Vol. 12, No. 10MOLECULAR AND CELLULAR BIOLOGY, OCt. 1992, p. 4562-45700270-7306/92/104562-09$02.00/0Copyright © 1992, American Society for Microbiology

Distinct Roles for the Two cGATA-1 Finger DomainsHENG-YIN YANG AND TODD EVANS*

Department ofBiological Sciences, Langley Hall, Room 518, University ofPittsburgh, Pittsburgh, Pennsylvania 15260

Received 1 June 1992/Accepted 17 July 1992

We have generated and analyzed by functional assays mutations of the chicken erythroid transcription factorGATA-1. The cGATA-1 protein contains two related finger domains highly conserved across species andcharacteristic of the family of GATA-binding factors. We find that mutations in the C-terminal finger oradjacent basic region abolish sequence-specific DNA binding, confirming that this region constitutes a novelDNA-binding domain sufficient to recognize the consensus WGATAR motif. At least three separate regionsoutside of this finger II domain contribute in a cooperative manner to the trans-activation potential of theprotein. As expected from previous results analyzing the mouse homolog, we find that the N-terminal fingerplays a role in DNA binding by affecting the stability of the DNA-protein complex. In addition, we findmutations of finger I subtly altered in DNA-binding function which greatly diminish trans-activation. Ourresults support the notion that the GATA-1 protein must be positioned precisely on the GATA cis element toenable the activation of target genes.

The DNA-binding protein GATA-1 is a positive-actingtranscription factor known to regulate most or all erythroidcell-specific genes (9, 22). The protein, found in erythroidcells of vertebrates at all stages of development (10, 28),functions by binding within regulatory regions of promotersand enhancers to activate transcription. Other transcriptionfactors must cooperate with GATA-1 to regulate sets ofgenes characteristic for specific stages during developmentor cellular differentiation. However, GATA-1 is the onlyhematopoietic cell-specific protein which has been generallyimplicated in the control of all such genes. By analyzingchimeric mice derived in part from embryonic stem cellsharboring a mutant GATA-1 gene, it was recently shown thatGATA-1 expression is required for the generation of a fullydifferentiated erythroid lineage (23). It has therefore beenproposed that GATA-1 plays a key role in regulating a switchfrom uncommitted erythroid precursor, resulting in a uniquecell-specific pattern of gene expression.The cDNA clones for GATA-1 have been isolated from

chick, mouse, human, and frog cells, and the primarystructures of the proteins have been determined (7, 25, 26,31, 32). The most striking feature of the deduced proteinsequence is a highly conserved region in the central third ofthe protein, consisting of two closely related but distinctrepeats. Each repeat contains the sequence CXNCX4TXLWRRX3GX3CNAC, followed by a basic domain which isalso well conserved among the vertebrates. By analogy towell-characterized cysteine-rich zinc-binding domains, thesetwo repeats have been called zinc fingers, and they werepredicted to play a role in DNA binding. Although we havesince found metal ion to be essential for DNA-bindingfunction (11), it is clear that the GATA-1 fingers are quitedifferent in sequence (and probably structure) from eitherthe steroid hormone receptor fingers or the TFIIIA class offinger. The vertebrate GATA-1 proteins are not well con-served between amphibian, avian, and mammalian speciesoutside of the finger regions.

Following the isolation of GATA-1 clones, a number ofother cDNAs that also contain the central conserved finger

* Corresponding author.

region were isolated by cross-hybridization to GATA-1 (6,30). These proteins (designated GATA-2, GATA-3, andGATA-4) also recognize and bind to a WGATAR motif inDNA and presumably activate sets of genes distinct fromGATA-1 in cells in which they are expressed (14, 16, 29).Several independently isolated nonvertebrate cDNAsclearly represent additional members of the growing GATAfamily of transcription factors (4, 12, 15). All of theseproteins are related in the finger region but do not showsequence similarities outside of this domain.We have previously shown that transient expression of the

GATA-1 cDNA is sufficient to trans-activate a reportera-globin promoter in fibroblasts (8). In this model system,expression of the globin reporter is entirely dependent onexpressed GATA-1 and a functional GATA-1 binding site.Avian or mammalian GATA-1 proteins function similarlywhen expressed in heterologous cell types (i.e., cGATAexpressed in human HeLa cells or hGATA-1 expressed inprimary chick fibroblasts), despite the lack of sequenceconservation between the factors outside the presumptiveDNA-binding domain (24). GATA-1 has been shown to be asufficient erythroid factor for the direct trans-activation innonerythroid cells of several promoters, including its ownand that of the erythropoietin receptor gene (2, 8, 13, 33). Inother cases, GATA-1 has not functioned to trans-activatecertain erythroid gene promoters, implying the importanceof other erythroid factors and necessitating the use ofartificial reporter constructs (18, 27).Understanding how GATA-1 functions to coordinately

regulate a diverse set of genes and establish a differentiatedstate will require a comprehensive knowledge of functionaldomains of the protein. This knowledge would assist indetermining potential cellular targets of GATA-1 within thecontext of an active transcription complex. With these goalsin mind, we have sought to identify domains of cGATA-1 byanalyzing mutated proteins in DNA-binding and transienttrans-activation experiments.

