope-tagged Mot1
derivatives. Cells were taken just prior to addition of 2% galactose to raffinose media (⫺), after 2% galactose induction for 60 min at 25°C (⫹),
or after galactose induction and another 45 min of incubation at 37°C (⫹) to inactivate the endogenous mot1-42 allele (not detectable in this assay).
The uniformity of the background bands just above the 121- and 54-kDa markers confirms approximately equal loading and transfer of samples.
this transient-replacement system appears to provide an adequate reflection of Mot1 dependency.
From these expression-profiling experiments, we find genes
that are negatively regulated by Mot1 (clusters 1 to 3 in Fig.
4A) and those that are positively regulated by Mot1 (cluster 5)
each require the TBP1, TBP2, and UK1 conserved domains of
Mot1 in that changes in gene expression were similar to that of
the null mutant (Fig. 4A, compare columns 3 to 6). Thus, in
general, Mot1 uses the same conserved domains to positively
and negatively regulate transcription. Genes in cluster 1 are
characterized as being stress induced, TATA containing,
SAGA dominated, and negatively regulated by a wide range of
TBP and chromatin regulators (Table 2). Cluster 2 genes have
the same characteristics as those of cluster 1, except that they
are not stress induced and are not inhibited by histones. Cluster 3 appears to be a mixture of cluster 1 and 2 genes. Genes
in cluster 5 are characterized as being stress repressed, TATA-
less, and TFIID dominated. In addition, the ribosomal protein
genes dominate this group. The positive contribution of Mot1
to cluster 5 expression is in line with reports proposing that
Mot1 positively regulates transcription by redistributing TBP
throughout the genome (15, 41) and/or by dismantling transcriptionally inactive TBP (18, 46). Taken together, Mot1 typically regulates 6% of all TFIID-dominated genes in a positive
manner, whereas 28% of all SAGA-dominated genes tend to
be negatively controlled by Mot1.
Deletion of the Mot1 ATPase domain causes ectopic gene
expression. The Mot1 ATP1 mutant, corresponding to a deletion of the ATPase domain, had mixed behavior. At most
analyzed genes, reflected by clusters 2 and 5 in Fig. 4A, negative and positive regulation by Mot1 required the ATPase
domain as much as it required the other conserved regions.
Cluster 1 expression displayed less dependence on the ATPase
domain of Mot1. The difference between clusters 1 and 2 may
FIG. 3. Effects of Mot1 mutants on cell growth. Strains carrying pMOT221 (mot1-42 LEU2) and mot1 mutant derivative alleles were grown at
25°C in YPR (3% raffinose) to mid-log phase. Then, 0.5 A600 unit of cells were removed, and 2.5 ␮l of 10-fold serial dilutions were spotted on three
sets of YPD (2% dextrose) and YPG (2% galactose) plates and incubated at 25°C, 30°C, or 37°C. The mot1 mutant strains are indicated at the
left of the plate images. The photographs were taken after 48 h.
2258
VENTERS ET AL.
FIG. 4. Genome-wide transcription profiling of Mot1 mutants.
(A) Cluster analysis of Mot1 mutant expression profiles. Strains harboring each of the indicated Mot1 mutants were harvested according
to the transient-replacement strategy (Fig. 2A). mRNA was isolated
and cohybridized, along with an untagged wild-type reference (WT), to
spotted full-length PCR-generated ORF microarrays. Changes in gene
expression (log2 scale) were clustered using Cluster software and visualized with Treeview (22). Membership required quality data in six
of the seven experiments and a log2 absolute value of 0.59 (1.5-fold
change) in at least one experiment and data in six of the seven clustered experiments. A total of 515 ORFs met these criteria, with FDRs
across all experiments of ⱕ4.3%. Rows were clustered by K means into
five clusters (n ⫽ 120, 120, 21, 49, and 209). The rows represent
individual genes, and fold changes in gene expression are reflected in
the color intensity. Columns representing an average of at least two
dye-swapped experiments were clustered hierarchically. The dendrogram relating them is depicted above the column labels. The table
below the cluster plot provides the median log2 ratio in each cluster in
each experiment. (B) Moving-average comparison of genome-wide
changes in expression for mot1-42 (null) with mot1-1 (4) and mot1-14
(17). Fold changes in gene expression (log2 scale) for mot1-42 were
sorted. The average changes in expression for mot1-42 in each sliding
200-gene window was plotted against an equivalent moving average
obtained from mot1-1 and mot1-14 strains.
be subtle, such as a greater rate-limiting dependency on TBP
binding versus ATP hydrolysis. Alternatively, since cluster 1
genes were generally induced and then partially repressed by
the temperature regime used here, it is possible that while
Mot1 binding is needed for this shutdown, the Mot1 ATPase
activity may be partially dispensable. In other words, Mot1
binding in the absence of ATPase activity is sufficient to elicit
some repression. This would be consistent with current models
of Mot1 function in which simply binding to TBP would be
sufficient to preclude binding to certain general transcription
factors.
