The Brachyury gene encodes a novel DNA binding protein

The EMBO Journal vol.12 no.8 pp.3211 -3220, 1993

The Brachyury gene encodes a novel DNA bindingprotein

Andreas Kispert and Bernhard G.Herrmann

Max-Planck-Institut fur Entwicklungsbiologie, Abt. Biochemie,Spemannstr. 35/11, 7400 Tubingen, Germany

Communicated by H.Lehrach

Brachyury (T) mutant embryos are deficient in mesodermformation and do not complete axial development. Thenotochord is most strongly affected. The T gene isexpressed transiently in primitive streak-derived nascentand migrating mesoderm cells and continuously in thenotochord. Ectopic expression of T protein in the aninalcap of Xenopus embryos results in ectopic mesodermformation. The T protein is located in the nucleus. Theseand other data suggested that the T gene might beinvolved in the control of transcriptional regulation. Inan attempt to demonstrate specific DNA binding of theT protein we have identified a consensus sequence amongDNA fragments selected from a mixture of randomoligomers. Under our experimental conditions T proteinbinds as a monomer to DNA. This property resides inthe N-terminal domain of 229 amino acid residues whichis strongly conserved between the mouse protein, and itsXenopus and zebrafi'sh homologues. The latter proteinsalso recognize the consensus DNA binding site. Wesuggest that the T protein is involved in the control ofgenes required for mesoderm formation, and for thedifferentiation and function of chorda mesoderm.Key words: Brachyury protein/DNA binding/embryogenesis/mesoderm/mouse

finding that the T protein is located in the nucleus (Schulte-Merker et al., 1992; A.Kispert and B.G.Herrmann,unpublished). In turn, the notochord is required for theorganization and differentiation of the axial embryonicstructures. Along the axis from anterior to posterior anincreasing amount of functional T protein is required fornotochord formation and axial development (MacMurray andShin, 1988; Yanagisawa, 1990). Increasing the dosage ofT in an otherwise tailless T/ + mutant mouse byintroducing one or two copies of the wild-type T gene ontransgenes into the genome of the mutant results in a dosedependent extension of the tail to almost normal length (Stottet al., 1993). In Xenopus it has been shown that expressionof the T gene occurs in response to mesoderm inducingfactors in the absence of protein synthesis (Smith et al.,1991), and that ectopic expression of the T gene can resultin mesoderm formation at ectopic sites (Cunliffe and Smith,1992).The molecular and embryological data suggest that the T

protein is involved in the transcriptional regulation of genesrequired in the formation and differentiation of mesoderm.Therefore we have used a protocol for the selection ofDNAbinding sites from a mixture of random-mer oligonucleotides(Pollock and Treisman, 1990) to demonstrate that T proteincan specifically bind to DNA, and to identify DNA sequencesrecognized by this protein. In addition we have determinedthe DNA binding domain and show that T binds as amonomer under our experimental conditions. We suggestthat the T gene encodes a novel DNA binding proteinrequired for the tissue specific control of gene expression.

IntroductionThe mouse Brachyury (1) gene has been discovered throughthe phenotype of its mutated form and has been describedby several authors (Dobrovolskaia-Zavadskaia, 1927;Chesley, 1935; Gliicksohn-Sch6nheimer, 1938; Gruneberg,1958; for review see Willison, 1990). Mice heterozygousfor the T mutation do not complete axial development andhave a short tail. Homozygous TIT embryos die in utero at

- 10 days of gestation due to the lack of the allantois, theprecursor of the umbilical cord (Gliicksohn-Schonheimer,1944). Posterior axial development in these embryos isdisrupted. The brain and a few abnormal somites are formed,but at - 8 days of gestation the notochord precursor failsto be elongated and subsequently mesoderm formation inthe primitive streak comes to a standstill. Cloning of the Tgene revealed that the Tmutation is a deletion of the Tgene(Herrmann et al., 1990). In normal embryos the T gene isexpressed in the tissues most affected by the lack of Tprotein, the notochord and the nascent and migratingmesoderm (Wilkinson et al., 1990; Herrmann, 1991).Notochord formation and differentiation requires T cell-autonomously (Rashbass et al., 1991), consistent with the

ResultsSelection of DNA fragments binding to T protein froma pool of random 26-mer oligonucleotidesTo isolate DNA sequences specifically recognized by theT protein, a protocol for the enrichment of target DNAfragments was applied (Figure 1) (Pollock and Treisman,1990). Similar procedures for binding site selection havebeen described (Oliphant et al., 1989; Thiesen and Bach,1990; Gogos et al., 1991; Howe and Watson, 1991). Inbrief, a mixture of random 26-mer oligonucleotides, flankedby defined primer sites, was incubated with T proteinsynthesized in vitro. The complex was immunoprecipitatedwith antibodies specific either for an N-terminal or a C-terminal portion of the T protein. The bound DNA wasextracted, amplified by polymerase chain reaction and analiquot subjected to another round of the selection procedure.The progress of the binding site selection was followed byan electrophoretic mobility shift assay. Pools of unselectedDNA fragments and of selected molecules after two, fourand six rounds of enrichment were labelled and used asprobes. After four enrichment cycles specific DNA bindingof the T protein was demonstrated by supershifting of theT-DNA complex with an antiserum against the T protein

