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Proc. Natl. Acad. Sci. USAVol. 88, pp. 8597-8601, October
1991Genetics
Regulated expression of the GAL4 activator gene in yeast
providesa sensitive genetic switch for glucose repression
(repressors/weak promoters/synergism)
DAVID W. GRIGGS AND MARK JOHNSTONDepartment of Genetics,
Washington University School of Medicine, St. Louis, MO 63110
Communicated by Ronald W. Davis, June 21, 1991
ABSTRACT Glucose (catabolite) repression is mediatedby multiple
mechanisms that combine to regulate transcriptionof the GAL genes
over at least a thousandfold range. We havedetermined that this is
due predominantly to modest glucoserepression (4- to 7-fold) of
expression of GAL4, the geneencoding the transcriptional activator
of the GAL genes. GALAregulation is affected by mutations in
several genes previouslyimplicated in the glucose repression
pathway; it is not depen-dent on GAL4 or GAL80 protein function.
GALA promotersequences that mediate glucose repression were found
to liedownstream of positively acting elements that may be
"TATAboxes." Two nearly identical sequences (10/12 base pairs)
inthis region that may be binding sites for the MIG1 protein
wereidentified as functional glucose-control elements. A
4-base-pairinsertion in one of these sites causes constitutive GAL4
syn-thesis and leads to substantial relief (50-fold) of glucose
repres-sion ofGAL] expression. Furthermore, promoter deletions
thatmodestly reduce GALA expression, and therefore presumablythe
amount of GAL4 protein synthesized, cause much greaterreductions in
GAL] expression. These results suggest thatGALA works
synergistically to activate GALl expression.Thus, glucose
repression of GAL) expression is due largely toa relatively small
reduction of GALA protein levels caused byreduced GALA
transcription. This illustrates how modest reg-ulation of a weakly
expressed regulatory gene can act as asensitive genetic switch to
produce greatly amplifiled responsesto environmental changes.
Expression of the GAL], -7, and -10 genes, which arerequired for
galactose catabolism in Saccharomyces cerevi-siae, is regulated at
two levels (1). (i) Galactose induces theirtranscription by
preventing GAL80 protein from inhibitingfunction of the GAL4
transcriptional activator. (ii) Glucosecauses severe repression of
GAL gene transcription by aprocess to which several different
mechanisms contribute.Some operate to reduce the amount of inducer
available toinactivate GAL80 by reducing expression of GAL3,
requiredfor inducer synthesis (2), and of GAL2, encoding the
galac-tose transporter (3), and by inactivation of preexisting
galac-tose permease in the cell (4). Other mechanisms of
repressionoperate through sites in the GAL promoters termed
theupstream activation sequence (UAS) and the upstream re-pression
sequence (URS) and, therefore, act more directly torepress
transcription.The UAS and URS regions from the GAL] promoter
are
capable of independently mediating glucose repression (5).The
repression that operates through the UAS region, whichcontains four
binding sites for the GAL4 activator, probablyreflects reduced
levels or reduced function of the GAL4protein in glucose-grown
cells. Repression mediated by theURS, which lies between the UAS
and the "TATA element,"
is presumably due to unidentified repressors that bind to
thisregion.UAS-mediated repression is characterized by the failure
of
GAL4 to bind the UAS in cells growing in the presence ofglucose
(6, 7). This could be due to glucose-induced modifi-cations ofGAL4
that affect DNA binding, to glucose-inducedproteolysis of the GAL4
protein, or to glucose repression ofGALA gene expression. We
describe experiments that showthat GALA expression is modestly
reduced by glucosethrough the action of specific negatively acting
elements inthe GALA promoter. The resulting reduction in
intracellularGAL4 activator levels leads to a greatly amplified
effect onexpression ofGAL] and accounts for a substantial portion
ofglucose repression of GAL] expression.
MATERIALS AND METHODSStrains and Growth Conditions. All yeast
strains used in
this study (except YM3322) contain ura3-52, Ahis3-200, ade2-101,
lys2-801, LEU2::pRY181 (GALJ/lacZ) (pRY181 is de-scribed in ref.
