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J. Biol. Chem., Vol. 277, Issue 41, 38095-38103, October 11, 2002
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From the Department of Biochemistry, University of Washington,
Seattle, Washington 98195-7350
Received for publication, June 20, 2002, and in revised form, July 25, 2002
The yeast transcriptional activator Adr1 controls
the expression of genes required for ethanol, glycerol, and fatty acid
utilization. We show that Adr1 acts directly on the promoters of
ADH2, ACS1, GUT1, CTA1,
and POT1 using chromatin immunoprecipitation assays. The
yeast homolog of the AMP-activated protein kinase, Snf1, promotes Adr1
chromatin binding in the absence of glucose, and the protein phosphatase complex, Glc7·Reg1, represses its binding in the
presence of glucose. A post-translational process is implicated in the regulation of Adr1 binding activity. Chromatin binding by Adr1 is not
the only step in ADH2 transcription that is regulated by glucose repression. Adr1 can bind to chromatin in repressed conditions in the presence of hyperacetylated histones. To study steps subsequent to promoter binding we utilized miniAdr1 transcription factors to
characterize Adr1-dependent transcription in
vitro. Yeast nuclear extracts prepared from glucose-repressed and
glucose-derepressed cells are equally capable of supporting
miniAdr1-dependent transcription and pre-initiation complex
formation. Nuclear extracts prepared from a snf1 mutant
support miniAdr1-dependent transcription but are partially
defective in the formation of pre-initiation complexes with Mediator
components being particularly depleted. We conclude that Snf1 regulates
Adr1-dependent transcription primarily at the level of
chromatin binding.
The absence of a fermentable carbon source signals yeast nuclei to
activate expression of genes controlling aerobic metabolism of
alternative carbon sources (1-3). Numerous transcription factors allow
the cell to generate energy and metabolites from non-glucose carbon
sources such as ethanol and glycerol, as well as from storage carbohydrates and lipids. Many of these transcription factors are
controlled positively by
Snf1,1 a yeast homolog of
AMP-activated protein kinase, and negatively by Glc7, a type-1 protein
phosphatase, and one of its regulatory subunits, Reg1 (4). Integration
of the metabolic pathways utilized in the absence of glucose is
mediated in part through combinatorial interaction of transcription
factors that regulate genes acting in more than one pathway.
Adr1 is an example of one such transcription factor. ADR1
was discovered by virtue of its activation of ADH2, encoding
the ADHII isozyme responsible for the first step in ethanol utilization (5). Adr1 synergistically activates expression of ADH2 and ACS1, encoding acetyl-CoA synthetase, by binding to UAS1 in
combination with a second glucose-regulated transcription factor
required for two-carbon metabolism, Cat8. Cat8 binds to the adjacent
UAS2/CSRE in the ADH2 and ACS1 promoters
(6-11). Adr1 is also thought to act in concert with the
oleate-regulated transcription factor Oaf1/Pip2 to activate expression
of genes such as CTA1, SPS19, POX1,
PEX11, and POT1, which are involved in
Adr1 is a large transcription factor containing a complex DNA binding
domain consisting of C2H2 zinc fingers and a proximal accessory region
(PAR). A nuclear localization signal is located near the amino terminus
(19), and four transcription activation domains (TADs) have been
identified by deletions and gene fusions to LexA (20) and
GAL4 (21). TADI (amino acids 1-220) is in the DNA binding
amino terminus. TADII is contained within amino acids 263-359. TADIII,
present within amino acids 420-462, is the best characterized of the
four TADs and is the only Adr1 TAD that is active when fused to both
LexA and Gal4. TADIII contains several copies of the conserved
activation motif FXX Regulation of Adr1 is complex, possibly involving transcriptional,
translational, and post-translational processes (27-32). BCY1, encoding the regulatory subunit of
cAMP-dependent protein kinase is a positive regulator of
ADR1 expression (33), and the catalytic subunits of the
kinase phosphorylate Ser-230 of Adr1 in vivo and
in vitro (28,
29).2 However,
phosphorylation of Ser-230 in vivo appears to modulate the
transcriptional activity of Adr1 rather than alter its sensitivity to
glucose repression (29, 31, 33). The signals acting upstream of
BCY1 in this pathway have not been determined. Glc7·Reg1
is also involved in transcriptional and post-translational regulation of Adr1 but the targets of the phosphatase activity that affect the
activity of Adr1 have not been identified (34, 35). The role of Snf1 in
ADH2 expression is also unclear. It appears to be involved
in both ADR1-dependent and
ADR1-independent ADH2 expression (36, 37).
Deletion of SNF1 does not affect ADR1 expression, suggesting that Snf1-dependent phosphorylation affects its
activity (33). In addition, a number of genes of unknown function have been implicated in regulating the synthesis or activity of
ADR1 (38, 39).
Glucose regulation acts through the Adr1 DNA binding domain (ABD, amino
acids 1-165). A protein fusion containing ABD and either the VP16 TAD
or TADIII is able to confer glucose-regulated expression on
ADH2 (31). A gene fusion encoding nearly the entire Adr1
open reading frame (amino acids 17-1323), and the DNA binding domain
of Gal4 is glucose-regulated at UAS1-containing but not at
UASG-containing promoters (31), suggesting that glucose
repression mediated by ABD is promoter-specific. Consistent with this
interpretation, chromatin immunoprecipitation (ChIP) studies
demonstrated that Adr1 binds to the ADH2 promoter only after
glucose has been depleted from the medium (18). The nuclear
localization of Adr1 is not regulated, suggesting that Adr1 is within
the nucleus in the presence of glucose but is unable to bind
ADH2 chromatin (31). Adr1 isolated from repressed and
derepressed cells is equally competent to bind DNA (32). Taken together
the results suggest that the access of Adr1 to DNA may be restricted by
chromatin, even though UAS1 appears to be in a nucleosome-free region
(24). Whether regulation acts directly through Adr1, for example by
modifying the chromatin binding activity of Adr1, or through
modification of chromatin to restrict access of Adr1 to the promoter,
or through some combination of these mechanisms is not known.
