Proc. Natl. Acad. Sci. USAVol. 90, pp. 6859-6863, July 1993Cell Biology

PEBP2/PEA2 represents a family of transcription factorshomologous to the products of the Drosophila runt geneand the human AMLI gene

[T-cell receptor/early development/acute myeloid leukemia/t(8;21)/DNA binding and dimerization domain]

EIKO OGAWA*, MITSUO MARUYAMA*, HIROSHI KAGOSHIMAt, MANABU INUZUKA*, JIE Lu*,MASANOBU SATAKE*, KATSUYA SHIGESADAt, AND YOSHIAKI ITO*tDepartments of *Viral Oncology and tGenetics and Molecular Biology, Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606, Japan

Communicated by Renato Dulbecco, March 17, 1993 (received for review January 18, 1993)

ABSTRACT cDNAs representing the a subunit of polyo-mavirus enhancer binding protein 2 (PEBP2; also cafled PEA2)were isolated. The products of the cDNAs are highly homolo-gous to that of Drosophila segmentation gene runt (run) for anN-proximal 128-amino acid region showing 66% identity. Therun homology region encompasses the domain capable ofbinding to a specific nucleotide sequence motif and of dimer-izing with the companion 13 subunit. The human AMLI generelated to t(8;21) acute myeloid leukemia also has a runhomology region. Together with the 18 subunit, which increasesthe affinity of the a subunit to DNA without binding to DNAby itself, PEBP2 represents a newly discovered family oftranscription factor. The major species ofPEBP2a mRNA wasexpressed in T-cell lines but not in B-cell lines tested. Evidenceindicated that PEBP2 functions as a transcriptional activatorand is,involved in regulation of T-ceil-specific gene expression.

Potential use of polyomavirus (Py) as a probe of mousedevelopment has been recognized since it was observed thatthe wild-type Py did not grow in embryonal carcinoma cellsbut that it grew when the cells were induced to differentiate(1-5). Through the analysis of the Py enhancer, whichdetermines the differentiation-dependent and cell-type-specific expression of the viral genes, a number of transcrip-tion factors involved in developmental regulation have beenidentified (6-10).PEBP2/PEA2, which binds to both the A and B cores of

the Py enhancer (11), is undetectable in embryonal carcinomaF9 cells and becomes detectable after the cells are induced todifferentiate (6, 7). It specifically recognizes a consensussequence, R/TACCRCA (R, purine), which was originallyidentified in the Py enhancer (11) and is also compatible withthe core motif of murine leukemia virus enhancers (12, 13).In addition, many T-cell-specific genes, such as T-cell re-ceptor (TCR) a, X3, yand 8 and CD3e genes, contain potentialPEBP2 binding sites, suggesting the possibility that PEBP2 isinvolved in T-cell-specific gene expression (13).While PEBP2 is expressed' in NIH 3T3 cells, a much

faster-migrating factor in electrophoresis mobility-shift assay(EMSA), termed PEBP3, is often expressed in NIH 3T3 cellstransformed by activated Ha-ras oncogene or Py middle-sized tumor antigen (14). The treatment of the nuclear extractfrom NIH 3T3 cells containing PEBP2 with acid phosphataseapparently converts PEBP2 to PEBP3 (15). PEBP3 is, there-fore, considered to be a subcomponent of PEBP2 represent-ing its DNA binding core. Thus, we used PEBP3 as a materialof choice for further analysis.

Purified PEBP3 is a heterodimer composed oftwo groups ofheterogeneous populations of polypeptides a (30-35 kDa) and

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

,B (20-25 kDa) (11). Only the a polypeptides have the DNAbinding moiety and the 8 polypeptides increase the affinity ofthe a polypeptide to DNA (16). Molecular cloning of the ,Bsubunit is described elsewhere (16). This paper describesmolecular cloning of cDNA for the a subunit.§

MATERIALS AND METHODSIsolation of cDNA Clones. A cDNA library prepared from

Ha-ras-transformed NIH 3T3 cells was screened using degen-erate oligonucleotides, 5'-AARGTNACNGTNGAYGG-3'(Y, pyrimidine), as deduced from the peptide sequence[(K)VTVDG], which was determined from purified PEBP3(16). Details of the procedure were essentially the same asdescribed for cloning of (-subunit cDNA (16).