MATERIALS AND METHODS

Construction of mutated cDNA clones. The deletion muta-tions were generated by excising DNA fragments from either

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DISTINCT FUNCITIONS FOR GATA-1 FINGERS 4563

TABLE 1. cGATA-1 deletion mutationsa

GATA-1 pBluescript Endpoint of RSVclone clone deletion expressionconstruct

WT p20-1 pHY15.15A6 pHY10.1 SfaNI (+58) K pHY18.15A25 pHY4.22 BstNI (+113) M pHYl9.15A50 pHY5.3 BstNI (+188) M pHY20.15457 pHY8.3 RsaI (+211) pHY21.15471 pll-2 b pRSV11-25477 pHY6.1 BstNI (+269) M pHY23.15484 pHY9.4 XmaI (+288) M pHY24.15A111 pHY3.8 HincII (+373) pHY25.1054121 pHY7.2 NarI (+403) K pHY26.25A158 pHYl.3 BssHII (+514) K pHY27.154180 pTE11.5 NcoI (+576) pHY28.13A283 pHY30.1 BspEI (+887) M pHY33.13A234 -c SacII (+741) pRSVSacII3A204 pHY31.1 FspI (+650) pHY34.234161 pHY32.1 BssHII (+514) K pHY35.15A82/3A227 _c XmaI (+288 to +712) pHY29/Xma

a The wild-type (WT) clone is a full-length cDNA descnibed previously (7).For each mutation, the following information is given: the name of thedeletion mutation as used in the text for clarity (GATA-1 clone), the plasmid(laboratory name) used for in vitro transcription-translation experiments(pBluescript clone), the restriction site used to create the mutation and theenzyme used (K, KNenow polymerase; M, mung bean nuclease) to generateblunted ends when necessary (endpoint of deletion), and the plasmid (labo-ratory name) of the resulting subclone after transfer of the mutated cDNA intoan RSV expression vector (RSV expression construct).

b The 5A71 mutation results from a randomly isolated truncated cDNAclone which uses for translation an internal ATG at position 71.

c Unlike the majority of clones, the 3A234 and 5482/34227 mutations wereconstructed by using specific oligomers to generate in-frame stop and/or startcodons.

the 5' or 3' end of the full-length (wild-type) cDNA clonep20-1 (7), using convenient restriction sites. To restore thenatural 5' and 3' flanking sequences, the full-length cDNAinsert was first isolated and purified from the pBluescript IISK- vector by BamHI and SalI digestion. For 5' deletions(54 clones; Table 1), following digestion (partial or com-plete) with an enzyme that cleaves internally (see footnote toTable 1), the fragment was blunted accordingly, digestedwith HindIII, and subcloned back into a p20-1-derivedvector. These vectors were made by partial digestion ofp20-1 with NcoI (which cuts once at the initiation methioninecodon and once internally); the resulting DNA was bluntedaccordingly, restricted to completion with HindIII, andpurified by gel electrophoresis to obtain a full-length vectorcontaining all 5' noncoding sequence to the initiation Met.For 3' deletions (3A clones), a similar strategy was used,employing a unique Bsu36I site at the 5' end of the insertsand a CelIl site near the stop codon. Following restriction togenerate a deletion, fragments were blunted, digested withBsu36I, and ligated into an appropriate Bsu36I-CelII-di-gested vector. Because of the use of the blunted CelII site,the 3A clones retain all 3' noncoding sequences in addition tothe last four amino acids. In all cases, the method used tocreate blunt ends was chosen to ensure the proper readingframe of the coding sequences. The resulting clones (pBlue-script clones; Table 1) were used for in vitro transcriptionassays.The double-point mutations within the DNA-binding do-

main were generated by site-directed mutagenesis, using akit from Amersham as instructed by the manufacturer. Asingle-stranded phagemid template was generated from the

p20-1 cDNA clone; four mutated oligomers were designed tospecifically hybridize to each of the four Cys-X-X-Cysmotifs. Each oligomer was degenerate to alter either Cyscodon (TGC) to either Gly (GGC) or Ser (AGC). Individualclones were picked randomly and sequenced to identify thechange.

In all cases, the wild-type or mutated cDNAs were sub-cloned into a BamHI-HindIII-digested pRSV-LTR expres-sion vector (pRSV-LTR clones; Table 1). This vector isderived from pRSE2-C(3-11) (3) and allows efficient expres-sion in avian tissue culture cells, regulated by viral elementsof Rous sarcoma virus (RSV). All plasmids in this studywere purified by banding twice in CsCl gradients.

In vitro transcription and translation. Each pBluescriptclone was linearized with HindIII and transcribed in vitro,using T`3 polymerase as instructed by the manufacturer(Stratagene). In a standard assay, 5 ,ug of RNA (previouslyanalyzed for integrity by formaldehyde 5gel electrophoresis)was translated in the presence of [3 S]methionine in acommercial (Promega) rabbit reticulocyte lysate. Transla-tion products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); gels werefixed in acetic acid prior to fluorography.

Cell culture and DNA transfection. QT6jl (QT6) cells (21)were maintained in Dulbecco modified Eagle medium con-taining 8% fetal bovine serum, 2% chick serum, and antibi-otics. trans-activation results with the full-length GATA-1cDNA in QT6 cells were identical to those obtained forprimary chicken embryo fibroblasts (8). Transfections wereperformed essentially as described previously (8); in a stan-dard assay, 5 x 105 cells (seeded 18 to 24 h, previously) wereincubated for 5 h with a reaction mixture containing 2.0 pg ofpRSV-LTR expression plasmid, 0.25 p,g of pCH110 internalcontrol 0-galactosidase expression vector, 3.0 ,ug of reporterchloramphenicol acetyltransferase (CAT) plasmid, and 30 ,ugof Lipofectin (Bethesda Research Laboratories). The re-porter CAT plasmids used in this study have been describedelsewhere (8).

After 40 h, QT6 cells were harvested and total cell extractwas prepared by freeze-thawing three times. Equal amountsof lysate were used in 4-methylumbelliferyl 3-galactosidaseassays, analyzed by fluorescent spectrophotometry. Lysatecontaining equal amounts of ,3-galactosidase activity (414optical density units read at 445 nm) was then used in CATassays as described previously (8) except that results werequantified by using an AMBIS Radioanalytic Imaging Sys-tem.