Recent studies suggest that binding of Adr1 to the ADH2
promoter in repressed conditions is not sufficient for activation of
transcription (18). Hyperacetylation of the ADH2 promoter caused by mutations in the histone deacetylases RPD3 and
HDA1 allows Adr1 to bind the ADH2 promoter in
glucose-repressed conditions, but recruitment of TBP does not occur.
The latter observation suggests that a second step in transcription
initiation after binding of Adr1 is regulated by glucose repression.
One possibility is that one or more of the TADs confer an additional
level of regulation that is required for TBP recruitment.
The present studies show that the ability of Adr1 to bind the
ADH2, ACS1, POT1,
CTA1, and GUT1 promoters in
vivo is glucose-regulated by Snf1 and Reg1. To study the possible
regulation of activation domain function, we employed an
Adr1-dependent nuclear extract transcription assay. In this
assay a simplified version of Adr1, called miniAdr1, is used as the
transcription activator. miniAdr1 contains ABD fused to a single
activation domain, TADIII. The nuclear extract supplemented with
recombinant miniAdr1 and an appropriate DNA template has many of the
transcriptional properties displayed by Adr1 in vivo.
However, recombinant miniAdr1 is active in nuclear extracts prepared
from glucose-repressed cultures, and is Snf1-independent, suggesting
that Snf1 functions only to facilitate chromatin binding by Adr1 and
not to enhance its activation potential.
Strains--
Yeast strains are listed in Table
I. Escherichia coli
strains used for plasmid propagation are DH5 Growth of Yeast Cultures--
Yeast strains were grown in YPD or
synthetic medium (SM) prepared according to standard methods (40).
Glucose-repressed cultures were started in YP or supplemented SM
containing 5% glucose. Derepressing cultures were inoculated into YP
or supplemented SM containing 0.05% glucose (and 3% ethanol when
indicated). Transformation of yeast used a modified LiAcetate protocol
(41).
Plasmid Construction--
The miniAdr1 activator and reporter
plasmids are similar to those described (see Refs. 21, 31, 42, and 43).
mini3Adr1 has a 2-amino acid spacer between ABD and TADIII. ABD ends at amino acid 160 of finger two, and the two copies of finger one are
separated by TNEKPY, the linker amino acids present between finger one
and finger two. It has a His6 tag at its amino terminus for
purification. The miniAdr1 genes are transcribed under the control of
the native ADR1 promoter on a centromere-containing plasmid. The reporter plasmid containing binding sites for miniAdr1 in
the HIS4 promoter was constructed from pSH515 obtained from S. Hahn (44) by inserting the double-stranded oligonucleotide SK half
(42) into KpnI- and XhoI-cut pSH515. This
introduced a single Adr1 binding site into the promoter. This sequence
was changed to the consensus-binding site for mini3Adr1, the
three-finger Adr1 miniprotein, by oligonucleotide mutagenesis. The
plasmids used as sources of promoter fragments for pre-initiation
complex formation are pSH515 (GAL4-UASG), pNK509
(mini3Adr1 (3F consensus)), and pNK510 (mini3Adr1 (3F consensus))-GAGA
mutation in the promoter. Mutation of TATA to GAGA used a QuikChange
mutagenesis kit from Stratagene.
Protein Purification--
All Adr1 miniproteins were expressed
from pQE31 (Qiagen)-based plasmids and purified from E. coli
MC1061 (pREP) using growth and purification procedures similar to those
previously described (42). For the Adr1 miniproteins containing two
fingers the purification was carried out on nickel-nitrilotriacetic
acid-agarose in denaturing conditions as recommended by Qiagen. The
protein was renatured on the column by a reverse urea gradient and
eluted with 0.25 M imidazole.
Enzyme Assays--
Chromatin-immunoprecipitation Assays--
Cells in 100 ml of
culture were treated for 5 min with 1% formaldehyde at 25 °C. The
cross-linking was stopped by addition of glycine to 0.125 M, and the cells were washed and processed for
immunoprecipitation as described previously (46). Immunoprecipitation of HA-tagged Adr1 bound to chromatin used rabbit polyclonal anti-HA antisera (F-7, Santa Cruz Biotechnology) and protein A-Sepharose beads.
Oligonucleotides for PCR amplification are listed in Table II. Real-time quantitative PCR was
performed on an Applied Biosystems AB7700 and analyzed using software
provided by the manufacturer. The slope of the curve of input DNA
versus PCR cycles was used to calculate the DNA
concentration of the PCR products generated by ACT1 and
ADH2 primers using DNA immunoprecipitated using anti-HA antisera. The amount of ADH2 product in each sample was
corrected for the amount of ACT1 product formed using the
same immunoprecipitated DNA. The ACT1 DNA is nonspecifically
immunoprecipitated by the antisera. This contamination serves as an
internal control for the PCR efficiency in each experimental DNA
sample.
Nuclear Transcription Extracts and Assays--
Nuclear extracts
were prepared and used as described (44, 47) from yeast spheroplasts
prepared with recombinant lyticase. Active nuclear transcription
extracts could be prepared from derepressed cells only if the cells had
been derepressed less than 6 h. If the cells are derepressed
12 h in the absence of glucose, they are difficult to convert to
spheroplasts and the resulting nuclear extracts have low
transcriptional activity.3
Six hours is a sufficient time to observe complete derepression of
ADH2 mRNA levels. Thus all of the important
modifications that ensure efficient transcription have been completed
before the extracts are prepared (25, 31).
Templates for in vitro transcription reactions were pHDY10
(ten Adr1 binding sites (43)); pNK509 (a single consensus binding site
for mini3Adr1); and pSH515 (a single Gal4 binding site (44)). To assay
transcription in vitro a primer extension assay was
performed using a laci oligonucleotide as described.
