Proc. Natl. Acad. Sci. USAVol. 92, pp. 11598-11602, December 1995Biochemistry

Alternative translation initiation site usage results in twofunctionally distinct forms of the GATA-1 transcription factorRAFFAELLA CALLIGARIS*t, STEFANIA BOTTARDI*t, SUSANNA COGOI*, ISABEL APEZTEGUIA*I,AND CLAUDIO SANTORO§*Laboratorio Nazionale-Consorzio Interuniversitario Biotecnologie, and §Laboratorio Nazionale-Consorizo Interuniversitario Biotecnologie, Dipartimento diBiofisica e Chimica delle Macromolecole, Padriciano 99, 34012 Trieste, Italy

Communicated by Robert Tjian, University of California, Berkeley, CA, August 16, 1995

ABSTRACT GATA-1 is a zinc-finger transcription factorthat plays a critical role in the normal development ofhematopoietic cell lineages. In human and murine erythroidcells a previously undescribed 40-kDa protein is detected withGATA-1-specific antibodies. We show that the 40-kDa GATA-1(GATA-1s) is produced by the use of an internal AUG initi-ation codon in the GATA-1 transcript. The GATA-1 proteinsshare identical binding activity and form heterodimers inerythroleukemic cells but differ in their transactivation po-tential and in their expression in developing mouse embryos.

GATA-1 is a member of a family of transcription factors(GATA factors) distinguished by the high degree of similarityof their DNA-binding domains. These domains contain typeIV zinc fingers and bind to cis-acting elements containing theWGATAR motif (1). GATA-1 was originally identified as anerythroid-specific factor involved in globin gene expression (2,3). Subsequently it has been found to be expressed in mega-karyocytes and mast cells, where it regulates lineage-specificgenes (4-6). More recently, GATA-1 expression has been ob-served in eosinophils (7). In mouse Sertoli cells, the GATA-1gene is transcribed by a distinct promoter (8). In spite of thedistribution in different cell types, several lines of evidencedemonstrate that GATA-1 is essential for erythroid development.Targeted disruption of the mouse gene establishes that GATA-1is indispensable for the terminal differentiation of erythroid cellsbut not for other lineages of hematopoietic cells in vivo (9) andin vitro (10). The ectopic expression of GATA-1 in nonerythroidhematopoietic cells can convert them into erythroblast-like cells(11). These findings suggest that GATA-1 functions through aconcerted interaction with other regulatory factors and thatmodulation of its expression plays a pivotol role in hematopoieticcell development. Several investigations have shown that at leasttwo GATA-1 domains are important in this respect: the N-terminal region, which confers full transcriptional activity toGATA-1 (12), and the C-terminal zinc finger, which mediates thebinding to DNA (13) and the physical interaction with otherzinc-finger-containing proteins such as the ubiquitous Spl, theerythroid-restricted EKLF (14), and other GATA factors (15).Here we show that at least two isoforms of GATA-1 exist inhuman and murine erythroid cells, and we demonstrate that theyresult from alternative translation initiation site usage. The twoisoforms show virtually identical binding specificities and arecapable of forming heterodimers. The previously undescribedisoform lacks the N-terminal domain and consequently shows adecreased transactivation potential. The two GATA-1 proteinsappear to be differentially expressed in mouse embryo tissues.

MATERIALS AND METHODSAntisera, Immunoprecipitation, and Western Blot Analysis.