Nuclear extract preparation. Extracts used in DNA-bind-ing assays were prepared from 6 x 107 QT6 cells transfectedas described above, using scaled-up reagents, by a modifiedprocedure of published methods (5). Cells were harvested bytrypsin treatment, washed well with phosphate-bufferedsaline, and collected by pelleting in a clinical centrifuge at1,500 rpm for 10 min. Pellets were resuspended in 1.0 ml ofice-cold buffer A and incubated for 10 min on ice. Subse-quent steps were performed at 4°C. Swelled cells werepelleted and then lysed in 0.5 ml of buffer A by Douncehomogenization; nuclei were pelleted at full speed for 10 minin a microcentrifuge. Proteins were extracted in 0.2 ml ofbuffer C for 10 min on ice, and chromatin was pelleted asdescribed above for 20 min. The resulting supernatant wasdialyzed versus 50 volumes of buffer D for 5 h, cleared bypelleting for 15 min in the microcentrifuge, and stored asnuclear extract at -80°C. Nuclear extract from chickenembryonic erythroid cells was prepared as described previ-ously (10).

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4564 YANG AND EVANS

DNA-protein interactions. All gel mobility shift assayswere performed as described previously (7). A typical reac-tion mixture contained 4 pl of QT6 nuclear extract or 5 pl ofin vitro translation reaction mixture, 2 p,g of poly(dI-dC),and 0.005 pmol of 32P-labeled oligomer probe. The se-quences of the probes used in this study (top strand only) areas follows, with the WGATAR motifs underlined: a-globin(TE72/73), AGCTTGCGGATAA AIAAGGCCGGAATTCA (derived from the chick aD-globin promoter; the samesequence as in the paLD3 reporter, containing a double,high-affinity GATA-1 binding site); rr-globin (TEPI3), AATTCAGCTCAAGGAGIAIAAGGGTCCGA (derived from thechick ir-globin promoter); control (TEHUM2), AATTCAGCTITTACCCTAICATAGGCCGA (derived from the hu-man 3-globin enhancer; binding to GATA-1 is extremelyweak [note that the GATA site is in reverse orientation]);and aD12 (TE74/75), AGCT[GCGCTGAAjATAAGGCCGGAATTCA (derived from the paD12 reporter; similar to thea-globin probe except that the 5' GATA motif is mutated,making this a lower-affinity binding site).The off-rate determination experiments were performed

and analyzed essentially as described previously (17). Astandard 20-pl mobility shift reaction was scaled up to 140 PI1and incubated on ice for 20 min. At the zero time point, a400-fold excess of nonradiolabeled double-stranded compet-itor (identical to the labeled probe) was added. At varioustimes (without stopping the electrophoretic current), 20-plaliquots were loaded onto standard 8% nondenaturing acryl-amide gels and electrophoresed at 150 V until the unboundprobe in the zero-time-point sample was near the bottom ofthe gel. Bound probe and unbound probe were quantified bythe AMBIS system. To measure association rates, weadopted a nitrocellulose filter-binding assay. Briefly, a stan-dard gel mobility shift assay was assembled on ice, using afivefold excess of labeled probe. After addition of 1 pl ofnuclear extract, aliquots were at various times aspiratedthrough nitrocellulose filters (type HA; Millipore). Boundprobe was determined by scintillation counting; controlprobes with a mutated GATA site were not retained on thefilters. Methylation interference experiments were per-formed by scaling up a standard gel mobility shift assayfivefold, using partially methylated end-labeled probe (20).Bound probe and free probe were electrophoresed from gelsonto an NA45 membrane (Schleicher & Schuell), rinsed offwith 1 M NaCl, ethanol precipitated, and cleaved by piperi-dine treatment (20) prior to denaturing gel electrophoresis.

RESULTS

Finger II and the adjacent basic domain are critical for DNAbinding. To define sequences of the cGATA-1 protein im-portant either for binding specifically to DNA or for regulat-ing transcriptional activity of target genes, we created aseries of mutations deleted progressively from either the 5'or 3' end of the full-length (wild-type) cDNA. We took thisrelatively unbiased approach because of the lack of aminoacid sequence conservation among vertebrate GATA factorsoutside of the cysteine-rich central domain.We used restriction sites within the cDNA to generate

mutated clones which retained (relative to the wild-typecDNA) all upstream 5' noncoding sequence, the originalinitiation methionine codon, the proper reading frame, andall 3' noncoding sequence (see Materials and Methods).Each construct was transcribed in vitro from a bacterialpromoter, and the resulting RNA was translated by using

A110 134 164 188 222

wr NFinger. I II

I0I+9 I+8KlKRR

304B C

-- DNABINDING

5A6

5A255A505A575A715A775A84

5A1215Al58

SA1803A283

1M%

+ 66%

+ 39%

+ 50%

+ 41%

+ 27%+ 28%+ 26%+ 6%

+ 13%

3%

+ 85%3A234 + 31%

3A2043Al61

5A8213A227

4%4%

+ 4%BASAL REPORTER ACTIVllY 4%

FIG. 1. Results of deletions on DNA binding and trans-activa-tion. (A) Schematic illustrating the locations of fingers I and II on thefull-length wild-type (WT) cDNA clone, the basic regions (+9 and+8), a putative nuclear localization signal (KKRR). Numbers at thetop correspond to amino acids. Black bars indicate sequences whichare present within each mutated clone. (B) DNA binding wasanalyzed as described in Materials and Methods. (C) Activityrepresents CAT activity derived from the trans-activated paD3reporter relative to that obtained from the full-length wild-typecDNA clone. The conditions of the assay were such that the wildtype yielded about 60% conversion to the acetylated forms. Controltransfections using a reverse full-length cDNA yielded a backgroundof4% relative activity. In at least four independent experiments, thestandard deviations were about 35% of the values shown.