8). All cultures were grown at 30'C in YPmedium (9) containing the
described carbon sources. Thepresence of 0.1% glucose in medium
with 5% (vol/vol)glycerol stimulated the growth of strains but
caused nodetectable glucose repression, as has been noted (5).
Plasmids Designed for Construction and Chromosomal In-tegration
of Modified Promoters and Fusions. A detaileddescription of the
construction of these plasmids will bepresented elsewhere. Briefly,
GALA and 1.5-2.0 kilobases ofDNA flanking each end were cloned into
a modified pBlue-script SK+ (Stratagene) vector. A 1.1-kilobase
HindIll frag-ment containing the selectable gene URA3 was then
insertedinto a HindIII site adjacent to the 3' end of the gal4
codingregion. Fusions were constructed by replacing an
internalrestriction fragment of GALA with fragments carrying
thegene for chloramphenicol acetyltransferase (CAT) or HIS3such
that GALA was fused in-frame at its Sph I site at codon11 to the
first codon of the reporter genes. Deletions andlinker insertions
in the GALA promoter were constructedusing PCR methodology, which
will be described in detailelsewhere. To integrate the various
mutations and fusions,yeast were transformed with the products of a
restrictiondigestion that releases the cloned insert from the
vector.Since the recipients usually contained a deletion
removingthe entire GALA coding region and since the ends of
DNAfragments are highly recombinogenic (10), URA+ transform-ants
arise by recombination between sequences flankingGALA. Southern
blot analysis confirmed the proper integra-tion of mutations in all
transformants tested.Enzyme Assays. For CAT assays, cells from 5-ml
cultures
grown to an A6w of 0.8-1.5 in YP medium with the appro-priate
carbon sources were washed with 0.5 ml of 0.25 MTris-HCl (pH 7.5)
and frozen in liquid nitrogen. To prepare
Abbreviations: UAS, upstream activation sequence; URS,
upstreamrepression sequence; CAT, chloramphenicol
acetyltransferase.
8597
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page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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extracts from thawed pellets in 1.5-ml microcentrifuge tubes,0.2
ml of ice-cold 0.25 M Tris-HCl (pH 7.5) was added toresuspend the
cells, acid-washed glass beads were added toa level 1-2 mm below
the meniscus, and the tubes wereshaken at maximum speed on the
6-inch platform head (1 inch= 2.54 cm) of a Vortex Genie 2
(Scientific Industries,Bohemia, NY) at 40C for eight 20-s periods,
with 20-s pausesbetween each period of shaking. The tubes were
centrifugedin a standard microcentrifuge at 40C for 5 min, and
samplesof the supernatants were stored at -700C. The
concentrationof protein in each extract was determined by the
method ofBradford (11). CAT activities were determined by the
phase-extraction method described by Seed and Sheen (12).
Typi-cally, 3-10 ,gg of protein were assayed in 100-jil
reactionvolumes. Units ofCAT activity are defined as cpm measuredin
the organic phase and expressed as a percentage of totalcpm (%
conversion) divided by the amount ofprotein assayed(pg) and the
time of incubation (min). Assay of f-galacto-sidase activity was
carried out on permeabilized cells asdescribed by Yocum et al.
(8).
RESULTSExpression of GAL4 Is Regulated by Glucose. GALA is
an
extremely weakly expressed gene (13). To provide a
sensitiveassay for measuring GALA expression, we constructed
plas-mids containing chimeric genes in which several kilobases
ofDNA upstream of and including codon 11 ofGAL4 are fusedto either
the CAT gene or HIS3. A single copy of thesefusions was integrated
into the yeast genome without anyassociated vector sequences by
recombination at the GALAlocus such that all sequences native to
the region upstream ofthe fusion junctions were retained.The data
in Table 1 show that expression of the GALA-
CAT fusion in glucose-grown cells was 5- to 7-fold lower thanin
cells grown on glycerol. This effect was similar in
strainscontaining wild-type and null alleles of GALA and
GAL80.These results confirm earlier work showing GAL4 does
notregulate its own synthesis (13) and suggest the existence of
amechanism for regulating the synthesis of GAL4 protein inresponse
to glucose.