Plasmids. T,33W4W-tkCAT plasmid was constructed byinsertion of the T183-T,34 fragment (nt 620-735 of mouseTCR(3 enhancer; ref. 17) containing two PEBP2 sites into theBamHI site of pBL2CAT vector plasmid (18). T,(3M4W-,T,83W4M-, and T(33M4M-tkCAT contain the mutations ineither or both of the PEBP2 sites ((33, TGTGGTT -*

TGTCGTa at nt 627-632; (4, AACCACA-CACGACA at nt708-714). The mutations are the same as the M2 mutation(14). For expression in mammalian cells, the coding regionsof al, a2, and 831 cDNAs were cloned into the Xho I site ofpCDMPy, in which the Py origin and enhancer region weredeleted from pCDM8 (Invitrogen), resulting in pCDMPy-al,pCDMPy-a2, and pCDMPy-31. The bacterial expressionplasmids pETal and pETa2, carrying the whole codingregions of al and a2 cDNAs, were constructed in the sameway as pETf82 (16). To produce deletion mutants of a2, thewhole coding region of a2 cDNA was inserted into theBamHI/HindIII fragment of plasmid pQE9 (Qiagen) result-ing in pQE9-a2 encoding a fusion protein tagged with anN-terminal histidine cluster. Various deletions were intro-duced into pQE9-a2 using appropriate restriction sites:N94C306 encodes a region of aa 94-306; N1C226 encodes aa1-226; N1C158 encodes aa 1-158; N80C226 encodes aa80-226. N1C226, N1C158, and N80C226 proteins have one,two, or one vector-derived C-terminal amino acid(s), respec-tively. For in vitro transcription and translation experiments,the Nde I/EcoRI fragment from pETal orpETa2 was clonedinto the HincII site of pSK- (Stratagene).

in Vitro Transcription and Translation. In vitro transcrip-tion reactions were carried out with a commercially availablekit (Stratagene) and in vitro translation reactions were per-

Abbreviations: EMSA, electrophoretic mobility-shift assay; TCR,T-cell receptor; CAT, chloramphenicol acetyltransferase; Py, poly-omavirus.qTo whom reprint requests should be addressed.§The sequences reported in this paper have been deposited in theGenBank data base [accession nos. D14636 (PEBP2al) and D14637(PEBP2a2)].

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0 1 2 3 4 5 6 7kbp

16 305513aaType 1 (al) AAAAA 5496 nt

3051 1306 aa

Type 2 (a2) A--MAAAAA 2228 nt

FIG. 1. Diagrammatic representation oftwo types ofthe PEBP2acDNAs. The longest cDNA clone of type 1 (al) and type 2 (a2)cDNA clone are represented by thick horizontal bars for untranslatedregions and by boxes for open reading frames. Nucleotide numberson the right of poly(A) tracts (AAAAA) represent length of cDNAs.Open boxes, hatched box, and solid box represent the segmentscommon to type 1 and type 2 cDNAs, unique to type 1 and uniqueto type 2, respectively. The 3' untranslated regions are unique toeach.

formed using a rabbit reticulocyte system (Promega) with[3H]leucine according to the manufacturer's instructions.

Expression in Escherchia coli and Purification of a andSubunits. al and a2 proteins were expressed as described forpET,B2 (16) and partially purified by heparin-Sepharose col-umn chromatography (11). Expression and purification of 82protein from pET82 was performed as described (16). His-tidine-tagged a2 and its deletion mutants were expressed andpurified using a nickel-chelate affinity resin (Qiagen) accord-ing to the manufacturer's instructions. About 10 ng ofproteinwas used per EMSA.

Transfection, Chloramphenicol Acetyltransferase (CAT) As-say and Nuclear Extract Preparation. COS-7 cells were trans-fected according to Chen and Okayama (19). L1210 andBW5147 cells were transfected by a modified DEAE-dextranmethod (20). As an internal control, 2 ,ug of pRSV,Bgalplasmid was cotransfected. CAT and ,B-galactosidase activ-ities were determined as described (21) using cell extractsprepared 48 h after transfection. Nuclear extracts wereprepared as described (14).

Northern Blot Analysis of PEBP2a mRNA. Five micro-grams of poly(A)+ RNA was prepared and subjected to

A976

11096

281216

681336108

1456148

1576188

1696228

1816268

1936308

2056348

2176388

2296428

2416468

2536508 S V W R P Y * (513 aa. 55.8 kD)

Northern blot analysis as described (16). The blot washybridized with 32P-labeled Nco I/HindIII fragment (nt 1293-1698) of al cDNA.