rabbit reticulocyte lysate in the presence of labeled methio-nine. RNA derived from each of the clones directs thetranslation of labeled protein of a size consistent with theextent of the deletion (data not shown). The mutated pro-teins do not all accumulate in vitro to the same level as doesthe wild-type clone, while certain deletions appear to in-crease the sensitivity of the translated protein to endogenousproteases present in the lysate.The synthesized proteins were analyzed for DNA-binding

activity in a gel mobility shift assay. The probe was either anoligomer containing a GATA-1 binding site derived from theccD-globin promoter or a similar control oligomer which doesnot bind GATA-1. The results are summarized in Fig. lB.Deletion of N-terminal sequences that include finger I (5A6to 5A158) does not eliminate specific DNA-binding activity,while an N-terminal deletion into finger II abolishes it(5A180). Surprisingly, a 3' deletion which includes part of thedownstream basic region but does not include sequencesbetween the cysteines of finger II (3A204) also eliminatesbinding activity.cGATA-1 contains distinct activation domains. We have

previously shown (8) that when expressed in nonerythroid

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DISTINCT FUNCTIONS FOR GATA-1 FINGERS 4565

00 C#~Rz- kr- 00 cn C.)

k¶)e,It- n Clfl)X. r --6 S I origin

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FIG. 2. Analysis of binding activity in nuclear extracts of trans-fected cells. Cells were transfected with the clones indicated abovethe lanes under the conditions used to measure trans-activation.Nuclear extracts were prepared, and equal amounts were analyzedby gel mobility shift assays, using either the aD-globin binding site(first lane of each pair) or the control oligomer which does not bindGATA-1 (second lane). In lane AS, the reverse cDNA was ex-pressed; this sample demonstrates nonspecific binding or the migra-tion of complexes formed with other GATA-binding proteinspresent in QT6 cells. In lane RBC, nuclear extract prepared fromchick erythroid cells was used to demonstrate the migration ofnatural GATA-1 complex. The positions of the well origin, GATA-1complexes, and free probe are indicated. WT, wild type.

cells, the wild-type cGATA-1 protein binds to and trans-activates an aD-globin promoter to express a reporter CATgene. trans-activation occurs by increasing transcriptionfrom the normal start site of this minimal promoter, strictlydependent on the natural single high-affinity GATA-1 bind-ing site. To determine regions of cGATA-1 required fortranscriptional activation, we transferred each of the dele-tion constructs into an avian expression vector and assayedfor the effect of the various deletions in this cotransfectionassay. In these experiments, expression of GATA-1 deriva-tives is directed by an RSV long terminal repeat; plasmidswere transfected into avian QT6 cells, and all transfectionsincluded an internal control plasmid directing expression of,B-galactosidase from a simian virus 40 promoter.Using conditions in which the expressed proteins are in

excess (analyzed in preliminary experiments; data notshown), we determined the relative trans-activation poten-tial for each of the mutated proteins. These results are givenin Fig. 1C. Given the apparent in vitro instability of certainGATA-1 mutations, we were concerned that the proteinsmight not accumulate stably in vivo. Therefore, nuclearextracts were prepared from transfected cells and assayedfor the presence of GATA-1 by using a polyclonal antibody.The results (not shown) demonstrated that the deleted pro-teins do not accumulate to the same level as and almost allare expressed less than the full-length protein. Therefore,some of the effects on trans-activation may be due to proteininstability. Using the antibody, we are able to confirm thatproteins which do not bind DNA are being expressed in vivoyet are completely unable to activate the reporter.Even precise estimates of GATA-1 abundance by Western

immunoblotting may not accurately reflect the percentage ofnondenatured and potentially active protein. By preparingnuclear extracts from transfected cells, we are able to betterestimate the relative abundance of accumulated DNA-bind-ing activity for each of the mutations. Gel mobility shiftassays using nuclear extracts from the corresponding trans-fected cells are shown in Fig. 2. Because the mutated

proteins are expressed at levels that are saturated for trans-activation of the reporter, and transfected cells accumulate aquantity of DNA-binding activity which, at least for many ofthe clones, is similar to that obtained when the wild-typeprotein is known to be in excess, we conclude that certaindeletions have removed sequences involved primarily intrans-activation rather than DNA binding or protein stabil-ity.

Deletion down to amino acid 71 progressively reduces thelevels of trans-activation to 30% of the level of the wild-typeprotein. Therefore, deletion of these sequences is likely tohave removed a trans-activation domain. Further deletionshave no significant effect until sequences including abouthalf of finger I are removed (5A121). This mutated protein isessentially nonactivating, although it retains DNA-bindingactivity. We can conclude that the sequences N terminal tothe DNA-binding domain contain at least two regions con-tributing to transcriptional activity. The first domain (act I)appears to reside within the first 71 amino acids but isdifficult to map precisely because of protein instability; thesecond domain (act II) maps near or within finger I.

Deleting between 17 and 70 amino acids from the 3' end ofthe protein reduces activity to 31% of the wild-type level andappears to define a third activation domain (act III). Asnoted above, those mutants which do not bind DNA (dele-tions into finger II or the adjacent basic region) do notactivate the reporter. Deletion of the first 82 and the last 77amino acids (deleting act I and III) abolishes trans-activationwithout altering the putative act II or affecting DNA-bindingactivity (5A82/3A227). We show below that act II seems to becritical, but it does not function in the absence of at least oneof the other domains.