Regulation of GALA was also apparent from the growth ofa strain
containing a GAL4-HIS3 fusion on minimal plateslacking histidine
(Fig. 1). When the carbon source wasraffinose, which does not cause
repression of the galactosemetabolizing pathway, GALA promoter
activity was suffi-cient to produce a His' phenotype; growth on
glucoseapparently reduced the expression of the hybrid gene to
alevel that was inadequate to support colony formation.
Trans-Acting Mutations Affecting GALA Regulation. Afterextended
incubation (4-6 days) of the GAL4-HIS3-containing strain in the
presence of glucose, His' mutantsresistant to glucose repression
arose at a frequency of 10-5-
Table 1. Regulation of GAL4-CAT expression by glucoseCAT
activity
FoldStrain Genotype Glycerol Glucose decreaseYM2632 gaiC ga180-
4.4 0.9 4.7 ± 1.1YM2631 gaiC GAL80 2.6 0.4 6.8 ± 1.2YM3544 GAL4+
ga180- 2.2 0.3 7.5 ± 1.5YM3543 GAL+ GAL80+ 2.5 0.4 5.9 ± 2.5
All strains contain the CAT gene fused to GALA at the GALA
locus.GAL4+ strains contain a single copy of the functional gene
integratedat L YS2. CAT assays were performed on exponentially
growing cellsin YP medium containing either 5% glycerol and 0.1%
glucose or 2%glucose as indicated by glycerol or glucose,
respectively. Activitiesrepresent the average calculated from at
least three experiments.Data for fold decrease are expressed as
mean ± SEM.
GAL4PROMOTER HIS3
RAF GLUHIS - HIS
FIG. 1. Carbon-source-dependent growth of a strain containing
aGALA-HIS3 fusion. Strain YM3182 containing the GAL4-HIS3fusion
integrated at GALA was streaked (upper plates) and spread(lower
plates at 1 x i07 cells) on SD plates lacking histidine
andincubated at 30TC for 3-5 days. RAF, raffinose; Glu,
glucose.
10-6 (Fig. 1). Analysis of several of these mutants revealedthat
some contained recessive defects that were comple-mented by GRRI,
SSN6, or TUPI, genes that have beendescribed and are required for
glucose repression of GALIand other genes (14-16). Furthermore,
glucose repression ofGALA-CAT activity was relieved in strains with
character-ized mutations in these genes and in several others
implicatedin glucose regulation (1, 14, 16-18) (Fig. 2A). The
GALl]gene, which is required for full expression of GAL) butappears
not to be involved in glucose repression (19), had noeffect on
either the level ofGALA expression or its regulation.Function
ofSNF1, a protein kinase essential for release fromrepression of
all glucose-regulated genes analyzed to date(20), was also required
for derepression of GAL4-CATactivity (Fig. 2B). A mutation in SSN6,
which is a suppressorof snfl mutations (15), resulted in
constitutive GALA expres-sion (Fig. 2B). Thus, all of these genes
affect GALA in thesame manner in which they affect other
glucose-repressedgenes.
Identification of a GALA Promoter Element ControllingGlucose
Repression. We tested for the existence of promotersequences
necessary for glucose regulation of GALA byexamining the effects of
internal deletions constructed up-stream of the GALA-CAT gene. As
shown in Fig. 3, a50-base-pair (bp) region (positions -77 to -25)
required forglucose repression was identified (line A). This region
lies=40 bp upstream from the most promoter-proximal site
fortranscription initiation (21) and lies downstream from
posi-tively acting elements that we have identified from a
moreextensive analysis of the GALA promoter. These positivelyacting
elements include two that we believe may be TATAboxes because (i)
at least one of them is required for anyGALA expression, (ii) their
sequences are A+T-rich, and (iii)they are in a location
characteristic of TATA elements (ourcomplete analysis of the GALA
promoter will be presentedelsewhere).Within the glucose control
region, we recognized a directly
repeated sequence (10/12-bp identity), each copy of whichshould
lie on the same face of the DNA helix (i.e., separatedby 21 bp;
Fig. 3). All deletions or linker insertions thatdisrupted the
upstream copy resulted in completely consti-tutive promoter
activity (constructs A, B, E, F, G, and H);a deletion removing the
downstream copy (construct C)eliminated most, though possibly not
all, repression. Muta-
Proc. Natl. Acad. Sci. USA 88 (1991)D
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Proc. NatL. Acad. Sci. USA 88 (1991) 8599
TATA BOXES?