The rabbit polyclonal antibody RahrGATA-1 was raised

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against bacterially expressed human GATA-1. This antiserumdoes not cross-react with mouse GATA-1 and recognizes theN terminus of GATA-1 (residues 1-67) as determined byepitope mapping of GATA-1 deletion mutants. RaFIF2 wasgenerated against the human recombinant GATA-1 zinc-finger domain from residue 191 to residue 317 (16). Bothantisera were affinity purified. The rat monoclonal antibodyN6 was commercially supplied (Santa Cruz Biotechnology).For immunoprecipitation, MEL (murine erythroleukemia)cells were labeled with [35S]methionine for 2 hr and subse-quently lysed in a buffer containing 1% Nonidet P-40, 20 mMTris HCl at pH 7.8, 2 mM EDTA, 500 mM NaCl, and proteaseinhibitors. Lysates were sonicated, cleared by centrifugation,and incubated with the monoclonal antibody N6 at a finaldilution of 1:500. The immunocomplexes were precipitated withprotein G-Sepharose (Pharmacia), resolved by SDS/polyacryl-amide gel electrophoresis, and visualized for fluorography. West-ern blot analysis was carried out by conventional methods and theantibody/antigen complexes were detected by enhanced chemi-luminescence (Amersham).Plasmid Constructs. To construct the expression vector

pGDwt, a full-length mouse GATA-1 cDNA was inserted intothe BamHI and Xba I sites of pGDSV7, a vector harboring thesimian virus 40 (SV40) and phage T7 promoters (17). ThecDNA clone was isolated by expression screening of a MELcell AZAPExpress cDNA library (S.B., unpublished results).pGDmutl and pGDmut84 were generated by PCR-mediatedsite-directed mutagenesis. Amplified fragments were sequencedand inserted into pGDwt. The reporter gene plasmid (2xGATA-CAT) was obtained by the insertion of a blunt-ended dimer of amouse al-globin gene-derived GATA oligonucleotide (5'-CCGGAATCCTTATCAGTTCCG-3') into the filled-in BamHIsite of pMT-CAT, a plasmid containing the human metal-lothionein IIA gene promoter from nucleotide +37 to nucleotide-69 and the chloramphenicol acetyltransferase (CAT) gene.In Vitro Transcription/Translation. pGD plasmid DNAs

were transcribed and translated by using the TNT T7 coupledreticulocyte lysate system (Promega) in the presence of[35S]methionine. Resulting translated products were resolvedby SDS/polyacrylamide gel electrophoresis and visualized byfluorography.

Cell Culture and DNA Transfection. K562, MEL, and U937cells were grown in RPMI 1640 medium, and HeLa, HepG2,JEG-3, SAOS, and COS cells were grown in Dulbecco'smodified Eagle's medium (DMEM). All media contained 10%fetal calf serum. SAOS and COS cells were transfected with 2jig of DNA by calcium phosphate or DEAE-dextran methods,respectively (18). COS cells were cotransfected with 0.4 ,ug ofreporter plasmid and 1.6 ,ug of expression plasmid DNAs.Forty-eight hours after transfection cells were harvested, andone half was processed for Western blotting to check the

Abbreviations: SV40, simian virus 40; CAT, chloramphenicol acetyl-transferase.tR.C. and S.B. contributed equally to this work.tPresent address: Centro Investigaciones Biologicas, Havana, Cuba.

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expression efficiency of GATA-1 constructs and the other halfwas processed for CAT assay as described (32). CAT activitywas calculated by image scanning or by direct scintillationcounting.

Nuclear Extracts and Gel-Shift Analysis. Nuclear extractswere prepared as described (19). hGATA-1 from K562 cellswas purified by DNA-affinity chromatography (3). Gel mo-bility-shift assays were as previously reported (16), using asprobe the GATA oligonucleotide derived from the mouseal-globin gene. Five or ten micrograms of nuclear extractproteins or 3 ,ul of reticulocyte lysates was typically tested. Forsupershift analysis, gel-shift reaction mixes containing MELextracts were challenged with 5 ,ul of N6 or RahrGATA-1antibodies.