Regions outside of finger H contribute to complex stability.Because of the significant effect upon deletion of act II (e.g.,5A121), we sought to determine whether loss of finger I mayhave subtle effects on stability of the protein-DNA complex.Mobility shift assays performed by using lysates from invitro- or in vivo-expressed proteins showed no significantoverall affinity differences between the wild-type protein andthe single-finger mutant 5A158 (actually, the latter appearedto have an even greater affinity for DNA under the condi-tions of the in vitro binding assay; data not shown). Previousresults for the mouse homolog (18) showed that loss of fingerI in mouse GATA-1 could lead to a significant (at leastfivefold) drop in complex stability, as measured by theprotein off rate, depending on the binding site. We per-formed off-rate analyses comparing the wild-type protein,mutation 5A158, which is deleted of finger I, and mutation3A234, in which the C-terminal act III is removed. Nuclearproteins were harvested from transfected QT6 cells, and offrates were measured by using either the high-affinity aDbinding site or a lower-affinity site derived from the embry-onic chicken a-globin gene, ir. After initial complex forma-tion, a vast excess of cold unlabeled probe DNA was added.At various time points, aliquots were removed and run on agel to analyze the ratio of free probe to complex remaining.The results are shown in Fig. 3 and summarized in Table

2. The data support the conclusions of Martin and Orkin (18)and indicate that the presence of finger I (or the N terminusincluding finger I) decreases the off rate for the protein-DNAcomplex; the wild-type protein forms a complex with a4.3-fold-greater half-life relative to the 5A158 protein on theaD probe. The single-finger protein-DNA complex shows asimilar half-life regardless of the DNA probe, while thewild-type protein has a 1.7-fold-greater half-life for thehigher-affinity aD_globin site relative to the Tr-globin-derived

k) O= r- r--

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4566 YANG AND EVANS

(I.-GGIobin Probe

IW

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FIG. 3. Off-rate analysis of wild-type (NW) and mutant GATA-1proteins. (A) Nuclear extract was prepared from cells transfectedwith the indicated expression clone. Following complex formationwith the labeledoD-globin probe, a vast excess (determined inpreliminary experiments) of unlabeled oligomer was added; aliquotswere loaded onto the gel at thetime points (minutes) indicated abovethe lanes. For the mutant clones, the lanes represent identical timepoints(0 to 24 min); the lanes corresponding to the 32-min time pointor the free probe are not included. Arrows indicate the position ofbound complexes. (B) The ratio of labeled probe bound in thecomplex at a given time point over the labeled probe bound incomplex at time zero is plotted as a function of time. The slope ofthe resulting curve is used to calculate the half-life of the complex(see Table 2) as described previously (19).

probe. It appears, therefore, that finger I may contribute tothe discrimination between high- and low-affinity sites. In-terestingly, the 3' deletion clone 3A234 (containing bothfingers) displays an off rate similar to that of the wild-typeprotein with theaD-globin probe, but the complex has a

significantly reduced half-life (relative to the wild type) withthe Tr probe. We conclude that regions both 5' and 3' to

finger II can influence the stability of protein-DNA interac-tions and thus contribute to the overall effects that we detecton transcriptional activity of these mutants.

Distinct roles of finger I in DNA binding and trans activa-tion. To distinguish more critically the function of the twozinc fingers, we constructed double-point mutants whichspecifically alter both cysteine residues, in either the 5' or 3'C-X-X-C cluster, within either finger I or finger II (Fig. 4A).

TABLE 2. Half-lives of the complexes formed by using theprotein and oligomer probesa

GATA-1 Half-life (min) with oligomer probe:protein o-Globin Tr-Globin aD12WT 10.3 6.0 6.35A158 2.4 2.1 NDb3A234 8.1 3.3 NDHB2 4.6 9.2 2.4HB3 3.7 9.2 2.3

a Dissociation rates measured described in Materials and Methods.

b ND, not determined.

When these mutated clones were expressed and assayed invitro (Fig. 4B to D), it was found that any mutation (of eithercluster) in finger II abolishes the DNA-binding activity of theprotein. These results confirm and extend the observationthat finger II is critical for binding, even in the presence of anotherwise intact molecule. The results also support thenotion that the cysteine residues might form a precisestructural unit via metal ion coordination. Mutations infinger I have little effect on binding, perhaps leading to a

slight increase in affinity (as measured by the amount ofcomplex formed under the conditions of the mobility shiftassay).When these mutated proteins were expressed in the

cotransfection assay in QT6 cells (Fig. 4A and D), a finger IImutant (pHB4) did not activate thepaD3 reporter, as

expected. The results concerning mutations within finger Iare particularly revealing. Mutation of the 5' C-X-X-C clus-ter led to a 50% drop in activity, regardless of the mutation(pHB1 or pHB2). Off-rate determination experiments (Fig.4E and Table 2) indicate that this drop may be related toeffects on complex stability; Western blots (not shown)indicate that these proteins accumulate identically to thewild-type protein. However, mutation of the 3' cluster(pHB3) results in a 10-fold drop in activity, with little effecton DNA-protein stability beyond the drop found with use ofpHB2. Note that the stability of complexes formed with theHB2 or HB3 protein is identical with use of the rr-globinprobe and has actually increased relative to the higher-affinity (for the wild-type protein) ctD-globin probe. Althoughthe HB3 mutant may be slightly less stable than HB2(confirmed by Western blots; also, less DNA-binding activ-ity accumulates [Fig. 4D]), titration experiments demon-strated that all proteins were expressed in excess (notshown).To further investigate the functional distinction between