RESULTSMolecular Cloning of cDNAs Encoding PEBP2a. Using a

mixed oligonucleotide probe derived from the peptide se-quence determined from PEBP3 (see Materials and Meth-ods), we screened a cDNA library prepared with RNA fromHa-ras-transformed NIH 3T3 cells. Several positive cloneswere obtained and classified into two types (Fig. 1). One typeof cDNA, comprising the major species (seven of eightclones) and designated al, contained one long open readingframe starting directly from the 5' terminus followed by an

extremely long 3' untranslated region (-'4000 nt) ending witha poly(A) tail. The other type was designated a2 (minorspecies; one of eight clones). The nucleotide sequence of thefirst 868 residues of the longest al cDNA (5496 nt) wasidentical to the sequence from nt 1061 of a2 cDNA (Fig. 1,open boxes). The 3'-terminal remainder of a2 was divergedfrom that of al. The exact matching between the two typesofcDNAs over such a long stretch strongly suggests that theyrepresent isoforms of PEBP2a mRNA generated throughalternative splicing. Furthermore, genomic DNA sequenceanalysis has indicated that the 5'-terminal sequence of a2cDNA including its N-proximal coding region beyond the 5'limit of al cDNA is wholly encoded by a single exon with nointernal splice donor and acceptor sites (data not shown).Thus, a presumptive complete cDNA sequence of al wasreconstituted by merging the first 1060 nt of a2 and thelongest obtained sequence of al (Fig. 2A).

Structural Features ofthe cDNA-Encoded Proteins. The openreading fiames of al and a2 cDNAs, respectively, encodepredicted proteins of 513 (Mr 55,800) and 306 (Mr 33,700) aa,whose sequences are identical for the first 304 residues (Fig.2A). Two peptide sequences derived from PEBP3 (16), in-

catccatccactccaccacgcc---- al (type 1)Xps i h 9 t t p

1921303 t L S * ( 306 aa. 33.7 kD )

FIG. 2. Nucleotide and deduced amino acid sequences ofthe PEBP2a cDNAs. (A) PEBP2al cDNA. Peptide sequences coinciding with thoseof the peptides obtained from purified PEBP3 (see ref. 16) are doubly underlined. Arrow points to the 5'-terminal-most of the longest al cDNA.(B) PEBP2a2 cDNA. Nucleotide sequence of the region unique to a2 cDNA is shown with the numbering following that of al cDNA. Arrowindicates branch point where the al and a2 cDNA sequences diverge. Capital letters represent sequence unique to a2 cDNA. Large parts ofthe noncoding regions of al and a2 cDNAs have been omitted and are available from GenBank data base.

CCCCCTCCCCCGGCCACTTCGCTAACTTGTGGCTGTGATGCGTATTCCTGTAGATCCGAGCACCAGCCGGCGCTTCAGCCCCCCCTCCAGCAGCCTGCAGCCCGGCAAGATGAGCGAM R I P V D P S T S R R P S P P S S S L Q P G R M S D

CGTGAGCCCGGTGGTGGCTGCGCAGCAGCAGCAACG=CAGCACGAACAGCAGCAGCAACAACAGCAACAGCAACAACAGCAGCAGCAGCAGCAGCAGCAGCAGGAGGCGGCCGCAGCV S P V V A A Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q B A A A AAGCAGCGGCGGCAGCGGCGGCGGCAGCAGCGGCGGCGGCCGCAGTGCCCCGATTGAGGCCGCCGCACGACAACCGCACCATGGTGGAGATCATCGCGGACCACCCGGCCGAACTGTCA A A A A A A A A A A A A A V P R L R P P H D N R T M V E I I A D H P A B L V RCACCGACAGTCCCAACTTCCTGTGCTCCGTGCTGCCCTCGCACTGGCGGTGCAACAAGACCCTGCCCGTGGCCTTCAAGGTTGTAGCCCTCGGAGAGGTACCAGATGGGACTGTGGTTACT D S P N P L C S V L P S H W R C N X T L P V A FK V V A L G E V P D G T V V TCGTCATGGCCGGGAATGATGAGAACTACTCCGCCGAGCTCCGAAATGCCTC;CGCTGTTATGAAAACAAGTAGCCAGGTTCAACGATCTGAGATTTGTGGGCCGGAGCGGACGAGGCAAV M A G N D E N Y S A B L R N A S A V M R N Q V A R P N D L R P V G R S G R G KGAGTTTCACCTTGACCATAACAGTCTTCACAAATCCTCCCCAAGTGGCCACTTACCACAGCTATTAAAGTGACAGTGGACGGTCCCCGGGAACCAAGAAGGCACAGACAGAAGCTTGAS F T L T I T V P T N P P Q V A T Y H R A I K V T V D G P R E P R R H R Q K L D