Cluster 4 genes appeared to be largely Mot1 independent,
since expression of the null or any of the Mot1 domain dele-
MOL. CELL. BIOL.
tions (except ATP1) had little effect on transcription (Fig. 4A,
columns 3 to 6 in cluster 4). The ATP1 mutant caused an
increase in transcription. Since the primary TBP binding regions of this protein are intact, conceivably the ATP1 mutant
might promote DNA binding of TBP at these genes but is
unable to dissociate TBP without the ATPase domain. In this
case, the binding of the Mot1 ATP1 mutant to TBP would
seem not to completely interfere with subsequent transcription
complex assembly. A plausible rationale for this lies in our
observation that cluster 4 genes tend to be repressed by the
SSN6-TUP1 complex (P ⫽ 10⫺39) (Table 2). In this context,
any enhancement of TBP binding by Mot1(ATP1), even in a
weakened state, would provide some increase in expression.
One implication is that stabilization of TBP binding at SSN6TUP1-repressed genes circumvents to some extent SSN6TUP1 repression.
Cluster 3 genes are generally inhibited by wild-type Mot1
but showed decreased expression when the ATPase domain
was removed (Fig. 4A, column 2, cluster 3), paradoxically suggesting that the Mot1 ATPase region plays an apparently positive role at these genes while Mot1 as a whole plays a negative
role. Two alternative explanations might account for the apparent paradox. First, as in cluster 4, the ATP1 mutant might
stabilize TBP binding at the promoter of cluster 3 genes. However, this TBP-Mot1(ATP1) mutant complex seems to interfere with productive transcription complex assembly at these
cluster 3 genes. Alternatively, the ATP1 mutant might promote
nonspecific binding of TBP to the genome, which would reduce
the amount of TBP that could be recruited to cluster 3 genes,
resulting in decreased expression of cluster 3 genes. This would
be consistent with the notion that Mot1 normally removes TBP
from nontargeted regions of the genome (41). We explore
these possibilities below.
Mot1(ATP1) directly interacts with TBP. Potential interpretations of the Mot1(ATP1) expression profile are predicated
upon the Mot1(ATP1) mutant maintaining the ability to interact with TBP. Thus, to test whether Mot1(ATP1) directly interacts with TBP in vivo, we performed coimmunoprecipitation
assays for TBP in the FHT-Mot1 mutant strains. Growth and
Mot1 replacement were performed as in the expression studies. Importantly, to maintain native protein-protein interactions, the cells were not cross-linked throughout the coimmunoprecipitation procedure. TBP was immunoprecipitated from
digested, soluble chromatin derived from Mot1 mutant cells.
The ability of Mot1 mutants to interact with TBP on chromatin
was revealed by immunoblot analysis (Fig. 5A). The positive
control, the tagged (WT1) inducible Mot1, showed an interaction with TBP (Fig. 5A, bottom). As in Fig. 2B, the untagged
Mot1 (WT) and null controls were not detected, because both
lack the FHT tag and thus are not recognized by the HA
antibody. Among the Mot1 mutant derivatives, the
Mot1(ATP1) mutant showed the strongest interaction in vivo
with TBP, while the other mutants (TBP1, TBP2, and UK1)
displayed either weak or no interaction. Therefore, the interaction between Mot1(ATP1) and TBP revealed by their coimmunoprecipitation supports one assertion of the interpretation
that expression changes in clusters 3 and 4 may be a direct
result of the Mot1(ATP1) mutant altering the DNA-binding
status of TBP at these genes.