POSITIVE ELEMENTS
-13_ -320...a-192 -1394 -120
GLUCOSE CONTROL_.-77-m2-
.-.-:7-SE,-4
-7 -i7 -56 i471 -36
30 60 90 120 150 180Time after glucose removal
FIG. 2. GALA expression in various glucose repression
mutants.(A) GALA-CAT activity measured in cells growing
exponentially inYP medium containing 5% glycerol and 0.1% glucose
or containing2% glucose. Strains: Wt (wild type, YM3216), gall)
(YM3220), grrl(YM3317), tup1 (YM3390), hxk2 (YM3313), regi
(YM3316), ga182(YM3314), ga183 (YM3315), and migi (YM3733). All
alleles exceptga182 and ga183 are gene disruptions. (B) Time course
of derepres-sion of GALA-CAT activity in wild-type (YM3216) (6),
Asnfl(YM3322) (e), and Assn6 (YM3319) (A) backgrounds. Cells grown
toearly logarithmic phase in YP containing 2% glucose were
centri-fuged and resuspended in YP containing 5% glycerol and
0.1%glucose. Incubation was continued and samples were removed
atvarious times (min) for assay.
tions in the region that left both copies intact (constructs
Dand I), or nearly intact (construct J), preserved normalglucose
regulation.The directly repeated element in GALA resembles a
re-
peated sequence. present in an inverted orientation in
theporbnoter of the glucose-repressed SUC2 gene (Fig. 3). InSUC2,
these sequences are binding sites for a protein knownas MIG1 -(18).
The presence of AMIG on a high copy numberplasmid causes reduced
expression of SUC2 and inhibitsgrowth on galactose, raffinose, and
other nonrepressingsugars; disruptions of MIG) relieve glucose
repression ofSUC2 (18). Thus,, MIG1 exhibits the properties of a
glucose-sensitive repressor. MIGI function is also required for
reg-ulation of GALA, since a mig) null mutation relieves
glucoserepression of GALA expression (Fig. 2A). Recent in
vitrofootprinting experiments have confirmed MIG1 binding tothe
upstream motif (site 1) in the GALA promoter (30).Although no MIG1
binding was detected at the downstreammotif (site 2), our results
show that its deletion does affectregulation in vivo (Fig. 3,
construct C). Nevertheless, theresidual repression observed in the
absence of the down-stream site (Fig. 3, construct C), the complete
loss ofrepression caused by mutation of the upstream site
(con-structs E and H), the footprinting experiments, and thegreater
sequence similarity of the upstream site to the MIG1binding sites
in SUC2 implicate the upstream motif (site 1) inGALA as the primary
binding site for MIG1.
A (-77/-25)
B (-64/-25)
C (-51/-25)
D (-38/-25)
E (-771-63)
F (-77/-52) --
5; (-77/-39)
II (-65L) -
I (-52L) -. --
J (-39L) ---__
'. iI.. ' 'iJ T .-C.ATL` S ATT :'IUC2 S ITTEh ':T'C 2 SIT'I-
i>'
-.
A ".