TGACTCTAAACCTAGTTTGTTCTCTGATCGCCTCAGTGATTTAGGGCGCATTCCTCATCCCAGTATGAGAGTAGGTGTCCCGCCTCAGAACCCACGGCCCTCCCTGAACTCTGCACCAAGD S X P S L P S D R L S D L G R I P H P S N R V G V P P Q N P R P S L N S A P S

TCCTTTTAATCCACAAGGACAGAGTCAGATTACAGATCCCAGGCAGGCACAGTCTTCCCCACCGTGGTCCTATGACCAGTCTTACCCCTCCTATCTGAGCCAGATGACATCCCCATCCATP P N P Q G Q S Q I T D P R Q A Q S S P P W S Y D Q S Y P S Y L S Q M T S P S I

CCACTCCACCACGCCGCTGTCTTCCACACGGGGCACCGGGCTACCTGCCATCACTGACGTGCCCAGGCGTATTTCAGATGATGACACTGCCACCTCTGACTTCTGCCTCTGGCCTTCCTCH S T T P L S S T R G T G L P A I T D V P R R I S D D D T A T S D P C L W P S STCTCAGTAAGAAGAGCCAGGCAGGTGCTTCAGACTGGCCCTTTTTCAGACCCCAGGCAGTTCCCAAGCATTTCATCCCTCACTGAGAGCCGCTTCTCCAACCCACGAATGCACTACCCL S X K S Q A G A 8 B L G P P S D P R Q P P S I S S L T B S R P S N P R M H Y PAGCCACCTTTACCTACACCCCGCCAGTCACGTCAGGCATGTCCCTCGGCATGTCCGCCACCACTCACTACCACACGTACCTGCCACCACCCTACCCCGGCTCTTCCCAAAGCCAGAGTGGA T P T Y T P P V T S G M S L G M S A TT N Y H T Y L P P P Y P G S S Q S Q S GACCCTTCCAGACCAGCAGCACTCCATATCTCTACTATGGTACTTCGTCAGCATCCTATCAGTTCCCAATGGTACCCGGGGGAGACCGGTCTCCTTCCAGGATGGTCCCACCATGCACCACP P Q T S S T P Y L Y Y G T S S A S Y Q F P M V P G G D R S P S R M V P P C T TCACCTCGAATGGCAGCACGCTATTAAATCCAAATLCCTAACCAGAATGATGGTGTTGACGCTGACGGAAGCCACAGCAGTTCCCCAACTGTTTTGAATTCTAGCGGCAGAATGGATGAT S N G S T L L N P N L P N Q N D G V D A D G S H S S S P T V L N S S G R M D EGTCTGTTTGGCGGCCATATTGAAATGTCAACCATGGCCCAGTGGCATGGGGGCCACATCCCGCATGTGTTAATATATACATATATAAAGAGAGTGCCTATATATGTATATTGATTAGC

gacatcc-&OrXTTGAGATGCTGATGGTGTC;TCCACCTCCAGTGGACTCCAGTGGACTTCACATATCTCTTCTTAAAGTCTCTTTAGGAAAACCAAATTGATTGTTTTTCTCATTTATAC

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cluding the one used for screening (nt 16within the region common to both prediBoth al and a2 proteins can recogniz

sequence (see below) but do not have amotifs known for previously charactefactors. A peculiar feature of their peplpresence of 28 consecutive glutamine restretch of 18 alanine residues. Highthreonine, and proline residues in the Cprotein are reminiscent of transactivatimany transcription factors.Sequence Comparison of the al ProteiI

A striking sequence similarity was foiprotein and the predicted products of atation gene, runt (run) (22), and a hum(Fig. 3). A human gene on chromosAMLI, has been identified within whict(8;21) chromosome translocation in acuare clustered (23).The high homology was confined to ti

94 and 220 of al and to the region betwerunt protein with 66% homology. This rerunt homology region in the remainder ofby Kania et al. (22), the runt homolog