Radioactive primer extension products were separated from
unincorporated nucleotides by electrophoresis and visualized using a
STORM imaging system and ImageQuaNT software both from Molecular
Dynamics. Pre-initiation complex (PIC) formation utilized promoter
templates synthesized with biotinylated oligonucleotides (44, 47). The
template for mini3Adr1-dependent PIC formation was derived
from pNK509 using oligonucleotides pNOT and pBIO (44). Binding,
washing, and elution steps were performed as described. Proteins bound to the promoter fragments were separated by SDS-PAGE and identified by
Western blotting. Quantification of proteins used a Molecular Dynamics
STORM imaging system and ImageQuaNT software.
Adr1-dependent Derepression Is Regulated by Binding to
Chromatin--
We used a HA-epitope-tagged version of Adr1 in a strain
containing three extra chromosomal copies of ADR1 (31) to
study glucose-regulated chromatin binding. In this strain derepression of ADH2 expression occurs more rapidly than in the presence
of a single copy of ADR1, but ADH2 expression
remains stringently glucose repressed (31) and
Snf1-dependent.3 Fig.
1A shows that in the multicopy
Adr1 strain chromatin binding occurs rapidly after glucose
depletion. Fig. 1B indicates that re-addition of glucose
leads to an equally rapid loss of binding by Adr1. Real-time
quantitative PCR analysis was used to quantify binding of Adr1-HA to
the ADH2 promoter. The quantitative data are shown
underneath the lanes in Fig. 1. Using this assay, binding is detected 5 min after glucose depletion (Fig. 1A) and increases 100-fold
by 30 min. Within this time interval there is no change in the level of
Adr1 (31), suggesting that chromatin binding by Adr1, and loss of
binding activity when glucose is added, are regulated at the
post-translational level. This interpretation is confirmed by the data
in Fig. 1C showing that Adr1 binds to chromatin when the
cells are derepressed in the absence of protein synthesis.
Unexpectedly, a low level of Adr1 binding to chromatin is detected in
the presence of glucose and cycloheximide. In the presence of
cycloheximide and glucose, a low level of ADH2 mRNA was
observed that prompted us to suggest that ADH2 mRNA
might be synthesized at a low rate in repressed conditions and
stabilized in the absence of protein synthesis (31). The observation
that chromatin binding by Adr1 is enhanced during cycloheximide
treatment of glucose-repressed cells makes it more likely that
ADH2 transcription, and by inference, Adr1 modification, is
directly affected by the absence of protein synthesis.
ADH2 is dependent on Snf1 protein kinase for derepression
(5, 33, 36). To determine whether Snf1 is required for chromatin binding, we performed ChIP of the ADH2 promoter in a strain
lacking SNF1. Adr1 binding was not detected, and real-time
quantitative PCR indicated that the level of binding was reduced at
least 20-fold in the absence of SNF1 (Fig. 1D).
Thus, Snf1 is essential for chromatin binding by Adr1.
The Reg1·Glc7 protein phosphatase is necessary for glucose repression
of ADH2 expression (34, 35). The
reg1-dependent constitutive expression is
dependent on Adr1, indicating that reduced Glc7 activity must allow
Adr1 to bind the promoter in repressed conditions. Activation of
ADH2 expression in a reg1 or a glc7
mutant in glucose-growth conditions is incomplete, suggesting that one
or more GLC7- and REG1-independent pathways
contributes to ADH2 repression. To determine whether this
putative REG1·GLC7-independent pathway acts
before or after chromatin binding, we examined Adr1 binding in a
reg1 mutant. If Adr1 occupies the ADH2 promoter
at the same level in a reg1 mutant grown in repressed
conditions as it does in derepressed conditions in a wild-type strain,
then the putative REG1·GLC7-independent pathway
must operate after Adr1 has bound the promoter. Alternatively, if the
REG1·GLC7-independent pathway contributes to
the ability of Adr1 to bind the promoter, we would expect to see
reduced binding of Adr1 to the promoter in a reg1 mutant
strain grown in repressed conditions. Real-time quantitative PCR
detected Adr1 binding significantly above the background level in a
reg1 mutant, but chromatin binding to the ADH2
promoter is still 30-fold below the level observed after derepression
(3.2 units relative to 100 units for fully derepressed wild-type
cells), consistent with the relative levels of ADH2 expression in wild-type derepressed versus reg1 mutant
repressed cultures. Adr1 levels are elevated in a reg1
strain (35) but not to the level observed in strains containing
multiple copies of Adr1 (31). Because no Adr1 is detected bound
to chromatin in the latter strain (Fig. 1A), the modest
elevation in Adr1 level in the reg1 strain cannot explain
chromatin binding. Thus, Adr1 binding to chromatin is regulated by
Glc7·Reg1 and by a second pathway that is independent of
REG1·GLC7. This putative second pathway is
Snf1-dependent, because no binding is detected in a snf1 mutant.
To determine the generality of glucose-regulated chromatin binding by
Adr1, we performed ChIP analysis of four other
ADR1-dependent genes representing three
metabolic pathways: ACS1 (acetyl-CoA synthetase),
POT1 (peroxisomal-CoA thiolase), CTA1
(peroxisomal catalase), and GUT1 (glycerol kinase). Adr1
directly regulates the expression of these genes by binding to the
promoters only in derepressed conditions (Fig. 1D). Thus,
regulation at the level of chromatin binding is a common property of
ADR1-dependent genes.
Characterization of miniAdr1--
We used miniAdr1, a small,
functional version of Adr1 (31) to develop an in vitro
transcription system. miniAdr1 contains the DNA binding domain and
nuclear localization signal (ABD, amino acids 1-172), and a
transcription activation domain, TAD III (amino acids 420-462) from
Adr1. One advantage of miniAdr1 is its small size, which allows usable
quantities of recombinant protein to be purified intact from E. coli.
ADH2 expression dependent on miniAdr1 is stringently
glucose-regulated, suggesting that it retains regulatory targets of
Snf1 and Reg1·Glc7 (31). To demonstrate this directly we transformed adr1-null strains that were wild-type or mutant for
SNF1 or REG1 with a miniAdr1 expression plasmid
and assayed ADH2 expression. ADH2 derepression
was Snf1-dependent, and constitutive ADH2
expression was observed in a reg1 mutant.3 Thus,
miniAdr1 appears to be regulated like the full-length Adr1 protein.