"ONS - N
FIG. 3. Delineation of sites in the GALA promoter required
forglucose repression. The first of three previously identified
(21)positions for transcription initiation is designated + 1. A
6-bp inser-tion containing aBamHI site lies between the indicated
end points ofall deletions. Vertical arrows designate insertion
mutations that forma new BamHI site beginning 1 bp downstream of
the nucleotideindicated at the left end of each line. Each modified
promoter wasfused to the CAT gene and integrated at the GALA locus
in YM2632.CAT activity was assayed as described in Table 1, and the
repressionratio was calculated as the activity on glycerol divided
by activity onglucose. The sequences of sites 1 and 2 (solid boxes)
are shown at thebottom and are compared to similar sites from the
SUC2 promoter.
S i ce of GALM Regulton in the Galactose CataboliteRepression
System. In gaI80 cells, where mechanisms ofrepression that operate
to reduce inducer levels are irrele-vant, transcription of the
GAL], -7, and -10 structural genesis reduced about a hundredfold by
glucose (22). Since theobserved effect of glucose is to reduce GALA
expression only-5-fold, it was conceivable that this regulation
would haveonly a minor role in the overall process of repression of
thegenes that GAL4 activates.To correlate changes in GALA
expression with their con-
sequent effects on expression of a gene activated by GAL4,we
employed internal deletions in the GALA promoter to varyits
strength and to measure the effects of reducing GAL4synthesis on
GAL) expression under nonrepressing condi'tions (5% glycerol). GALA
expression was evaluated bydetermining the effect of each deletion
on GAA-CAT ac-tivity; GAL) expression was measured by assaying
activity ofa GAL-lacZ gene in an isogenic background with the
samealtered promoters driving wild-type GALA synthesis. Theresults
of this analysis show that modest reductions in GALexpression lead
to much larger reductions in expression ofGAL]. In fact, a decrease
in GAL expression comparable tothat mediated by glucose (en-fold,
15% ofwild type) appearsto cause at least a 40-fold reduction in
GAL) expression( 0.0C0
* 4.0
p3.0
0
2.0
1.0
PEPRESS O0RATIO
4.9
0.90.9'0 I). .i
0.71-00. 6B
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-O -*o~,~.0I . * .1- . .
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13.4
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Genetics: Griggs and Johnston
A .'- :..1", A " .:.. ''' '.", :"' f". !,
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G)
Z-
C,)co()0.
x0)
X.Ji
1 20
80 a
40 I"
0 1,. 0
0~~~~~O&4 o-o-,i20 40 60 80
GAL4 expression, % wild type100 120
FIG. 4. Effect of reduction ofGAL4 expression on GAL]
expres-sion. For GALA expression, the CAT activity produced from
aGAL4-CAT fusion gene carrying various deletions that weaken
theGAL4 promoter is plotted. For GAL) expression, the
0-galactosidaseactivity produced from a GALJ-lacZ fusion in the
same geneticbackground (YM2632) with GAL4 expression being driven
by thesame altered promoters is plotted. All assays were performed
usingcultures growing exponentially under nonrepressing conditions
(YPcontaining 5% glycerol and 0.1% glucose). Each point represents
theaverage of at least three assays of each enzyme's activity.
Standarddeviations for all data were -22 except for the assay ofCAT
activityfor the point marked e, which had a standard deviation of
32.Standard deviations for points between 0 and 40%6 ofwild-type
GAL4activity were 50 times higher in the strain with
constitutiveGAL4 expression than in the same strain with normal
GALregulation (Table 2). Similar relief of GAL) repression
wasobserved when GALA was expressed constitutively due to amutation
in mig) (data not shown). The magnitude of theeffect is such that
there remains only a residual 4-fold effectthat must be accounted
for by other mechanisms, such as theURS-mediated system of
repression (5) or possibly post-translational modifications
affecting GALA protein function(23).
