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.

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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-

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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>'

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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

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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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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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