VOL. 31, 2011
IN VIVO STRUCTURE-FUNCTION STUDY OF Mot1
2259
TABLE 2. Relationship of Mot1 expression profiles with published microarray data
P value for clustera:
Category
Group/property
Expression
Stress response
Regulatory factors
Nucleosome
Protein/characteristic
% rank
TATA box
SAGA dominated
TATA box and SAGA dominated
ESRb upregulated
ESR downregulated
Rap1 ChIP and ribosomal protein
High transcription frequency
Heat shock
Salt stress
Alkali stress
Diauxic shift
Oxidative stress
Amino acid starvation
mot1-14
mot1-14
mot1-1
mot1-1
bur6-1 (NC2)
tup1⌬ (SSN6-TUP1)
TBP K145E (Mot1 binding)
TBP F182V (NC2 binding)
H3⌬1-28
H4⌬2-26
htz1⌬
Top 10
Top 10
Top 10
Top 10
Top 10
Top 10
Top 10
Bottom 10
Top 10
Bottom 10
Top 10
Top 10
Top 10
Top 10
Top 10
Top 10
Top 10
Reference
1
2
3
4
5
52
73
85
69
–
–
–
26
33
26
–
–
–
–
10
13
18
7
–
–
–
9
–
–
–
–
–
–
–
–
–
–
52
101
40
8
32
32
23
23
37
30
68
35
36
29
25
23
59
–
33
–
39
14
24
76
8
31
26
6
–
12
–
–
11
45
–
19
–
17
–
35
30
–
–
–
7
–
–
–
–
–
19
–
–
–
17
7
–
–
6
–
–
–
–
–
–
–
–
–
–
–
–
–
39
–
–
8
–
–
–
–
–
–
–
–
–
81
–
14
–
–
–
–
–
–
–
51
11
11
20
11
28
17
17
4
4
10
31
33
33
42
43
40
a
The ⫺log10(P value) for overlap between membership in each of the five Mot1 expression clusters and the top/bottom 10th percentile expression change or
group/property membership from published genomic data sets. The P value was calculated using the chi-square test and returns the probability that the overlap between
the two data sets occurs by chance. Thus, the most statistically significant relationships have the largest ⫺log10(P value). For clarity ⫺log10 (P value) values of less than
or equal to 5 were replaced with dashes.
b
ESR, environmental stress response.
Deletion of the Mot1 ATPase domain causes TBP redistribution in the genome. To further understand how the
Mot1(ATP1) mutant might affect TBP recruitment to promoters, we used genome-wide location analysis (ChIP-chip) to
monitor the changes in TBP, Mot1, and TFIID (Taf1 and Taf4
subunits) occupancy at every yeast gene in the Mot1(ATP1)
mutant. Growth and Mot1 replacement were performed as in
the expression studies. Each factor was immunoprecipitated
from sheared soluble chromatin derived from formaldehyde
cross-linked wild-type (WT1) and Mot1(ATP1) mutant cells.
Bound DNA was differentially labeled and cohybridized to
microarrays containing all intergenic regions. Median log2
changes in TBP and Mot1 occupancy for each of the clusters
defined in Fig. 4A are plotted in Fig. 5B and compared to
changes in gene expression for the Mot1(ATP1) and null mutants.
TBP and Mot1 occupancy changes at cluster 3 and 4 genes
mirrored the expression output for these genes in the ATP1
mutant (a decrease at cluster 3 and an increase at cluster 4),
suggesting that the expression change is a direct result of the
Mot1(ATP1) mutant altering the DNA-binding status of TBP
at these genes. The loss of TBP at cluster 3 genes suggests that
when Mot1 lacks its ATPase domain, the corresponding loss in
transcription is not due to stabilization of an inactive form of
TBP at these promoters. Instead, the results are more consistent with the loss of TBP possibly being due to stabilization of
TBP bound to other sites in the genome (e.g., cluster 4 genes)
by the Mot1(ATP1) mutant. Indeed, TBP and Mot1 occupancy
increased in the Mot1(ATP1) mutant at cluster 4 genes. Because cluster 3 genes normally have more TBP than cluster 4
genes (Fig. 5C), they have more to lose if TBP is distributed
nonspecifically throughout the genome in the Mot1(ATP1)
mutant.
One interpretation of the positive activity of Mot1 on expression at cluster 5 genes is that it removes an inactive form
of TBP, allowing productive binding of TFIID. Accordingly a
Mot1(ATP1) mutant might “lock down” TBP at cluster 5
genes, thereby preventing TFIID from binding. To test this
hypothesis, we conducted ChIP-chip on the Taf1 and Taf4
subunits of TFIID, comparing its occupancy at cluster 5 genes
in the wild type versus a Mot1(ATP1) mutant. Consistent with
this hypothesis, Taf1 and Taf4 occupancy levels decreased at
cluster 5 genes in the Mot1(ATP1) mutant, whereas TBP levels
remained largely unchanged (Fig. 5D). A constant level of TBP
is consistent with the hypothesis, in that one type of TBP (TAF
free) replaces another type of TBP (TAF bound; TFIID).
DISCUSSION
The binding of TBP to DNA is generally considered to be a
primary nucleating event in transcription complex assembly.
TBP-DNA binding is therefore subjected to substantial positive and negative regulation. TBP not only binds to the TATA
box located in promoters, it also binds to TATA-less promoter
regions, and it binds to nonspecific DNA with fairly high affinity. Since nonspecific DNA binding by TBP can nevertheless
2260
VENTERS ET AL.
FIG. 5. TBP coimmunoprecipitation (CoIP) and location profiling
of TBP, Mot1, and TFIID subunits in an ATPase-defective Mot1
strain. (A) TBP was immunoprecipitated from chromatin extracts and
then probed by Western blotting for both TBP (top) and FHT-Mot1
(anti-HA antibodies) (bottom). (B) ChIP-chip was performed in an
ATPase-defective Mot1 mutant (ATP1) and wild-type Mot1 strain
(WT1) background. The median log2 changes in occupancy for each of
the five clusters in Fig. 4A were calculated. For comparison, the median log2 expression changes for the null and ATP1 expression profiles
from Fig. 4A are also shown. (C) The median TBP occupancy at 25°C
from a previous study (50) for each cluster is shown as a histogram.