DISCUSSIONWe have determined that expression of the GALA
activatorgene is repressed modestly by glucose and that this
regulationis critical for glucose repression of galactose
metabolism.GALA regulation was evident from the activities of
GAL4-
CAT and GAIA-HIS3 gene fusions integrated by recombi-nation at
the GALA locus. The magnitude of the effect issimilar to the
reduction in GALA mRNA levels that Laughonand Gesteland (13)
observed in glucose-grown cells. Com-pelling confirmation of the
glucose repression of GALAexpression was provided by our ability to
select mutants withdefects in genes previously shown to be involved
in glucoserepression (GRRI, SSN6, and TUPI) by using a
straincontaining a GAIL-HIS3 gene fusion. Furthermore, all
ofthegenes required for glucose regulation that we tested were
alsorequired for GALA regulation (Fig. 2). Thus, GALA is a
typicalglucose-repressed gene.Two key results suggest that the
modest 5-fold regulation
of GAL expression accounts for a substantial amount of
theglucose repression of GAL). (i) A mutation in the GALApromoter
that abolishes its regulation by glucose relievesmost ofthe glucose
repression ofGAL) expression (Table 2).(it) Small reductions in
GAL4 expression are sufficient toaccount for much greater
reductions of GAL) expression(Fig. 4). In this experiment, it is
significant that when GAL4levels are reduced by promoter deletions,
independently ofglucose repression but by an amount similar to that
caused bygrowth on glucose (5- to 6-fold), the resulting reduction
ofGALl expression is of a magnitude comparable to that whichwe
observe to be caused by UAS-mediated glucose repres-sion (-40-fold)
(data not shown).The relationship between GAL4 and GAL)
expression
would be most simply explained by cooperative binding ofGALA to
its four binding sites in the GAL) promoter. Ginigerand Ptashne
(24) have established that GAL4 protein bindscooperatively to this
promoter in vivo. The promoters ofother GAL genes (e.g., GALJO,
GAL2, and GAL7) that areseverely repressed by glucose also contain
multiple sites forGALA binding (1), so GALA repression may affect
expressionof these genes similarly to GAL). Interestingly, the
activityof GAL80, which has only one GALA binding site, is
notrepressed significantly by glucose (25).We propose that the
combination of GALA regulation and
cooperative GALA action constitutes a genetic switch mech-anism
mediating transition in a two-state system. This isreminiscent of
the genetic switch that controls bacteriophageA development (26j.
In the derepressed state, the intracellularconcentration of GALA
protein would be sufficient to stabi-lize binding to multiple
adjacent sites in the GAL) promoterwith the aid of cooperative
interactions; in the presence ofglucose, the slightly reduced
expression ofGALA would dropthe activator concentration below a
narrow threshold levelrequired for occupancy of at least the weaker
sites. Alterna-tively, the effect of GALA regulation could be
amplified if,after GALA proteins are bound, they then function
cooper-atively at another level to activate transcription (27).The
experiments of Mylin et al. (23) have demonstrated a
correlation between GAL4 function and the presence of
Table 2. Effect of constitutive GALA expression on regulation of
GAL)GALA expression GAL) expression
GALA Fold Foldregulation Glycerol Glucose decrease Glycerol
Glucose decreaseWild type 6.1 ± 0.8 1.2 ± 0.1 5.3 1194 ± 250 7 ± 1
170Constitutive 6.4 ± 0.5 5.5 ± 1.4 1.2 1266 ± 95 360 ± 11 4
Strains exhibiting either normal glucose-regulated expression
ofGALA (strains YM3216 and YM3106)or constitutive expression of
GALA (strains YM3747 and YM3756) due to a BamHI linker insertion
atposition -65 of the GALA promoter (Fig. 3, construct H) were
analyzed. All strains are isogenic andnre gaI80. GALA expression
was ietermined by assaying GALA-CAT activity in strains YM3216
andYM3747. GAL) expression was determined by assaying GAL1-acZ
activity in isogenic strains(YM3106 and YM3756) in which GALA
synthesis was driven by the same wild-type or mutant promotersthat
were used to drive CAT synthesis. Assays were performed on
exponentially growing cells in YPmedium containing either 5%
glycerol and 0.1%6 glucose or 2% glucose as indicated by glycerol
orglucose, respectively. Data are expressed as mean ± SEM.
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Proc. NatL. Acad. Sci. USA 88 (1991) 8601
phosphorylated forms of the GAL4 protein in the cell.Addition of
glucose to cells growing under inducing condi-tions caused a rapid
shift to the nonphosphorylated form.However, our results suggest
such a mechanism can onlyaccount for a minor amount of glucose
repression of GAL]expression since constitutive GAL4 transcription
relievedmost of the glucose repression of GAL] (Table 2).