RESULTSTwo Forms of GATA-1 Are Present in Human K562 and

Murine MEL Cells. To test the specificity of antibodies raisedagainst GATA-1, we analyzed several cell line lysates byWestern blotting using the RahrGATA-1 antiserum, specificfor the N terminus of the human GATA-1, the rat monoclonalantibody N6, specific for the N terminus of the mouse GATA-1(20), and the RaFIF2 antiserum, raised against the zinc-fingerdomain of the human GATA-1 (16). As expected, RahrGATA-1and N6 detected a single GATA-1 protein only in human andmouse erythroid cell lysates, respectively (Fig. 1, lanes 11, 12, and14). This polypeptide shows an apparent molecular mass of 47kDa when referred to the standards used. Surprisingly, RaF1F2identified two proteins in both K562 and MEL cell lysates: the47-kDa GATA-1 protein and a second species, 40 kDa (Fig. 1,lanes 5, 6, and 13). The presence of the 40-kDa protein in theDNA-affinity-purified GATA-1 suggested that it is a GATAfactor.Due to the preservation of the zinc-finger domain among the

GATA factors, the 40-kDa form could represent a new mem-ber of the family. However, RaF1F2 failed to detect GATA-2and GATA-3 in JEG-3 cell lysate (Fig. 1, lane 1; ref. 21). Sincethese proteins are highly related to GATA-1 in their zinc-finger domains, we considered it unlikely that the 40-kDapolypeptide represents a new GATA factor. Thus, the RaFjF2antiserum presumably identifies a form of GATA-1 (GATA-ls) that lacks the N-terminal region (it is not detected byRahrGATA-1 and N6 antibodies) and that appears to be acommon feature of human and mouse erythroid cells.The 47- and 40-kDa Forms of GATA-1 Result from Alter-

native Translation Initiation Site Usage. The GATA-1 geneencodes a unique 1.8-kb mRNA (3). The only reportedexception is in mouse Sertoli cells, where an alternative firstuntranslated exon is transcribed (8). It is therefore probablethat the 40-kDa form of GATA-1 is either a proteolyticfragment of the complete protein or an alternative translationproduct. To test the latter possibility, we examined the humanand mouse cDNA coding sequences and observed the presenceof a potential translation initiation site at codon 84 (Fig. 2A).

The sequence surrounding this ATG triplet exhibits an overallhomology to the Kozak consensus cassette (22). Moreover,initiation from methionine-84 could be predicted to yield a40-kDa polypeptide. To address this issue, we constructedexpression vectors carrying the wild-type (pGDwt) or mutatedversions of the mouse GATA-1 cDNA. In these mutants ATGat either codon 1 (pGDmutl) or codon 84 (pGDmut84) wastransformed into the TTG codon for leucine by site-directedmutagenesis (Fig. 2A). TTG was chosen because the replace-ment of methionine with leucine is not anticipated to changethe functional properties of the mutated GATA-1 and becauseof its poor initiation potential (23). When these constructswere tested by in vitro transcription/translation, pGDwt gaverise to both 47- and 40-kDa polypeptides, while pGDmutl andpGDmut84 gave rise to the 40- and the 47-kDa proteins,respectively (Fig. 2B). These results confirm that the singleGATA-1 mRNA is alternatively translated at methioninecodons 1 and 84. The pGD constructs were also used to tran-siently transfect human osteosarcoma SAOS cells, and theirproducts were detected by Western blotting (Fig. 2B). Consistentwith the in vitro translation results, the wild-type construct yieldsthe 47- and 40-kDa proteins and the mutants are each restrictedin their expression to a single form. Because pGDmut84 gives risejust to the 47-kDa GATA-1, we exclude the possibility thatGATA-ls arises from proteolytic cleavage of the complete pro-tein. These results confirm that the two forms of GATA-1 dependupon translation initiation events that take place at methioninecodons 1 and 84 of the predicted GATA-1 coding sequence.GATA-ls Binds DNA as Monomer and Dimer. Previous