© Oxford University Press 3211

A.Kispert and B.G.Herrmann

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excess of a weakly binding DNA fragment (BS.4-35)obtained by random site selection or a 1000-fold excess ofunrelated DNA (Figure 3B). EMSA was also used to assessthe binding of T to a perfect palindrome (BS.p; see Materialsand methods) and one half-site of the palindromic consensussequence (BS.p/2). Figure 3C demonstrates that T proteinbinds strongly to the perfect palindrome BS.p but that onehalf-site is not sufficient to support T binding. Thepalindromic fragment was also bound by the proteinsencoded by the Xenopus and zebrafish homologues of theTgene (Smith et al., 1991; Schulte-Merker et al., 1992) asrevealed by specific shifts and supershifts in a gel-shift assay(Figure 3D).

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(Figure 2A). DNA fragments recovered after four roundsof the selection protocol using the N-terminal and the C-terminal antisera, respectively, were isolated by molecularcloning. Twenty cloned DNA fragments from either poolwere sequenced and compared. In each pool someoligonucleotides were represented several-fold indicating thatthe complexity of the selected pool has decreased at this stagein favour of strong binding sites. All oligonucleotidesanalysed had the core sequence AGGTG in common whichwas utilized for a best fit alignment. A consensus targetsequence with a nearly palindromic structure was identifiedthrough the compilation of all sequences (Figure 2B).

Selected target fragments bind specifically to TproteinThe strength of T protein binding to individual targetfragments was assayed using the electrophoretic mobilityshift assay (Figure 3A). Of the 11 fragments tested nine werestrongly bound by T protein. They all contain sequenceshighly similar to the consensus sequence with variationsmainly in the left half-site. BS.3-1, BS.3-5, BS.3-6 andBS.3-14 harbour a perfect palindrome of 16 bases. Weakbinding was observed with fragments BS.3-4 and BS.4-35.The comparison of the sequences of these two fragments withthose of strong binding sites and interference analyses (seebelow) suggest that the T-C change at position 6 of the righthalf-site (AGGTGT-AGGTGC) is responsible for thedecrease of the binding affinity. The strength ofDNA -protein interaction is positively affected by bindingof antibodies directed against a C-terminal polypeptide ofT to the complex suggesting that the DNA-protein complexis stabilized. The specificity of DNA-protein complexeswas confirmed by successful competition for T protein withincreasing amounts of unlabelled specific DNA. The strongbinding of T protein to BS.3-6 was not affected by a 100-fold

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The T protein predominantly recognizes pyrimidines inthe palindromic target siteTo determine the contribution of all individual bases withinand around the binding site we performed variousinterference analyses. For that purpose a 117 bp end-labelledXhoI-SacI fragment from the plasmid pBS.p harbouringthe 24 bp palindromic binding site BS.p was used. Due tothe symmetric nature of this binding site we analysed onlyone strand since the second half-site represents thecomplementary strand of the other half-site and the T proteincan bind to that fragment in both orientations. DNase Ifootprinting analysis (Figure 4A) showed that the T proteinprotects the entire palindromic sequence and some flankingbases from digestion by DNase I. All the contact pointsdetermined by interference analyses map within thatprotected region. The most distant base interfering with Tprotein binding is the thymidine residue at position -10

demonstrating that the region of contact is smaller than theregion of protection. Methylation interference analysisexamines the steric inhibition of protein-DNA interactionby the addition of methyl groups on guanosine and moreweakly on adenosine residues. The methylation of the N-7position of guanines and the N-3 position of adenines isindicative of major and minor groove contacts, respectively.In our experiment (Figure 4C) only the guanosine residueat position +5 of the palindrome exhibits strong interferenceafter methylation. The importance of this major groovecontact point is confirmed by a depurination interferenceanalysis (Figure 4B) of the same fragment. Again theguanine at position +5 shows the strongest interference effectwhile the removal of neighbouring purines at positions + 1,+2, +3 and +7 interferes much more weakly with Tbinding. Hydrazine treatment of DNA leads todepyrimidination and therefore allows us to probe thecontribution of thymidine and cytidine residues to the bindingaffinity. In our hands (Figure 4D) depyrimidination of allbases of the palindrome except the flanking thymines atpositions +11 and + 12 interfere with T protein bindingsuggesting that T protein mainly contacts pyrimidines in atarget site. Of particular importance are the thymidineresidues at positions +4 and +6 which flank the importantG residue at position +5 defining together the TGT tripletas a major contact site. The importance of the thymine at+6 is also demonstrated by the fact that BS.34 and BS.4-35which have an exchange at this position are only very weaklybound by T protein (Figure 3A). The contribution of flankingsequences was assessed by EMSA using palindromic DNAfragments of decreasing length as probes (Figure 4E).BS.p-5 is only very poorly bound by T protein whereasBS.p-4, a 22 bp palindromic fragment, is strongly bound.