MIIPAGPITMVANNTQQVL1AAAAAAAAAAVAQGPGP(MRIPVDPSTSRRFSPPSSSLQPGMiSDVSPVVAAQQ(MRIPVDASTSRRFPPSTALSPGKMSEALPLGAPDAC

61 THSASSTGSSTPDLSTNNTSSSSNATTSPQNSAKMP61 QQEAAAAAAA AAA------AVPR]48 -------------------------------------

121 GSPSILCSALPNHWRSNKSLPGAFKVIALDDW?DC109 DSPNLCSPSHWRCNKTLPVAFKVAIEVIPDGTN66 DSPNFLCSVLPTHWRCNKTLPI VALGDVPDGTI

181 TIQVAKFNDLRF R GLT TIATYPVQIASY169 IQVARENDLRFSGRGKS TI TNPPQVATY126 NQVARFNDLRF IR0GTLTVFTNPPQVATY

240 -PHP-GA-FNPFMLNPAWLDAAYMTY----GYADYFI229 -SKP-SI-FSDRLSDLGRIPHPSMRV----GVPPQNP186 OTKPGSLSFSERLSELEQLRRTAMRVSPHHPAPTPNI

284 LAXSSASSVSPNPNPSVATSSSSAVQPSEYP-HPAAA282 QAQSSPPWSYDQSYPSYLSQMTSPSIHSTTPLSSTRG241 QEEDTAPWRC

343 YAIPQFPFNHVAAAAAAKAATPHAFHPYNFAAAAGLt342 CLWPSSLSKKSQAGASELGPFSDPRQFPSISSLTESF

4J3 SSSPTQQHVLLKLNTSIETSSIHEQSASDGDSDDEQI402 LGMSATTHYHTYLPPPYPGSS-QSQSGPFQTSSTPYI

459 MRCDLKAPSSMKPLYHESGP--GAVANSRQPSPEITT461 MVPPCTTTSNGSTLLNPNLPNQNDGVDADGSHSsSPT

Brunt

PEBP2 al

AML1

66% homology1 18 30 106 233

/%

I: FE7)I~~ ~I =\\

1 2 \ 94_-_ 1

-

- i221 229

I ~_ ,

p ._

\\ Abox|127 51 17 1816

[ 2~~~~~2!92% homology

FIG. 3. Amino acid sequence comparisonand AMLi (23). (A) Alignment of amino ac

proteins. Amino acids matching in at least tware shaded. Hyphens indicate gaps. Consentbox of the ATP binding site is boxed. (B) Charproteins are diagrammatically shown. Hatch4indicate the runt homology region and thebetween al and either of the other two proteiland PST indicate the regions of a glutamirstretch; and the proline-, serine-, and threoniitively. Horizontal bars under hatched segmenof the A box shown in (A).

46-1690), were found putative ATP binding site (GRSGRGKS; boxed) conformingicted proteins. to the A box (24). In addition, the 5-aa sequence of the:e the PEBP2 binding extreme C terminus (VWRPY) was identical in two proteins.iny typical structural The runt protein also has an alanine cluster and regions rich-rized DNA binding in proline, serine, and threonine on both sides of the runttide sequences is the homology region. The AMLJ-encoded protein also containstsidues followed by a a runt homology region (aa 51-177), which matches that ofcontents of serine, PEBP2a by up to 92%. Furthermore, these proteins share-terminal half of the highly conserved sequences at two additional regions as

on domains found in indicated by solid segments in Fig. 3B.The Products of a cDNAs Recognize the PEM3P2 DNA

iwith run andAMLI. Binding Site. The proteins encoded by the a cDNAs were

und between the al translated in vitro (Fig. 4A). Two discrete bands were ob-Drosophila segmen- served in SDS/polyacrylamide gel electrophoresis in bothan gene, AMLJ (23) cases, probably due to two different usages of the initiation,ome 21, designated codon (aa 1 and 25 in Fig. 2A). The slower-migrating bandsrhbreakpoints of the in both cases corresponded well to the expected size of theLte myeloid leukemia two products: 56 kDa for al and 34 kDa for a2.itemyeloid leukemia The al and a2 proteins both produced specific shifted

he region between aa bands in EMSA (Fig. 4B, lanes 2 and 6). When /32 proteinZen aa 106 and 232 of expressed in E. coli was mixed with the al and a2 proteins,tgion is referred to as the bands were shifted to higher positions with concomitantfthis paper. As noted enhancements in their intensities (lanes 4 and 8). SuchJy region contains a upward shift and strengthening of the band upon addition of

the /8 protein are characteristic of the a polypeptide (11, 16).These results established that the isolated cDNAs truly code