reports have shown that extracts from erythroid cells or fromcells transfected with the wild-type GATA-1 gene give rise tomultiple complexes when tested in a gel-shift assay (3, 12). Atypical pattern obtained with the GATA sequence derivedfrom the mouse al-globin promoter is shown in Fig. 3. Themajor band is due to the GATA-1 monomer/DNA complex,while the slowly migrating band is due to the homodimericGATA-1 (24). The faster-migrating complexes have oftenbeen ascribed to the binding of proteolytic cleavage products.To check whether GATA-ls is responsible for some of thesecomplexes, we performed gel-shift assays with the products ofthe pGD constructs obtained in vitro or by COS cell transfec-tions. As expected, the product of pGDwt gives rise to agel-shift pattern very similar to that observed with MELextract. In particular, the band migrating slightly faster thanthe GATA-1 monomer/DNA complex (Fig. 3A, lanes 1,3, and6) comigrates with the major complex obtained with theproduct of pGDmutl (Fig. 3A, lanes 4 and 7), suggesting thatthis complex is due to GATA-is. In fact, this complex is absentin the band shift carried out with the product of pGDmut84,which lacks the GATA-ls protein (Fig. 3A, lanes 5 and 8).Moreover, the presence of a slowly migrating complex inpGDmutl samples suggests that GATA-ls binds as monomeras well as dimer. The monomeric and dimeric forms ofGATA-ls are also present in erythroid cell extracts as con-firmed by supershift assay. In Fig. 3B, it is shown that the

N <M CZ N U0. < C146 -j m 'ICw

a) ON C.)u Lf)I= .:) = -(-. .z

47 kDa -

31 kDa -

___m 'ICAl

-* *.hGATA-ls

_ _o 47 kDa - .,m mGATA-1

31 KDa - mGATA-1s

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 1. Two GATA-1 products are present in erythroid cell lines. SDS lysates from the human cell lines JEG-3 (choriocarcinoma), HeLa(epithelial carcinoma), U937 (histiocytic lymphoma), HepG2 (hepatoma), and K562 (erythroleukemia) or from the murine erythroleukemic MELcell line were assayed by immunoblotting with RahrGATA-1 (lanes 1-6), RaFjF2 (lanes 7-13), or the monoclonal antibody N6 (lane 14).DNA-affinity-purified human GATA-1 was included as positive control (lanes 5 and 11). Positions of molecular mass standards are indicated. Alllysates samples were normalized by cell number (1 x 104 cells per line sampled).

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A

pGD wt ccATGG AGTATGG

met 1

pGD mutt 1 7 C

leu

pCD mut 84 1s140 T

met 1

-7-~~~~~~~~~~~~~~~~~~~~~~Inmet 84

ATG

met 84

TTG

B

> CA. C~. C.

47 kDa - _

31 kDa * -

ICu 1 2 3 4

G r C) C)

UC- Ca.

47kDa - _

31 kDa _

5 6 7 8

FIG. 2. The GATA-1 isoforms arise from alternative translation initiation. (A) The three expression vectors pGDwt, pGDmut84, and pGDmutlare schematically represented. The nucleotide sequences surrounding the two in-frame methionine codons I and 84 of the mouse GATA-1 cDNAare shown. The nucleotides that fit the Kozak consensus cassette are underlined. (B) The pGDSV7 vector and the pGD constructs were assayedby in vitro transcription/translation (lanes 1-4) or by transient transfection in human osteosarcoma SAOS cells followed by immunoblotting withRaFIF2 (lanes 6-8).

antibody N6 quantitatively supershifts the monomeric anddimeric GATA-1/DNA complexes, while it does not supershiftthe complexes containing the GATA-Is isoform. The complexa observed with MEL extract is not consistently reproducible(compare lanes 1 and 11 in Fig. 3) and is likely due to aproteolytic cleavage product. The complex b is typically ob-served in any extract containing GATA-1. We cannot excludethat this complex also is due to proteolytic products, eventhough it comigrates with the complex generated by a GATA-1protein initiated at the methionine codon 234 (data notshown). We have not further proceeded in the analysis of this

complex since no other isoforms have been detected byWestern blot analysis of erythroid cell lysates.The Two Isoforms of GATA-1 Form Heterodimers in MEL