Development of a miniAdr1-dependent in Vitro
Transcription System--
miniAdr1 purified from E. coli
was tested in an unfractionated nuclear extract transcription system
using a primer extension assay to measure transcription. The nuclear
extract transcription system contains all of the components needed for
transcription by the pol II holoenzyme and is capable of multiround,
activator-, TAF-, and mediator-dependent transcription (44,
47). miniAdr1 is active in this system using a template containing
multiple UAS1 elements (ten binding sites). The RNA product initiates
at the correct site based on its size, and transcription is dependent on pol II as demonstrated by its sensitivity to
However, the activity of miniAdr1 in the nuclear transcription extract
is low compared with a Gal4-VP16 activator, and it is inactive using a
template containing a single Adr1 consensus binding site, or two
perfect binding sites in inverted orientation, UAS1, the preferred
sequence in vivo (43). The relatively low DNA binding
affinity of Adr1 compared with Gal4 (~Kd of
10 miniAdr1 Containing an Additional Zinc Finger Is
Glucose-repressed--
If low DNA binding affinity and low Specificity
cause reduced transcriptional activity of miniAdr1 in vitro,
we reasoned that addition of a third finger might enhance its activity
as a transcription factor in vitro. We made and tested a
modified version of miniAdr1 containing an additional finger 1 (mini3Adr1) with the same activation domain (TADIII). A related
three-finger Adr1 protein binds its specific DNA site with about
20-fold higher affinity and has a 5-fold higher Specificity than
miniAdr1 containing two fingers (42).
To ensure that mini3Adr1 is regulated in the same manner as wild-type
Adr1, we tested its activity in vivo by expressing it from
the ADR1 promoter on a low copy number,
centromere-containing plasmid. A reporter gene was constructed by
inserting a single consensus-binding site, GGGGGGGTG, for mini3Adr1
upstream of two promoter fusions, CYC1/lacZ and
HIS4/lacZ. The former is the promoter used for
previous reporter studies with miniAdr1 (42, 43). The latter is a
modified HIS4 promoter that is identical to the promoter
used as the template for the in vitro transcription studies. After transformation of appropriate yeast strains with activator and
reporter plasmids, Recombinant mini3Adr1 Activates Transcription in Vitro and Depends
on TBP, TADIII, and High Affinity DNA Binding--
When recombinant
mini3Adr1 is added to a nuclear extract transcription system containing
a template with a single optimal binding site, a transcript of the
appropriate size is observed (90 nucleotides, Fig.
2A). In the experiment shown
mini3Adr1 had lower activity than Gal4-VP16. In other experiments their
relative activities were more similar. Both basal and
mini3Adr1-dependent transcription are dependent on the HIS4
TATA box, indicating that TBP is required for
miniAdr1-dependent transcription in vitro and
that the template has a single functional TATA sequence (Fig. 2B).
To test the dependence of mini3Adr1 on TADIII, we performed
transcription in vitro using mini3Adr1 lacking TADIII or
containing mutated versions of TADIII. The HA2 activator contains
mutations in two activation motifs (21). Activator CR9 has mutations
that reduce the negative charge of the acidic patch at the carboxyl terminus of TADIII. Both mutants are weak activators in vivo
(21). No activation could be detected above the basal level in the
absence of TADIII (Fig. 2C, ABD). The two mutant
activators have lower activity than wild-type mini3Adr1 (Fig.
2C) but higher activity than ABD alone, indicating that
their activity is impaired but not abolished.
Mutations in the DNA binding domain also reduce in vitro
transcriptional activity. The mutations tested alter the DNA binding domain by deletion of PAR (42, 48) from miniAdr1 containing either two
or three fingers. The two-finger miniAdr1 used in these experiments has
a change-of-specificity mutation in the second finger, Leu-146 to His,
that enhances DNA binding about 70-fold (42). miniAdr1 (L146H,
F1H in Fig. 2D) is a weaker activator than
mini3Adr1 (3F), despite its higher binding affinity. Deleting PAR
(
In summary, mini3Adr1 behaves in most respects as a classic activator
in vitro: it requires a high affinity DNA binding domain and
a strong activation domain outside of the DNA binding domain. Transcription requires a TATA sequence in the promoter, implying a
dependence on TBP for initiation. An importance difference between mini3Adr1 and most other eukaryotic transcription activators is the
requirement for a region of the protein within the DNA binding domain,
the PAR region, for maximal transcription activation both in
vivo and in vitro.
Recombinant mini3Adr1 Is Active in Nuclear Transcription Extracts
Prepared from Repressed Cells--
The initial in vitro
transcription experiments were performed using nuclear extracts
prepared from glucose-grown cells. Because mini3Adr1 is most active
in vivo in cells depleted of glucose, we prepared and tested
nuclear extracts from both repressed and derepressed cells. As shown in
Fig. 3, transcription extracts prepared
from derepressed cells are as active as those prepared from repressed
cells using mini3Adr1 as activator. The activity of the transcription
extracts prepared from repressed cells was tested at several protein
concentrations with the same results. Thus, repression of recombinant
mini3Adr1 activity is not observed in nuclear transcription extracts
prepared from glucose-repressed yeast cells.
Recombinant mini3Adr1 Is Active in Nuclear Extracts Prepared from
snf1 Mutant Cells--
Because mini3Adr1 is unable to activate
transcription in a snf1 mutant (Table III), mini3Adr1
activity in snf1 nuclear transcription extracts was tested.
Fig. 4 shows that nuclear extracts
isolated from a snf1 mutant support
mini3Adr1-dependent transcription as well as wild-type
nuclear extracts, independent of growth conditions prior to making the
extracts (low or high glucose). The level of basal transcription is
also similar in all four extracts. These results suggest that
Snf1-dependent modification is not essential to allow
recombinant mini3Adr1 to activate transcription in vitro nor
for basal transcription.