the HB2 and HB3 mutations, an additional set of experi-ments was performed by using a different reporter containinga distinct GATA-1 binding site. The paD12 reporter (8) issimilar to paD3 except that the single high-affinity GATA-1binding site (containing two overlapping WGATAR motifs)is mutated in the upstream motif. The resulting site still bindsGATA-1 to activate the reporter, but the site is of loweraffinity, leading to a drop in trans-activation by expressedGATA-1 relative to paD3. As shown in Fig. 4A, the resultswith mutant HB2 and HB3 proteins in assays using thepaD12 reporter confirm the results for assays using paD3described above. The HB2 protein activates the reporterabout threefold less than does wild-type GATA-1 and dis-plays a corresponding loss of complex stability, as measuredby off-rate analysis using the paD12 binding-site oligomerprobe (Table 2). The HB3 protein is transcriptionally inac-tive while displaying no further decrease in complex stabilityrelative to HB2.Although HB2 and HB3 form complexes of similar stabil-

ities and the in vitro data indicate that they bind with similaraffinities to the GATA motif, we considered whether theproteins generated in vivo might be altered in their rates ofDNA association. We found that the gel mobility shift assay

was not useful for measuring the Ka of the protein-DNAinteractions. Therefore, we adopted a filter-binding assay

and measured the Ka of the wild-type, HB2, and HB3proteins, using the paD3 binding site. In all cases (data not

shown), the rates were equal (tj.2 of about 60 s), slightlyslower than that measured by using the endogenouscGATA-1 in crude erythroid cell lysates (tj.2 of about 40 s).

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DISTINCT FUNCTIONS FOR GATA-1 FINGERS 4567

Finger I Finger IIAN. .:IN _ _ =3C

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+ HB3- HB4

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FIG. 4. Distinct trans-activation potential of finger I point mu-tants. (A) Schematic illustrating the position of C-X-X-C motifs andthe amino acid changes in four of the finger mutants (HB1 to HB4).The results of DNA-binding experiments (see panel B) and trans-activation experiments using these mutants are also shown. In fourindependent experiments, the standard deviations were at most15%. The actual CAT activity derived from the paD12 reporter isapproximately one-third that of the paD3 reporter with use of thefull-length cDNA; results are expressed relative to those obtainedfor each reporter with use of the wild-type (WT) clone. (B) In vitrotranslation products (arrow) of the point mutants analyzed by gelelectrophoresis. RNA was derived from the following clones: lane 1,wild type; lane 2, HB1; lane 3, HB2; lane 4, HB3; lane 5, HB4; andlanes 6 to 8, other clones mutated in finger II cysteines. (C) Thetranslation products were analyzed by gel mobility shift assays forDNA-binding activity. Lanes are as in panel B except for lanes -

(control lysate with no RNA added) and 9 (chick erythroid nuclearextract used to demonstrate the migration of natural cGATA-1 [ar-row]). The probe (P) was labeled a-globin oligomer; similar resultswere obtained with use of other GATA-1 binding sites (not shown). (D)

12 3 4 1 2 3 4

v ,,,v ,

FIG. 5. Methylation interference of complex formation, usingthe wild-type or finger I mutated protein. (A) Autoradiograph of a15% denaturing gel separating the piperidine-cleaved products ofpartially methylated a oligomer probe, 5' labeled on the top orbottom strand. Bound protein-DNA complexes (lanes B) were

separated from free probe (lanes F) following a preparative mobilityshift assay using the following proteins: 1, wild-type cGATA-1 inembryonic erythroid cell lysates; 2, wild-type cGATA-1 expressedfrom the full-length cDNA in QT6 cells; 3, HB2 expressed in QT6cells; and 4, HB3 expressed in QT6 cells. Brackets indicate theregion on each strand where residues are underrepresented in thebound fraction. Arrowheads mark a G residue on the top strandwhich specifically inhibits binding to HB2 or HB3 when methylated;arrows indicate the methylated A residues which specifically do notinterfere with HB3 binding. (B) Sequence of the probe. Trianglesmark the top-strand methylated residues enriched in the free frac-tion for all complexes; the large filled arrowhead and the openarrowheads correspond to the arrowheads and arrows, respectively,in panel A.

Therefore, the difference in trans-activation potential for theHB2 and HB3 proteins is not related to DNA affinity.

Finally, we considered whether the HB2 or HB3 proteinmight make distinct interactions with the DNA, unrelated tobinding affinity. Methylation interference assays were per-formed, using a partially methylated paD3 binding-site probeand nuclear extracts derived from QT6 transfections con-taining wild-type or mutated cGATA-1. Following separa-tion of bound and free fractions, DNA was purified from thegel, cleaved with piperidine, and analyzed by denaturingpolyacrylamide gel electrophoresis. The resulting patterns

Analysis of binding activity. Nuclear extract prepared from cellstransfected with the indicated clone and analyzed for bindingactivity as in Fig. 2. The arrow indicates the migration of GATA-1complexes. (E) Off-rate analyses performed with nuclear extractfrom cells transfected with HB2 or HB3. Probes used are indicatedat the left, and arrows point to the GATA-1 complexes. Time pointswere (left to right) 0, 1, 2, 4, 8, 16, 24, 32, and 40 min for each. Thegels using HB2 contain an additional lane with free probe at the farright. The half-life for each complex is given in Table 2.

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4568 YANG AND EVANS

are shown in Fig. 5A; fragments missing from the boundfraction indicate those residues in which methylation inter-fered with productive binding, implying a site of closeinteraction.On the top strand, we detect no significant differences in

interference patterns for bound complexes with use of eryth-roid or wild-type cGATA-1 expressed in QT6. Interestingly,both the HB2 and HB3 proteins show a modest relativedecrease in bound complex when methylated at a single Gresidue (arrowheads in Fig. 5A). This result indicates thatthe proteins with a single functional finger make additionalclose interactions with DNA, which inhibits the productionof a more stable complex. The most revealing difference inpatterns is found on the bottom strand, where methylatedresidues that interfere with binding to the wild-type or HB2protein do not interfere with binding to the HB3 protein(arrows in Fig. SA). Therefore, the difference in trans-activation potential between HB2 and HB3 is best explainedby distinct positions that the two proteins might occupy onthe bound DNA site. The result indicates that the HB3protein fails to make critical contacts on the bottom strand(Fig. SB) which may be required for GATA-1 to activatetranscription.