Cells. The results described above led us to investigate whetherthe two isoforms of GATA-1 could form heterodimers inerythroid cells. To test this, lysates from pulse-labeled MELcells were incubated with the monoclonal antibody N6, and thenewly synthesized GATA-1 proteins were immunoprecipi-tated. The results of these experiments show that the twoisoforms heterodimerize (Fig. 4). The ratio of GATA-1 toGATA-Is appears to be higher than that observed by Western

A

dim. GATA-1- .

dirn. GATA-ls -

GATA-1 -

GATA-Is F-a B-p.

b -

free probe

B

dim. GATA-ls 1.

free probe

dim. GATA-1-<dim. GATA-Is4 GATA-1< GATA-ls

.*- b

1 2 3 4 5 6 7 8 9 1 0 1 1

FIG. 3. GATA-Is binds DNA as monomer and dimer. (A) Gel-mobility-shift assay. Lane 1, nuclear extracts from MEL cells; lanes 2-5, nuclearextracts from monkey kidney COS cells transfected with pGDSV7 (lane 2), pGDwt (lane 3), pGDmutl (lane 4), and pGDmut84 (lane 5); lanes6-9, in vitro translation products of pGDwt (lane 6), pGDmutl (lane 7), pGDmut84 (lane 8), and pGDSV7 (lane 9). The observed complexes areindicated (see text). dim., Dimer. (B) Supershift assay performed with MEL nuclear extract incubated in presence of N6 (lane 10) or RahrGATA-1(lane 11) antibodies. RahrGATA-1 was used as a negative control, being specific for the human GATA-1.

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Proc. Natl. Acad. Sci. USA 92 (1995) 11601

4mGATA-ls

1 2 3

FIG. 4. GATA-1 forms heterodimers with GATA-1s. Immunopre-cipitation of GATA-1 proteins from MEL cells labeled with [35S]me-thionine. Lane 1, the cell lysate was incubated with the monoclonalantibody N6 and protein G-Sepharose. Lane 2, the lysate was incu-bated only with protein G-Sepharose. Lane 3, products of the in vitrotranscribed and translated pGDwt DNA.

blotting, suggesting that only a fraction of GATA-is formsheterodimers with the full-length protein. This is consistent

AReporter CAT

GATA-1

. GATA-1s

0

C)0)u

00sI.e

(3

B

vector f

pGD wt

pGD mut 84

pGDmutl

50 100 150% wild type induction

FIG. 5. GATA-1 and GATA-Is have different transactivationpotentials. (A) (Upper) Representative CAT assay of COS cellscotransfected with the reporter gene and pGDSV7 (vector) or thepGD expression constructs. (Lower) Immunoblots of transfected celllysates were probed with RaFIF2 to check the synthesis of GATA-1proteins. (B) Histogram representing the average values of six inde-pendent experiments carried out in duplicate. The values are referredto the value obtained with pGDwt.

with the gel-shift data, which show that most of the GATA-1proteins are monomeric (Fig. 3A).GATA-1 and GATA-ls Have Different Transactivation Po-

tentials. It has been shown, using domain transfer and deletionmapping experiments, that the amino-terminal region ofGATA-1 encodes a transactivation domain (12, 25). We thusanticipated that the natural GATA-ls protein would be a lessefficient activator of transcription than GATA-1. Cotransfec-tion experiments in COS cells, using the pGD constructs asexpression vectors and a GATA-dependent reporter gene,confirmed this idea. Fig. 5 shows that transfection of pGDwtvector resulted in stimulated target gene expression, butconsistently to a lesser extent than transfection with pGD-mut84. The pGDmutl vector, which expresses only GATA-1s,was less effective in its ability to transactivate the reportergene. To exclude the possibility that these differences couldreflect variations in translation efficiency of the three con-structs, we checked transfected cells by Western blotting withRaFIF2 antibody. The expressions of the different constructswere comparable (Fig. SA Lower). From these results, itappears that GATA-1 can have different transcriptional ac-tivities, depending on the relative ratio of the two isoforms.