Pre-initiation Complex Formation Dependent on mini3Adr1 Is Not
Affected by Growth State of the Cells--
In a further attempt to
detect a difference between nuclear transcription extracts prepared
from cells in which miniAdr1 exhibits different levels of transcription
activation in vivo, we analyzed pre-initiation complexes
(PICs) formed on a mini3Adr1-dependent promoter.
Formation of PICs is dependent on intact mini3Adr1. This is most clear
for the recruitment of TFIIB,
TOA2,4 and members of the
Mediator and SAGA complexes: Med6, Srb2, Srb4,4 Srb10, and
Gcn5, all of which show a 2- to 10-fold dependence on mini3Adr1 for PIC
formation (Fig. 5). As has been observed previously (44, 47), some components of the PIC show less activator
dependence than do others, presumably because they are bound more
tightly and perhaps nonspecifically to DNA in the absence of
activator.
In the absence of TADIII (ABD alone) PIC formation is reduced to a
level lower than that observed in the absence of activator (Fig. 5).
The apparent inhibition of PIC formation suggests that ABD may have a
negative effect on PIC formation in the absence of a transcription
activation domain. These data are consistent with the transcription
studies that failed to detect a transcription activation function
associated with the DNA binding domain alone. In parallel with its
reduced activity in the transcription assay and in vivo, the
TADIII-HA2 mutant reduced PIC formation 2- to 5-fold.
Surprisingly, PIC formation was not significantly affected by mutation
of the TATA box (Fig. 5). The same mutation reduced transcription
in vitro (Fig. 2B) and in vivo (Table
III) to very low levels. Thus, mini3Adr1 is able to form PICs in
the absence of a TATA sequence, but the TATA box is nevertheless
required for transcription.
snf1 Mutant Nuclear Extracts Are Partially Defective in PIC
Formation--
We analyzed PIC formation in nuclear extracts prepared
from cells grown in high and low glucose. No reproducible difference was detected as long as extracts that were active for in
vitro transcription were compared.4 However, we detect
a lower level of PIC formation using a snf1 nuclear
transcription extract (Fig. 6). This is
particularly true using an extract prepared from derepressed
snf1 mutant cells. A similar difference in PIC formation is
observed using Gal4-VP16 activator.4 The defect is
primarily in PIC formation in the presence of activator, although there
is a decrease in basal PIC formation as well. Mediator components
appeared to be affected most. For example Srb2, Srb4, and Med6 were
decreased 15-, 6-, and 8-fold, respectively, when comparing PICs formed
in wild-type versus snf1 mutant extracts. The same nuclear
transcription extracts were tested for transcription activity, and no
differences were observed (Fig. 4).
A lower level of PIC formation might be observed if some of the PIC
components are present at reduced levels in the nuclear extracts. This
explanation seems unlikely because the transcriptional activities of
the wild-type and snf1 extracts are similar. To test this
possibility directly, we analyzed the nuclear extracts by Western
blotting for several of the components that were analyzed in the PICs.
No significant differences in the total levels of the proteins were
observed in the nuclear extracts.4 These results suggest
that PIC formation on the mini3Adr1-dependent promoter is
reduced in rate or extent when the nuclear transcription extract is
prepared from a snf1 mutant. The relative insensitivity of
the transcription assay to this deficiency could be due to different
templates being used (plasmid DNA versus a DNA fragment attached to a bead) or to different steps being rate-limiting in the
two assays.
Activation of Adr1-dependent genes appears to be
regulated by glucose repression at two steps prior to initiation of
transcription. The first step is DNA binding. Five
Adr1-dependent promoters, ADH2, CTA1,
ACS1, GUT1, and POT1, are bound by
Adr1 in vivo only when glucose is absent from the media.
Regulated Adr1 binding to the ADH2 promoter requires Snf1
protein kinase and is inhibited by Reg1·Glc7 protein phosphatase and
perhaps by a second pathway not involving Reg1.
The roles of Snf1 and Reg1·Glc7 in mediating chromatin
binding by Adr1 are unknown. One possibility is that Snf1
phosphorylates and activates Adr1, and Reg1·Glc7 dephosphorylates and
inactivates Adr1. Alternatively, targets of Snf1 and Reg1·Glc7
may include proteins that interact with Adr1 and affect its binding to
chromatin, or they may include chromatin itself.
Snf1 regulates gene expression in derepressing growth conditions in
multiple ways. These include inactivation of the repressor Mig1
(49), activation of the transcription factors Sip4 and Cat8 (7,
50-52), acting through the holoenzyme (53), and modification of
chromatin (54). Snf1 activates transcription of INO1 by
phosphorylating Ser-10 of histone H3. S10 phosphorylation is necessary
for subsequent acetylation of Lys-14 of the same histone (54).
These observations suggest a mechanism for Snf1-dependent
chromatin binding by Adr1. The regulatory region of the ADH2
promoter is nucleosome-free, but the proximal Adr1 binding site is
immediately adjacent to the upstream border of the TATA-box-containing
The hypothesis that Snf1 acts on Adr1-dependent promoters,
rather than on Adr1 itself, is consistent with several observations. Adr1 isolated from repressed cells is competent to bind DNA in vitro, and its apparent affinity for DNA is not increased after derepression (32). These observations suggest that Adr1 itself may not
be in an inactive form in repressed conditions. Rather, the chromatin
form of the ADH2 promoter may preclude efficient binding in
repressed conditions. Increased levels of Adr1 can partially overcome
glucose repression and the requirement for Snf1 (36, 55), suggesting
that Adr1 acts downstream of Snf1. In both of these situations
constitutive activation is incomplete; removal of glucose enhances
expression further. Thus, Snf1-dependent modification
required for complete derepression has not been bypassed by increasing
the level of the activator. Instead, a low level of activation occurs
despite the absence of the modification. This explanation assumes that
Adr1 has low affinity for its binding site in vivo in
repressed conditions. By increasing the level of Adr1 in glucose-grown
cells, mass action would partially compensate for its reduced affinity.