DISCUSSION

By analyzing mutations of the cGATA-1 cDNA, we havemapped, using DNA-binding and transient cotransfectionassays, the general regions of the protein that contribute toits function as a transcription factor of erythroid cell-specificgenes. Our results are summarized in Fig. 6A, with theapproximate boundaries of functional domains illustratedbelow the schematic. It is clear that the C-terminal finger IIis the critical domain required for sequence-specific DNA-binding activity, as deletion into this conserved region, orpoint mutants of the cysteine residues, abolishes binding. Itis interesting that the highly related finger I is unable to bindDNA by itself; mutations in this region appear to havedeleterious affects on the stability of protein-DNA interac-tions. Similar conclusions were reached upon studying spe-cific mutations of the mouse GATA-1 DNA-binding domain(18).The two zinc fingers are located on separate exons (13);

after duplication, the two regions have apparently divergedto incorporate discrete functions. It is not surprising to findthat finger II is the critical binding domain, as this region isconserved in single-finger proteins that also specificallyrecognize GATA motifs (12, 15). Our data indicate that thebasic region downstream of the cysteines (located on thesame exon) may be the DNA interaction domain, as (i) thisregion is well conserved among species and is not wellconserved between fingers I and II and (ii) mutations in thisregion that do not alter the cysteines (e.g., 3A204) abolishbinding activity.A wide array of distinct GATA binding sites (differing in

flanking sequences outside of the conserved WGATARmotif) exist in control regions of erythroid genes which aredifferentially regulated, depending on developmnental stageor state of differentiation. It seems likely that finger I isinvolved in discrimination among different sites and may becritical for subtle regulation among genes which are allinfluenced by the same factor. The stabilities of complexesthat we measure by the off-rate assay show that finger Imutants can bind more or less stably, depending on theparticular GATA site (Table 2). Perhaps more direct evi-dence implicating the first finger in site discrimination comes

A 110 134 164 188 222I1 1 1

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IN +8K cIAct I Act II Act III

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Relative

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FIG. 6. Locations of putative functional domains withincGATA-1. (A) Schematic illustrating the approximate locations ofactivation domains (act I to III) and regions implicated in proteinstability (St). Finger II and the adjacent basic region (+8) constitutethe DNA-binding domain (Db), while finger I also plays a role indetermining DNA affinity (Af). We predict, but have no directevidence, that a KKRR motif is part of a nuclear localization signal(Nl). All mutants which bound DNA were found to be predomi-nantly nuclear; for those mutants that did not generate nuclearDNA-binding activity, we checked but also did not find bindingactivity in cytoplasmic extracts. (B) Schematic showing thatcGATA-1 activation domains must function cooperatively, as anytwo activation domains generate only 10 to 30% of the full activity.

from a mutant of the Aspergillus protein AreA, in which thesingle C-terminal finger is duplicated, leading to increasesand decreases in expression of the structural genes that thisfactor regulates (1).

It appears that the three activation domains that we haveroughly defined exert a cooperative effect on transcriptionalactivation (Fig. 6B). Single domains alone do not contributemore than 10% of wild-type activity (although act I has notbeen tested by itself, the combination of act I plus act IIIyields only 10% activity, identical to the activity of act IIIalone). The three domains might act synergistically on acommon target or provide cooperative interactions with twoor more distinct cellular proteins.

It is interesting that three activation domains were alsomapped by structure-function studies on the mouse GATA-1homolog (18). The mammalian proteins were predicted (25)to contain three structural units distinct from the DNA-binding domain as a result of the presence of three copies ofa divergent repeat (the R repeat). These repeats are notconserved in the primary sequence of the chicken protein,and the activation domains that we map do not obviouslymap to similar structural domains of the mouse. Deletion ofcertain short regions of sequence similarity that can bedetected in cGATA-1 (e.g., amino acids 76 to 86 ofcGATA-1) do not have deleterious effects on activation (inthis case, compare 5A77 with 5A84). On the other hand, veryshort stretches of sequence conservation can be identified

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DISTINCT FUNCTIONS FOR GATA-1 FINGERS 4569

within both the N- and C-terminal activation domains, whilethe putative act II within the finger region would be wellconserved.Our work has established a dual function for the finger I

domain. In addition to its role in specificity and stability ofbinding, we find that it has a role in transcriptional activationas a result of the presence of the act II domain. Because thisregion is well conserved among GATA family members, actII is a good candidate for a general GATA protein activationdomain, perhaps interacting with specific cellular proteins.However, deletion of finger I did not affect the activity of themouse homolog (18), while a point mutant (L230F) reducedactivity about 50%. Perhaps the activation domains alsocooperate in the case of the mouse homolog, but a differentregion (e.g., N terminal) is more dominant. Also, differencesin reporter constructs or cellular environments may revealdistinct functions for different regions of the GATA-1 pro-teins.

Subtle alterations in the way that GATA-1 interacts withits cognate site may have a dramatic influence on how otherregions of the factor make protein-protein contacts. Accord-ing to this latter view, classical activation domains may notbe present in cGATA-1 (which does not contain obviousacidic or other prototypic domains). Instead, the regions thatwe have identified as act I, act II, and act III may function byproperly positioning the protein to make a precise interac-tion with the DNA sequence. Our data certainly indicatesuch a role for act II within the first finger. Obviously, theimportance of various regions might therefore depend on theparticular regulatory site bound by the factor. Our data areso far consistent with this notion, as we can detect at leastsmall effects on some aspect of DNA binding by using manyof our mutants, depending on the binding site and conditionsof the assay. In fact, evidence for this interpretation isprovided by studies of a natural human hemoglobinopathy,in which a mutant GATA-1 binding site leads to a very subtlealteration in the DNA-protein interaction but results ininappropriately high levels of globin gene expression (19).