Mutational analysis of the GALA promoter allowed thedelineation
of two nearly identical sequence elements nec-essary for glucose
regulation (Fig. 3). These sites are similarto MIG1 binding sites
in the glucose-repressed SUC2 (inver-tase) promoter, and mutation
of migi relieved repression ofGALA. Disruption of site 1 in the
GALA promoter completelydestroyed regulation; disruption of site 2,
the sequence ofwhich differs at only one position from the
consensus se-quence for MIG1 binding, only moderately affected
repres-sion (Fig. 3). This suggests that site 1 mediates
strongerbinding in vivo and is consistent with results of in
vitrofootprinting experiments showing MIG1 binding at site 1
butapparently not to site 2 (30). Thus the function of
thedownstream site (site 2) may be to stabilize binding of MIG1at
the other site through cooperative interactions. Althoughthe two
MIG1 binding sites in SUC2 are inverted with respectto one another
and are separated by 45-50 bp, the GALA sitesare directly repeated
and are separated by only 10 bp.Therefore, no specific
configuration of the sites with regardto orientation or intervening
distance seems to be required forrepressor function.The location of
the repressor binding sites is unusual.
There are no apparent TATA elements in the 38 bp betweenthe
downstream element (site 2) and the site of
transcriptioninitiation. However, a region just upstream of element
1 isabsolutely essential for basal promoter activity and
containstwo weak TATA-like motifs (data to be presented
elsewhere,see Fig. 3). Thus one potential mechanism for regulation
isthat binding of MIG1 during growth on glucose interfereswith the
assembly or the activity of the basic transcriptionapparatus at the
TATA box. The positioning of elementssuggests MIG1 may repress GALA
expression differentlythan it represses SUC2, where it appears more
likely thatMIGI competes for binding of an activator to a UAS
siteoverlapping the MIG1 binding site (18). In addition,
sequencecomparisons (J. Flick and M.J., unpublished data)
suggestthat MIG1 binds directly to the URS element (5)
locatedbetween the UAS and TATA box in the GAL] promoter.Hence, in
the three promoters in which MIG1 is likely tooperate, the binding
sites appear to reside in different loca-tions. It is interesting
that MIGI appears to operate on boththe GALA and GAL] promoters.
Thus, MIG1 may contributeto regulation of GALl expression at two
levels: it regulatesthe amount of GAL4 activator produced and may
modulateGAL4 function at the GAL] promoter.MIG1 has been shown to
contain two C2H2 zinc-finger
motifs that share considerable homology with fingers fromthree
mammalian proteins proposed to be involved in controlof mitogenesis
and in developmental regulation (18). Two ofthese, Egr-l (NGFI-A or
Krox-24) and Egr-2 (Krox-20), bindto sites similar to those
recognized by MIG1 and may beregulators of genes of the mammalian
early growth response,including one that encodes a glucose
transporter (28). Thethird gene encodes the Wilms tumor suppressor
protein,which probably acts to repress the expression
oftransforminggenes (29). Thus these proteins appear to make up a
family of
proteins whose DNA binding domains have been highlyconserved and
whose function is to adapt cells for rapidgrowth. The finding that
MIG1 is involved in regulation ofGALA, itselfa regulator
oftranscription, raises the possibilitythat the Egr or Wilms tumor
proteins might also regulatesynthesis of DNA binding proteins in
mammals. Such sys-tems, in which the regulation offunction ofone
DNA bindingprotein affects transcription of another, provide the
potentialfor great flexibility and complexity in cellular
responses.
We thank Jan OlofNehlin and Hans Ronne for providing
importantunpublished information and for supplying plasmids for
MIGI dis-ruption. This work was supported by Public Health Service
GrantGM32540 from the National Institutes of Health. D.W.G. was
aidedby Postdoctoral Fellowship Grant PF-4009 from the American
Can-cer Society.
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