Log2 values are relative to nonpromoter intergenic regions (⬎1,800),
which are regions located between two convergently transcribed genes.
(D) ChIP-chip was performed on two subunits of TFIID, Taf1 and
Taf4. Shown is a histogram of the median log2 changes in occupancy
for each of the five clusters in Fig. 4A for Taf1 and Taf4 occupancy
changes in an ATPase-defective Mot1 strain relative to wild-type Mot1
(TBP is also shown for comparison).
nucleate transcription (14), robust mechanisms exist in vivo to
prevent promiscuous assembly.
Mot1 plays an important role in removing TBP from inappropriate genomic sites (18, 41), which would free up TBP
and/or the underlying DNA to engage in productive interactions (46). In this way, Mot1 would play a positive role at
promoters that are rate limited either by the availability of TBP
or by dissociation of an inactive TBP-promoter complex. Such
an arrangement might predominate at ribosomal protein
genes, where Mot1 plays a positive role (cluster 5 in Fig. 4A).
Conceivably, a Mot1-inaccessible form of TBP (i.e., TFIID)
binds to ribosomal promoters (12, 39). Nonproductive binding
of a TFIID-independent form of TBP might antagonize TFIID
recruitment or any other stage in transcription initiation. Removal of this nonproductive TBP would therefore positively
impact transcription at these genes. Indeed, we find that stabilizing a TBP-Mot1 promoter interaction at Mot1-upregulated genes has the effect of displacing TFIID.
Stress-induced promoters often are highly transcribed even
MOL. CELL. BIOL.
under nonstress conditions (11, 23). These promoters tend to
rely more on a free form of TBP that does not involve TFIID.
This form of TBP may be directed to the appropriate promoters via the SAGA complex, and since it is not TFIID, it may be
more accessible to Mot1. Indeed, the abundance of accessible
TBP at such promoters attracts Mot1, where it downregulates
expression (cluster 1 in Fig. 4A). A moderately high level of
expression is achieved through this balance of positive SAGATBP action (among other factors) and negative Mot1-TBP
action (among other factors). Indeed, by using multidimensional chromatography coupled to mass spectrometry (5), a
recent study found that Mot1 interacts with a variety of activators and transcriptional coregulators, such as Msn2, Hsf1,
and RSC.
Since Mot1 has the ability to bind both TBP and DNA (44),
it potentially can stabilize TBP-DNA interactions. However,
this is not realized in general because Mot1 uses the energy of
ATP hydrolysis to remove TBP from DNA. A form of Mot1
that lacks the ATPase domain provides a window into how
potential stabilization of TBP-DNA interactions through Mot1
affects the distribution of TBP genome-wide. If the
Mot1(ATP1) mutant were to increase TBP-DNA stability in an
undirected way, then genomic loci that normally lack TBP
should see an increase in TBP in the mutant. Those loci that
normally have higher TBP levels should suffer a decrease in
occupancy as TBP is sequestered at the vast number of nonspecific loci. Indeed we identified a set of genes (cluster 4) that
had lower levels of TBP and whose levels of TBP, Mot1, and
transcription increased in the Mot1(ATP1) mutant. We identified another set of genes (cluster 3) that had higher levels of
TBP, which decreased in the Mot1(ATP1) mutant.
Taken together, our findings suggest that the four conserved
regions of Mot1 are essential for viability and required for
proper regulation of most Mot1-regulated genes. Loss of the
ATPase domain, however, imparts some unexpected regulation on certain genes. Although only a fraction of all yeast
genes are overtly regulated by Mot1, essentially all yeast genes
require TBP and thus are potential targets for Mot1. Genes
that are insensitive to loss of Mot1 likely reflect those that are
relatively quiescent and thus lack TBP or those that involve
TFIID, which is a Mot1-insensitive form of TBP. Allowing
TBP to redistribute genome-wide in a potentially more nonspecific manner results in a net gain of TBP at TBP-deficient
genes and a net loss at TBP-enriched genes. Thus, one consequence of Mot1 using ATP hydrolysis to remove TBP from
DNA may be an increase in promoter selectivity.
ACKNOWLEDGMENTS
We are grateful to David Auble for plasmids pMOT221 (mot1-42
LEU2), pMR13 (MOT1 WT URA3), and MOT1 WT (pAV20 LEU2).
We thank Joe Reese for kindly providing the Taf1 and Taf4 antibodies.
This work was supported by National Institutes of Health grant
GM059055.
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