Increasing the affinity of Adr1 for its DNA binding site can partially
overcome glucose repression, as shown by the constitutive activity of
mini3Adr1 in Table III. This explanation is also consistent with the
observation that Adr1 is limiting for activation in repressed cells but
is present at a saturating level in derepressed cells (33, 36).
The studies presented here show that Adr1 is not bound to chromatin in
glucose-repressed conditions. This presents an apparent conundrum for
understanding the mechanism of actions of ADR1c, a
constitutively acting, hyperactive allele of ADR1 (5, 28, 29, 32, 33). ADR1c mutations map outside the DNA
binding domain, and the mutant protein does not show enhanced DNA
binding using gel shift assays (32). How does the mutant protein gain
access to chromatin in repressed conditions? Enhanced affinity for DNA
by Adr1c is an unlikely explanation. One possibility is
that Adr1 always has access to the chromatinized promoter but that its
affinity in repressed conditions is weak, as discussed above. We assume that the weak affinity for chromatin can be enhanced by protein-protein contacts, and that Adr1c makes such contacts more
effectively than wild-type Adr1. In this model the Ser-230 region would
modulate the amount and not the activity of PICs by more effectively
recruiting a rate-limiting component of the transcriptional machinery.
The second step at which Adr1 activity appears to be regulated is
post-chromatin binding. When Adr1 is bound to the ADH2
promoter in repressed growth conditions due to hyperacetylation,
transcription does not occur, because TBP is not recruited (18). The
failure to recruit TBP could be due to additional modifications, of
either Adr1 or histones, that are important for PIC formation in
vivo. Expression of Adr1 at high levels (36, 55), or expression of
the constitutive allele, ADR1c (5, 33) partially
overcomes glucose repression, suggesting that the block to TBP
recruitment in repressed conditions is not absolute. The binding but
apparent lack of activity of chromatin-bound Adr1 in cells with
hyperacetylated histones and the activity of Adr1 in cells
overexpressing Adr1, or Adr1c, seem like contradictory
observations. They could be reconciled if high levels of Adr1, or
Adr1c, enhanced the recruitment of TBP, or if
hyperacetylation negatively affected the binding of TBP to the
ADH2 promoter.
A miniature version of Adr1, retaining the nuclear localization region
and DNA binding domain, amino acids 1-160, together with a single
ADR1-encoded activation domain, TADIII, is able to activate
transcription and assemble PICs in vitro. Surprisingly, miniAdr1 can recruit TBP and assemble PICs in the absence of a TATA
box. A similar observation has been made using a Gal4-VP16 activator,
whereas a weaker activator, Gal4-AH, requires a TATA sequence in the
promoter for efficient recruitment (44). Apparently TADIII and VP16 can
recruit TBP and other components in the absence of a high affinity TATA
binding site due to other protein-DNA and/or protein-protein
interactions, but the PIC formed is inactive. Ranish et al.
(44) discuss the possibility that TBP plays a dual role in
initiation, one before and another after PIC formation.
The studies suggest a post-DNA binding role in transcription activation
for PAR, a sub-domain of the DNA binding region. Deletion of PAR from
miniAdr1 containing three fingers does not affect DNA binding (42), but
it reduces transcription from a reporter gene in vivo and
reduces activated transcription in vitro. PAR apparently
contains an activation function that cannot be detected in the absence
of a traditional activation domain, because ABD alone is inactive both
in vivo and in vitro. However, in certain mutant
yeast strains ABD suffices for transcription activation (39). It seems
likely that PAR is responsible for this activity.
miniAdr1 is stringently glucose-regulated by Snf1 and Reg1 in
vivo. However, recombinant miniAdr1 activates transcription and
forms PICs equally well in nuclear extracts prepared from glucose
repressed and derepressed cells and activates transcription in extracts
prepared from Snf1 mutant cells. These results are consistent with our
hypothesis that the role of Snf1 in derepression of ADH2
expression is to modify chromatin to allow Adr1 to bind. However, an
additional post-binding role for Snf1 in ADH2 derepression cannot be ruled out by the transcription studies. Modifications to Adr1
or other components may not occur or may not be preserved in the
nuclear extract. For example, Snf1 might be required in vivo
to overcome a modification that inactivated Adr1-dependent transcription in repressed conditions. If this modification were absent, for example, because recombinant Adr1 is used, no requirement for Snf1 would be evident. Similarly, if the inhibitory modification were labile in the nuclear extract, Snf1 would not be required to
overcome its negative influence on transcription.
PIC formation in nuclear extracts prepared from snf1 is
partially defective, although no defect in transcription activity is
detected in the same extracts. Recruitment of Mediator components appears to be affected more strongly than recruitment of general transcription factors or pol II. This defect is not specific to transcription factors that are glucose-regulated, because PIC formation
dependent on Gal4-VP16 activator is also partially defective. Snf1
shows genetic interactions with Mediator components Srb10 and Srb11,
suggesting a role for Snf1 in overcoming holoenzyme-mediated repression
of transcription (53). The defects we observe in PIC formation are
consistent with such a role. Alternatively, the defect in PIC formation
may be a nonspecific consequence of a general metabolic slow-down that
occurs after derepression of the snf1 mutant, which is
unable to grow in the absence of a fermentable carbon source.
Snf1 regulates transcription at multiple levels even at a single locus.
Both Adr1 and Cat8 regulate ADH2 expression (10). Snf1
regulates Cat8 expression and its transcription activation function
(7), and it regulates chromatin binding by Adr1. We could not identify
a defect in transcription activation in vitro in Snf1 mutant
extracts, but a partial defect in PIC formation was observed. Thus,
Snf1 might also regulate a second step in Adr1 activation. Having
multiple roles to activate a single gene or a single transcription
factor provides the cell with finer control of gene expression in
response to different environmental conditions while still maintaining
strict glucose repression.
We thank Ken Dombek for constructive comments
on the manuscript, Natalia Yudkovsky for help in developing the
transcription assays, and Steve Hahn for allowing one of us
(E. T. Y.) to work in his laboratory.