ACKNOWLEDGMENTS

We thank C.-M. Chiang for the pRSE2-C expression vector, H.Blumberg for excellent technical assistance, and L. Engler foradvice concerning the on-rate experiments.T.E. is a Searle Scholar and gratefully acknowledges support from

the Chicago Community Trust. In addition, parts of this work wereaided by Basil O'Connor Starter Scholar Research Award 5-91-0521and PHS grant DK44167 from the National Institutes of Health (bothto T.E.).

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Aspergillus: are two fingers better than one? Gene 95:123-127.2. Chiba, T., Y. Ikawa, and K. Todokoro. 1991. GATA-1 transac-

tivates erythropoietin receptor gene, and erythropoietin recep-tor-mediated signals enhance GATA-1 gene expression. NucleicAcids Res. 19:3843-3848.

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4. Cunningham, T. S., and T. G. Cooper. 1991. Expression of theDAL80 gene, whose product is homologous to the GATAfactors and is a negative regulator of multiple nitrogen catabolicgenes in Saccharomyces cerevisiae, is sensitive to nitrogencatabolite repression. Mol. Cell. Biol. 11:6205-6215.

5. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983.Accurate transcription initiation by RNA polymerase II in a

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scription factor Eryfl: a new finger protein. Cell 58:877-885.8. Evans, T., and G. Felsenfeld. 1991. Trans-activation of a globin

promoter in nonerythroid cells. Mol. Cell. Biol. 11:843-853.9. Evans, T., G. Felsenfeld, and M. Reitman. 1990. Control of

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11. Felsenfeld, G., and T. Evans. Unpublished data.12. Fu, Y.-H., and G. A. Marzluf. 1990. Nit-2, the major positive-

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14. Ko, L. J., M. Yamamoto, M. W. Leonard, K. M. George, P.Ting, and J. D. Engel. 1991. Murine and human T-lymphocyteGATA-3 factors mediate transcription through a cis-regulatoryelement within the human T-cell receptor delta gene enhancer.Mol. Cell. Biol. 11:2778-2784.

15. Kudla, B., M. X. Caddick, T. Langdon, N. M. Martinez-Rossi,C. F. Bennett, S. Sibley, R. W. Davies, and H. N. Arst, Jr. 1990.The regulatory gene areA mediating nitrogen metabolite repres-sion in Aspergillus nidulans. Mutations affecting specificity ofgene activation alter a loop residue of a putative zinc finger.EMBO J. 9:1355-1364.

16. Lee, M.-E., D. H. Temizer, J. A. Clifford, and T. Quertermous.1991. Cloning of the GATA-binding protein that regulates en-dothelin-1 gene expression in endothelial cells. J. Biol. Chem.266:16188-16192.

17. Li, R., J. Knight, G. Bream, A. Stenlund, and M. Botchan. 1989.Specific recognition nucleotides and their DNA context deter-mine the affinity of E2 protein for 17 binding sites in the BPV-1genome. Genes Dev. 3:510-526.

18. Martin, D. I. K., and S. H. Orkin. 1990. Transcriptionalactivation and DNA binding by the erythroid factor GF-1/NF-El/Eryfl. Genes Dev. 4:1886-1898.

19. Martin, D. I. K., S.-F. Tsai, and S. H. Orkin. 1989. Increasedgamma-globin expression in a nondeletion HPFH mediated byan erythroid-specific DNA-binding factor. Nature (London)338:435-438.

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21. Moscovici, C., G. M. Moscovici, H. Jimenez, M. M. C. Lai, M. J.Hayman, and P. K. Vogt. 1977. Continuous tissue culture celllines derived from chemically induced tumors of Japanese quail.Cell 11:95-103.

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1990. Structure and evolution of a human erythroid transcrip-tion factor. Nature (London) 343:92-96.

26. Tsai, S.-F., D. I. K. Martin, L. I. Zon, A. D. D'Andrea, G. G.Wong, and S. H. Orkin. 1989. Cloning of cDNA for the majorDNA-binding protein of the erythroid lineage through expres-sion cloning in mammalian cells. Nature (London) 339:446-451.

27. Tsai, S.-F., E. Strauss, and S. H. Orkin. 1991. Functionalanalysis and in vivo footprinting implicate the erythroid tran-scription factor GATA-1 as a positive regulator of its own

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29. Wilson, D. B., D. M. Dorfnan, and S. H. Orkin. 1990. Anonerythroid GATA-binding protein is required for function ofthe human preproendothelin-1 promoter in endothelial cells.Mol. Cell. Biol. 10:4854-4862.

30. Yamamoto, M., L. J. Ko, M. W. Leonard, H. Beug, S. H. Orkin,and J. D. Engel. 1990. Activity and tissue-specific expression ofthe transcription factor NF-E1 multigene family. Genes Dev.4:1650-1662.

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32. Zon, L. I., S.-F. Tsai, S. Burgess, P. Matsudaira, G. A. P. Bruns,and S. H. Orldn. 1990. The major human erythroid DNA-binding protein (GF-1): primary sequence and localization of thegene to the X chromosome. Proc. Natl. Acad. Sci. USA87:668-672.

33. Zon, L. I., H. Youssoufian, C. Mather, H. F. Lodish, and S. H.Orkin. 1991. Activation of the erythropoietin receptor promoterby transcription factor GATA-1. Proc. Natl. Acad. Sci. USA88:10638-10641.

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