Expression of GATA-1 Proteins in Mouse Embryo Tissues.To check if the expression of the GATA-1 proteins variesduring mouse development, we analyzed embryo tissues byWestern blotting using the RcaFIF2 antibody. We examinedwhole embryos at 8.5 days of gestation (inclusive of yolk sacs)and heads (negative control) or livers of 11.5-day fetuses. Wedetected both GATA-1 isoforms in the mouse fetal liver (Fig.6, lane 3). Interestingly, the 47-kDa protein was not detectablein the 8.5-day embryo, whereas a band comigrating with theGATA-ls protein was visible (Fig. 6, lane 4). The absence ofthe full-length protein in the 8.5-day embryo was also con-firmed by probing the immunoblots with the antibody N6 (datanot shown). These results show that the two GATA-1 proteinsare present in mouse embryo tissues and imply a developmen-tal modulation of their expression.

DISCUSSIONHere we show that at least two proteins arise from thealternative usage of translation initiation sites present inGATA-1 mRNA. GATA-1 and GATA-ls differ in the pres-ence or absence of 83 amino acids at the N terminus. Thesynthesis of either form is strictly dependent on the integrityof the AUG 1 and 84 triplets as shown by in vitro and in vivofunctional analyses of mutants in the respective methioninecodons. The functional data are strengthened by the immu-nological analyses, carried out with specific antibodies for theN-terminal region (N6 and RahrGATA-1) or the zinc-fingerdomain (RaFIF2). The GATA-1 isoforms show virtually iden-tical DNA-binding activity and can form homodimers andheterodimers. GATA-ls is less active than the full-length

mGATA-1

g - | * i !mGATA 1s

1 2 3 4

FIG. 6. Differential expression of GATA-1 proteins in mouseembryo tissues. Western blot analysis of lysates derived from MELcells (lane 1), 11.5-day fetal heads (lane 2), 11.5-day fetal livers (lane3), and 8.5-day whole embryos (lane 4). The antibody was RaFIF2.Lanes 1-3 were exposed for 15 min; lane 4, for 30 min.

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protein in transactivating a GATA-dependent reporter gene.The two isoforms are expressed in mouse embryo tissues andappear to be developmentally modulated.There is growing evidence that GATA-1 exerts its function

by interacting with other transcriptionally active partners. Atleast two domains play an important role in this context: theC-terminal zinc finger, which mediates the association withother zinc-finger-containing proteins such as Spl, EKLF, andother GATA factors (14, 15), and the N-terminal region, whichis essential for full transcriptional activity (this report and refs.12 and 25). Thus, it is possible that the transcriptional activityof GATA-1-containing complexes could be modulated byvarying the ratio of the two isoforms.

Internal initiation has been described for other genes en-coding for transcription factors such as Pit-1 (26), CREMa/P3(27), c/EBPa and , (28), N-Oct 3 (29), Oct-4 (30), and Myc(31), and appears to be an efficient and rapid means tomodulate their activity. Moreover, in most of the reportedcases, this mechanism is evolutionarily preserved in rodentsand human. Thus, it is conceivable that GATA-1-expressingcells have selected such a tool in order to fine-tune theexpression of GATA-1-dependent genes.

We thank S. Philipsen (Erasmus University) for mouse embryosamples, Giannino Del Sal for pGDSV7 vector, and Claudio Schneiderand Claudio Brancolini for helpful suggestions. We are grateful toStefania Marzinotto for cell culture aid. This work has been supportedby grants from the European Economic Community (BI02 CT93-0315) and TeleThon (E116).

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Proc. Natl. Acad. Sci. USA 92 (1995)