*
This work was supported by Research Grant GM26079 from
NIGMS, National Institutes of Health (to E. T. Y.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, August 6, 2002, DOI 10.1074/jbc.M206158200
2
E. T. Young and K. M. Dombek, unpublished observations.
3
E. T. Young, unpublished observations.
4
N. Kacherovsky and E. T. Young, unpublished observations.
The abbreviations used are:
Snf1, proteins are
indicated by gene names with a large first letter followed by small
letters;
SNF1, genotypes and gene names are capitalized in
italics;
ABD, Adr1 DNA binding domain, amino acids 17-165;
TAD, transcription activation domain;
TBP, TATA binding protein;
TAF, TBP-associated factor;
PAR, proximal accessory region (of Adr1);
ADH, alcohol dehydrogenase;
PIC, pre-initiation complex;
ChIP, chromatin
immunoprecipitation;
UAS, upstream activation sequence;
WT, wild-type;
pol, polymerase;
SM, synthetic medium;
HA, hemagglutinin;
CSRE, carbon
source response element.
Snf1 Protein Kinase Regulates Adr1 Binding to Chromatin but Not
Transcription Activation*
,
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-oxidation of fatty acids and peroxisome biogenesis (12-16). A
third example of this combinatorial control may occur at the GUT1
promoter, where both Adr1 and Ino2/Ino4 play important roles in
derepression (17). However, evidence of the in vivo binding
of Adr1 to the promoters of these genes exists only for ADH2
(18).
that are important for its function. TADIV
is located between amino acids 642 and 704. In vitro binding
studies as well as in vivo co-immunoprecipitation suggest
that these TADs interact with TFIIB, TFIID, Ada2, and Gcn5 (22, 23).
Thus, Adr1 may play an important role in recruiting an active pol II
complex to promoters. In addition, Adr1 plays an essential role in
mediating chromatin remodeling at the ADH2 promoter prior to
derepression (24, 25), and TADIII is sufficient for this remodeling
(26).
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and XL1.
Escherichia coli MC1061 (pREP) is used for
preparation of recombinant miniAdr1 proteins.
Saccharomyces cerevisiae strains used in this study
-Galactosidase assays were performed on
protein extracts or on permeabilized cells as described by Guarente
(45). ADH activity assays were performed as described previously
(35).
Oligonucleotides
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Fig. 1.
Adr1-HA binds to ADH2,
CTA1, ACS1, and POT1
chromatin only in derepressed cells and binding requires the Snf1
protein kinase. R, cultures grown in YPD (5%) glucose;
DR, cultures grown in YP-0.05% glucose; I, input
DNA. PCR was performed on a dilution of total, non-immunoprecipitated
DNA; WT, wild-type with respect to SNF1. ChIP was
performed as described under "Experimental Procedures" using
polyclonal anti-HA antisera. Real-time quantitative PCR was used to
obtain the values shown below the lanes. Primers used for
PCR analysis are listed under "Experimental Procedures."
A, kinetics of Adr1-HA binding to the ADH2
promoter determined by ChIP assay. Strain KVRY12 (relevant genotype
YIpADR1-HA3:KAN::LEU2 SNF1) was grown in YPD for
repressed (R) samples and shifted to YP-0.05% glucose for
the times indicated for derepressed (DR) samples. The amount
of ADH2 DNA obtained after 30 min of derepression was
assigned a value of 100. The concentration of ADH2 product
present in other samples is expressed relative to this value. The zero
time value for KVRY12 in repressed growth conditions corresponds to the
average value using immunoprecipitated DNA obtained from four
independent samples of repressed wild-type cultures. B,
kinetics of Adr1-HA binding and release. Strain KVRY12 was grown, and
samples were taken as described above. Thirty minutes after glucose
removal, it was added back to a concentration of 5%, and samples were
taken for ChIP analysis. C, Adr1-HA binding occurs after
derepression in the absence of protein synthesis. Strain KVRY9 was
grown in YPD and treated with 10 µg/ml cycloheximide for 10 min and
then shifted to YP-0.05% glucose by centrifugation and resuspension.
Sixty minutes later cells were collected and prepared for ChIP.
D, regulated binding of Adr1-HA to yeast promoters
ADH2, ACS1, CTA1, GUT1, and
POT1. Strains KVRY12 (SNF1), KVRY9-1 ( 
snf1),
and KVRY9-2 (reg1, data not shown, see text) were grown as
described above in YPD (R) or shifted to YP-0.05% glucose
(DR) for 6 h. Strain KVRY12 was derepressed for 60 min.
Strain KVRY12-1 was derepressed for 6 h.
-amanitin.3
7 versus 109, respectively
(42)) could explain its low activity in vitro. miniAdr1 also
has low DNA binding specificity, a measure of its specific to
nonspecific DNA binding affinity (42).
-galactosidase activity was measured to assess
the regulation of mini3Adr1. The data in Table
III show that both promoters are glucose
repressed and mini3Adr1-dependent for derepression. The
HIS4 promoter is more tightly repressed (20-fold) than the
CYC1 promoter (8-fold) is. Although UAS1
(3F)/HIS4lacZ is strongly glucose-repressed, its activity on
glucose is stimulated 14-fold by mini3Adr1, suggesting that mini3Adr1
is able to activate transcription weakly in repressed conditions. Table
III shows that mini3Adr1-dependent transcription in
vivo is dependent on the HIS4 TATA sequence, because
mutation to GAGA abolished activity. Table III also shows that
mini3Adr1 is SNF1-dependent. Thus, mini3Adr1 displays the regulated properties of intact Adr1 and is an appropriate activator to use for in vitro studies.
mini3Adr1-dependent gene expression is glucose-repressed,
dependent on Snf1 for derepression, and requires a TATA box
::URA3, respectively, containing the
indicated plasmids, were grown in selective media
(trpura) plus 5% glucose (Repressed) to about
2 × 107 cells/ml. A portion of the culture was assayed
for -galactosidase activity as described under "Experimental
Procedures," and cells from another portion were collected by
centrifugation and resuspended in selective media plus 0.05% glucose
(Derepressed) and grown for 18 h at 30 °C. A portion of the
culture was removed and assayed for -galactosidase activity. The
-galactosidase activities are Miller units. Activator plasmids are
YCpmini3Adr1172 (ABD) and YCpmini3Adr1 reporter plasmids are pNK 101 and pNK102 (YEp(UAS1-3Fconsensus)HIS4/lacZ and
YEp(UAS1-3Fconsensus)CYC1/lacZ, respectively.