DQSSNATTASAIAINPAQSLAWS for the a subunit of PEBP2.3AALAGKLRSG- The position of the shifted band due to the putative al-13ISMTNMFASLHEQEYHG3IAQI or a2-,3 complex was considerably higher than that ofLRPPH1DNTMVEIIADIWAELVRT authentic PEBP3. In particular, the al-/3 band closely coin--DRSI.IVEVADHPGE.VR cided with that of PEBP2 as detected in the nuclear extractLVSIKCGNDENYCGE.RNCITrM from untransformed NIH 3T3 cells (14). Thus, the possibilityLVTVM=ENYSAELRNATAAMK emerges that a proteolytic truncation of the primary productYSKAIKVTVDGPREPRS-KQSYGY of al underlies the apparent conversion of PEBP2 to PEBP3tHRAIKVTVDGPREPRRRRtQKIDO previously observed (14, 15). That such a proteolytic modi-

fication could occur in vivo was suggested from the followingREiQAAAQANP-A4VHHPR experiment. The nuclear extracts from COS-7 cells trans-PRASLN-HSTAFNPQPQSQM---- fected with al or a2 cDNA exhibited two broad bands (Fig.WVAAAGQPSAMMPSPPGAATP 4C, lanes 3 and 4)-an upper less-intense one and a lower;TGLPAITDVPRRISDDDTATSDF denser one, which roughly paralleled those of PEBP3 (lane 1)

and free a subunits (11), respectively. When these nuclearRARNALUQSEPVHVSPASSRPSRFSNPRMHYPATFTYTPPVTSG4S extracts were mixed with that from cells expressing the /1:DVVKS----EFDLDKSLWVAPR protein (another isoform of the / subunit; ref. 16), the upperXYYGTSSASYQFPMVPGGDRSPSR band became predominant with a concomitant attenuation ofrKIKSAAVQQKTVWRPY the lower one (lanes 6 and 7). These results suggested thatrVLNSSGRMDESVWRPY both the al and a2 proteins were largely converted to

truncated forms equivalent to those contained in PEBP3.Similar proteolytic cleavage of the al and a2 proteins was

505 also observed when expressed in E. coli (Fig. 4D).F7 i09 aa The runt Homology Region Is Responsible for DNA Binding

and Heterodimerizing Abilities. In view of its remarkableconservation, the runt homology region must bear on thecrucial molecular function(s) of PEBP2a, such as DNA bind-

513 aa ing and dimerization activities. To test this possibility, weproduced a series of truncated a proteins in E. coli andsubjected them to EMSA in the presence and absence of /2

50 aa (Fig. 5). Large deletions on either or both sides of the runthomology region did not affect the abilities to bind to DNAandthe /3 subunit (lanes 3-6, 9, and 10). However, the DNA

of PEBP2a1, runt (22), binding activity was abolished when the runt homology region-id sequences of three was partly destroyed (lanes 7 and 8). These results indicateto of the three proteins that the runt homology region constitutes the core domain forsus sequence of the A both DNA binding and heterodimerization with the /3 subunit.racteristics of the three Expression ofPEBP2al in Various Cell Lines. Northern bloted and solid segments analysis with the al cDNA as a probe revealed two major

nsregions homologous mRNA species of approximately 7.4 and 6.3 kb in roughly

ne stretch: an alanine equal amounts in Ha-ras-transformed NIH 3T3 cells fromne-rich region, respec- which PEBP3 was purified (Fig. 6, lane 1). The two speciests indicate the position were also detected in parental NIH 3T3 cells, although at

significantly lower levels (lane 2). On the other hand, they

Arunt 1PEBP2a1 1AML1 1

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were not detected in F9 teratocarcinoma stem cells (lane 7)in accordance with the previous observation that PEBP2 wasnot detected in F9 cells (7). These mRNA species were alsoobserved in T-cell lines EL4 and BW5147 (lanes 3 and 4) butnot in B-cell lines L1210 and M12 (lanes 5 and 6). The mRNAspecies corresponding to the a2 protein was not detected,presumably because it is a minor species.

A B

kDa c- competitor:- + - + - ++

93 -

69- a46 -

30 -

22 -

1 2 3

PEBP3-0*

1 2 3 4 5 6 7 8 9

C P+ C+ROe(',pa~ &,,a ,D at

PEBP2-P tL9 |

PEBP34P Al* a

1 2 3 4 5 6 7 8 9 10

IU,H(x2 a1x

D B I P Bcompetitor: - -WM - - -W M -

PEBP3 -0P W .1 .