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Fig. 2.
miniAdr1 activates transcription in
vitro from a single binding site and is dependent on TBP,
TADIII, and the PAR domain. Nuclear transcription extracts
prepared from strain TYY202 were supplemented with mini3Adr1 and mutant
derivatives as indicated in the figure and assayed as described under
"Experimental Procedures." A, mini3Adr1 is an efficient
activator from a single binding site. The indicated amounts of
Gal4-VP16 or mini3Adr1 were added to transcription extracts containing
pSH515 or pNK509, respectively, plasmids that contain one
UASG or one UAS1 (3F consensus) binding sites,
respectively. B, mini3Adr1 requires a TATA box for
activation of transcription in vitro. Plasmids pNK509 (TATA)
and pNK509m1 (GAGA) were incubated with or without mini3Adr1 and
assayed for transcript production as usual. C, mutations in
TADIII reduce transcription in vitro. mini3Adr1 mutants CR9,
containing multiple mutations in the acidic portion of TADIII, and HA2,
containing two altered transcription activation motifs (21) were added
at the indicated concentrations. D, the PAR domain is
required for efficient transcription in vitro. mini3Adr1
(3F) plus or minus ( N) residues amino-terminal
to the zinc fingers (the PAR domain (48)) and miniAdr1 (L146H;
F1H)) plus or minus (N) the PAR domain were
added at the indicated concentrations to nuclear transcription
extracts, and transcription was measured by primer extension assays.
MW, molecular weight markers in nucleotides; +1,
expected initiation site.
N) reduces the activity of both miniactivators.
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Fig. 3.
Glucose repression of transcription is not
observed in vitro. Nuclear transcription extracts
were prepared from strain TYY202 grown in YPD containing 5% glucose
(R) or grown in YPD-5% glucose and then transferred to
YPD-0.05% glucose (DR) for 6 h. mini3Adr1-dependent
transcription was measured by primer extension assays. 
, no
activator; +, mini3Adr1; +1, expected transcription
initiation site (44); MW, molecular weight markers in
nucleotides.
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Fig. 4.
mini3Adr1-dependent transcription
in vitro is independent of the Snf1 protein
kinase. Nuclear transcription extracts were prepared from strains
TYY202 (SNF1) and TYY390 ( snf1) grown in
YPD-5% glucose (R) or shifted to YPD-0.05% (DR)
for 6 h. Basal and mini3Adr1-dependent transcription
were assayed by primer extension. , no activator; +, mini3Adr1;
+1, expected transcription initiation site; MW,
molecular weight markers in nucleotides.
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Fig. 5.
mini3Adr1-dependent
pre-initiation complex (PIC) formation is independent of a TATA
sequence in the promoter but requires TADIII. PIC formation was
assayed on DNA templates immobilized on magnetic beads as described
under "Experimental Procedures." TATA, wild-type
promoter template; GAGA, mutant promoter template in which
the HIS4 TATA sequence is changed to GAGA (44);
WT, wild-type mini3Adr1 activator; HA2, mutant
mini3Adr1 activator containing mutations in TADIII (21);
ABD, mini3Adr1 lacking TADIII. The Western blot was probed
sequentially for the indicated proteins as well as for mini3Adr1 (not
shown). mini3Adr1 was present at similar levels in all of the
supplemented reactions.
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Fig. 6.
Pre-initiation complex formation is reduced
in snf1 mutant extracts. Nuclear transcription
extracts were prepared from wild-type SNF1 and
snf1 mutant strains, TYY202 and TYY390, respectively, grown
in derepressed conditions (see legend to Fig. 4). mini3Adr1 was used to
direct PIC formation on DNA templates immobilized on magnetic beads as
described under "Experimental Procedures." After polyacrylamide gel
electrophoresis and Western blotting to identify specific proteins,
antibody signals were quantified by fluorometry. The intensity of the
signal for each protein was normalized to the value observed for basal
transcription in wild-type SNF1 extracts. A,
example of a Western blot probed for Rpb1, Rpb3, and mini3Adr1.
B, quantification of antibody signals for: 1,
Swi3; 2, Srb2; 3, Srb4; 4, Med6;
5, TBP; 6, TFIIB; 7, TOA2;
8, Rpb1 on a scale normalized to the amount of each protein
present in SNF1 wild-type PICs formed in the absence of
activator mini3Adr1.
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1 nucleosome (24, 25). After Adr1-dependent remodeling
the 1 nucleosome is displaced toward the promoter (26) away from
UAS1. We hypothesize that the proximity of the 1 nucleosome to UAS1 inhibits Adr1 binding in repressed growth conditions. After glucose depletion, we imagine that phosphorylation of S10 on histone H3 by Snf1
at the ADH2 promoter, and subsequent acetylation of the 1
nucleosome, "loosens" the nucleosome on the DNA and enhances Adr1
binding, leading to nucleosome movement and subsequent PIC formation.
Reg1·Glc7 might affect the phosphorylation of histone H3 Ser-10
indirectly, by regulating the activity of Snf1 (4). Alternatively,
Reg1·Glc7 might dephosphorylate histone H3 S10-phosphate. The
putative Reg1·Glc7-independent pathway of ADH2 repression could act in the same pathway to affect phosphorylation of S10 on
histone H3.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Washington, Box 357350, Seattle, WA 98195-7350. Tel.:
206-543-6517; Fax: 206-685-1792; E-mail: ety@u.washington.edu.
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