I1 .2

l 2 3 4 5 6 7 8 9 10

FIG. 4. Binding ofthe PEBP2a products to the PEBP2 recognitionsequence. (A) SDS/polyacrylamide gel electrophoresis of the in vitrotranslation products. A 5-y4 aliquot of the in vitro translation productwas analyzed by electrophoresis in a 10-20o SDS/polyacrylamidegradient gel followed by fluorography. Lanes: 1, a2; 2, al; 3, no RNAadded. (B)DNA binding and a-,Bdimerformation ofin vitro translatedproducts of al and a2. Using 32P-labeled AF9-5000 element (7) as aprobe, a 0.6-1l aliquot of each translation mixture was subjected toEMSA (lanes 2-5, a2; lanes 6-9, al) with (lanes 4, 5, 8, and 9) orwithout (lanes 2, 3, 6, and 7) the E. coli-made (2 protein. As acompetitor, unlabeled AF9-5000 element (lanes 3, 5, 7, and 9) was usedin a 50-fold molar excess over the probe. Authentic PEBP3 fromHa-ras-transformed NIH 3T3 cells is shown in lane 1. Arrowhead inlane 6 indicates position of the band detected. (C) DNA binding anda-(3 dimer formation of the PEBP2a1 and -a2 proteins expressed inCOS-7 cells. Nuclear extracts from pCDMPy-al and pCDMPy-a2transfected cells (3 and 1 ,ug, respectively) were analyzed by EMSAeither alone (lane 3, a2; lane 4, al) or together with 1 ,g of extractsfrom cells transfected with pCDMPy-(31 (lane 6, a2; lane 7, al) or withpCDMPy as a control designated as C (lane 8, a2; lane 9, al). PurifiedPEBP3 (lane 1) and nuclear extracts from NIH 3T3 cells (lane 2),pCDMPy-(3l transfected cells (lane 5), and pCDMPy-transfected cells(lane 10) were also analyzed in parallel. (D) DNA binding and a-,(dimer formation ofthe PEBP2a1 and -a2 proteins produced in E. coli.The a proteins were analyzed by EMSA (lanes 1-4, a2; lanes 6-9, al)with the (32 protein (lanes 2-4 and 7-9) or with bovine serum albumin(lanes 1 and 6). As competitor, AF9-5000 element (lanes 3 and 8), orthe DNA fragment carrying the mutant PEBP2 site (M2A) (14) (lanes4 and 9), was used in 50- or 1000-fold molar excess over the probe,respectively. Authentic PEBP3 from Ha-ras-transformed NIH 3T3cells (lane 5) and bovine serum albumin (B) alone (lane 10) are shownas controls.

7>797,N+jGv

1 2 3 4 5 6 7 8 9 10

FIG. 5. DNA binding and dimerizing activities of deletion mu-tants of the a subunit. Indicated a proteins produced in E. coli weresubjected to EMSA in the presence (even-numbered lanes) orabsence (odd-numbered lanes) of the (32 protein. Lanes: 1 and 2, al;3 and 4, N94C306; 5 and 6, N1C226; 7 and 8, N1C158; 9 and 10,N80C226.

PEBP2ai Activates Transription in Vivo via the PEBP2 Site.The preferential expression ofPEBP2a mRNA in T-cell linescompared to B-cell lines prompted us to examine whetherPEBP2a is involved in the regulation ofT-cell-specific geneexpression. To this end, we chose TCRf3 enhancer, whichcontained two potential PEBP2-binding sites in its coreregions termed Tf33 and T,34 (T/33-T.34) (25). Transienttransfection assays showed that the enhancer activity ofT,83-T/84 was, on the average, 12-fold higher in T-cell lineBW5147 than in B-cell line L1210 (Table 1). The mutations inT(33 and T/34 reduced the transcriptional activity to 33% and76% of the wild-type control, respectively. The mutations ofboth sites caused a further reduction to 22%. This analysisrevealed that the PEBP2 sites significantly contributed to theT-cell-specific enhancer activity of T/33-Tf34. Furthermore,the exogenous expression of the al protein in the B-cell lineL1210 resulted in the activation oftranscription dependent onthe T(83-Tp84 enhancer (Table 2). The al also activatedtranscription from the Py A element in a PEBP2 site-dependent manner. The a2 showed a weaker activity than theal (data not shown).

DISCUSSIONThe PEBP2a subunit was found to be related to the productofDrosophila segmentation gene run and of human leukemia

: -+ - + + +

a1( .S.a*P .. ...`P

- 28S

-18S

1 2 3 4 5 6 7

,-actin-_ .*

FIG. 6. Northern blot analysis of PEBP2a mRNA in various celllines. Poly(A)+ RNA (5 jg) from indicated cells was subjected toNorthern blot analysis with 32P-labeled al cDNA as a probe. Lanes:1, Ha-ras-transformed NIH 3T3; 2, NIH 3T3; 3, EL4 (T-cell line); 4,BW5147 (T-cell line); 5, L1210 (B-cell line); 6, M12 (B-cell line); 7,F9 teratocarcinoma stem cells.

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Proc. Natl. Acad. Sci. USA 90 (1993) 6863

Table 1. Effect of mutations in the PEBP2 sites on activity ofT83-T,B4 enhancer core regions

Reporter

Cell line T)33W4W TP3M4W T/33W4M TP3M4M pBL2CATBW5147 26.4 t 3.6 8.6 t 1.1 20.7 t 7.5 6.0 t 1.3 1.0L1210 2.0 t 1.1 ND ND ND 1.0CAT activities of the constructs were assayed in BW5147 and

L1210 cells. Relative CAT activity is indicated with the value for thebase construct pBL2CAT taken as reference. Averages of resultsobtained from two independent experiments are shown. ND, notdetermined.

gene AMLI. The run-encoded protein is known to be anuclear protein and to be involved in the regulation ofdevelopmental gene expression, although its mechanism offunction has been obscure (22). The present finding predictsthat the run-encoded protein as well as the AMLI-encodedprotein also must be a transcriptional activator, probablyhaving the same DNA binding specificity as PEBP2.The 147-aa fragment ofPEBP2a including the runt homol-

ogy region is sufficient for both binding to the PEBP2 site andheterodimerizing with the ,B subunit. This region does notcontain known motifs for DNA binding. We have previouslyshown that the 13 subunit, which also has no significanthomology to any known proteins, increases the affinity of thea subunit to DNA but that it does not bind to DNA by itself(16). These results establish that PEBP2/PEA2 represents anewly discovered family of transcription factor. We haveshown that the a mRNAs are expressed in a highly cell-type-specific manner. We also observed that they become detect-able in P19 embryonal carcinoma cells after, but not before,retinoic acid treatment of the cells (data not shown). There-fore, PEBP2, like the runt protein in Drosophila, may beinvolved in the regulation of early mouse development.Peculiarly, however, the expression of the companion sub-unit, 13, was shown to be ubiquitous, detectable even in F9cells (16). This raises an intriguing question as to how theoverall activity of PEBP2 is modulated by such differentialexpressions of the a and 13 subunits.

Transfection assays demonstrated that PEBP2 can activatetranscription in vivo from the TCRP enhancer. Furthermore, theenhancer activity ofthe TCRP enhancer depends on the PEBP2sites in T cells. This is consistent with the preferential expressionof PEBP2a mRNA in T cells. Another group has also reportedthat mutations in the human TCR6 enhancer coincidentallyfailing within its PEBP2-compatible site, cause great reduction inits enhancer activities (26). These observations taken togethersuggest that one crucial role of PEBP2a is to regulate T-cell-specific gene expression.

Table 2. Activation of transcription through the T,83-T,84 coreregion by exogenously expressed PEBP2al in B cells

-fold activation

Exp. 1 Exp. 2

Reporter 0 ig 8 ,g 16 jug 0 ug 4jg 8,ug 16 g

pTf33W4W-tkCAT 1.0 3.6 6.3 3.2 8.3 9.1 18.4pT33M4M-tkCAT 1.2 1.5 1.3 ND ND ND NDFour micrograms of reporter plasmid and the indicated dose of

effector plasmid pCDMPy-al were cotransfected into L1210 cellsand CAT activities were assayed. CAT activities are displayed as theratio to the control, in which pCDMPy as an effector and pBL2CATas a reporter were used.

The pattern ofAMLI mRNA expression (23) appears to besignificantly different from that ofPEBP2a (Fig. 6), implyingthat AMLI and PEBP2a are related but distinct genes.Clarifying the exact relationships between the PEBP2a andAMLI genes would be vital to understand their roles inleukemogenesis as well as normal cellular gene expression.

Note. After submission of this manuscript, we identified the secondgene, PEBP2aB, encoding the runt homology region. Thus, the genedescribed in this paper will be referred to as PEBP2aA.

We thank Dr. K. Nakai (Kyoto University) for the homologysearch, Drs. Y. Teranishi and J. Kondo (Mitsubishi-kasei Co.,Yokohama, Japan) for sequence analysis of peptides, and M. Maedaand N. Sawada for technical assistance. This work was supported byGrants-in-Aid for Special Project Research on Cancer Bio-Sciencefor Cancer Research.

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