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Fig. 2. Isolation of DNA fragments bound by T protein. (A) Electrophoretic mobility shift assay of DNA pools isolated after two, four or six roundsof the binding site selection procedure. The number of selection cycles is indicated above each lane, 0 refers to the R76 random-mer pool beforeselection of binding sites. Binding reactions included 1 ,Al of rabbit reticulocyte lysate (RRL) or lysate programmed with RNA encoding the full-length T protein (RRL-T) and 1 Al of antiserum (RRL+Ab)/(RRL-T+Ab) specifically recognizing a polypeptide in the C-terminal (a-TC328-420)half of the T protein. After 4 or 6 cycles of binding site selection supershifts of DNA-T protein complexes with either antiserum were detected,indicating specific binding of pools of DNA fragments to T protein. (B) Sequences of 20 DNA fragments each, isolated after four rounds of bindingsite selection with a-TNI-123 or a-TC328-420 and picked randomly were aligned at the invariant core sequence AGGTG. Linker sequences areshown in lower case letters, in some cases (e.g. BS.3-4, BS.4-35) they might have contributed to the binding site. In either experiment somefragments were isolated several times. The occurrence of the bases from the 40 aligned sequences is listed for 24 positions and the percentage givenfor the base occurring most frequently. A consensus sequence of 20 bases was derived by taking into consideration only bases which were found in>60% of the cases. The consensus sequence is very similar to a perfect palindrome of eight bases. The left half-site (eight bases) seems to beshorter and less well conserved than the right half-site (12 bases).

This result and the interference data define a 20 bppalindrome as the region which is contacted by the T protein.A summary of the interference data on this 20 bp palindromeis presented in Figure 4F.

The entire N-terminal half of the T protein is requiredfor DNA bindingIn order to delineate the DNA binding domain of the Tprotein, subregions of the T protein were translated in vitro(Figure 5A) and assayed for binding to the palindromic targetfragment BS.p in a gel-shift assay (Figure 5B and C). TheN-terminal half of 229 amino acid residues was required forDNA binding under our experimental conditions. Very weakbinding was conferred by a polypeptide containing aminoacids 18-229, but a further deletion of amino acids fromthe N-terminal or C-terminal end completely abolishedcomplex formation. DNA binding is also inhibited by an

excess of antibodies specifically recognizing a portion of theN-terminal T domain, but is unaffected by an excess ofantibodies to the C-terminal half of T (Figure 5D).

A monomer of T protein is sufficient for DNA bindingTo test whether the T protein binds DNA as a monomer ordimer, decreasing amounts of the complete protein weremixed with increasing amounts of the N-terminal DNAbinding domain and assayed for binding to the palindromictarget DNA fragment BS.p (Figure 6A). If a protein dimerwere required for DNA binding under these conditions, acomplex of a size between that of the N-terminal half andthat of the complete T protein bound to DNA, would beexpected, provided that dimerization occurred between theN-terminal regions. However, only the two sizes ofDNA -protein complex were observed. Dimerization withthe C-terminal half of T was not detected either (data notshown). In addition, the complete T protein was co-translatedand incubated with a T-glutathione-S-transferase fusionprotein (GST-T) in the presence of target DNA(Figure 6B). Glutathione-S-transferase-T fusion protein wasbound to glutathione beads, precipitated, denatured andseparated on a SDS -polyacrylamide gel. If T proteindimerized, it should bind to the fusion protein and be co-

3213

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Fig. 3. Analysis of the binding of T protein to selected and artificial fragments by electrophoretic mobility shift assay (EMSA). (A) EMSA ofcomplexes of T protein with DNA fragments derived by binding site selection with a-TNI-123 (BS.3-1, BS.3-4, BS.3-5, BS.3-6, BS.3-8, BS.3-10,BS.3-13, BS.3-14) and with ca-TC328-420 (BS.4-1, BS.4-5, BS.4-35). Binding reactions included buffer only (probe), unprogrammed rabbitreticulocyte lysate (RRL) and rabbit reticulocyte lysate programmed with T RNA encoding the full-length T protein (RRL-T) with (+Ab) or withoutthe addition of an antiserum against a C-terminal portion of the T protein, ai-TC328-420. Strong binding was obtained with BS.3-1, BS.3-5, BS.3-6,BS.3-8, BS.3-10, BS.3-13, BS.3-14, BS.4-5, intermediate binding with BS.4-1 and weak binding with BS.3-4 and BS.4-35. Fragments bindingstrongly are very similar to the palindromic consensus sequence and show variations mainly in the left half-site. With BS.34 and BS.4-35 the T-Cexchange at position 6 of the right half-site might be responsible for the weak binding to T protein (see Results). Addition of the antiserum abolishedthe T shift and created a supershift of more slowly migrating large complexes of probe-T protein and antibodies. The antiserum increases thestability of the DNA-protein complex as revealed by the fact that more probe is found in the bound fraction compared with the binding reactionwithout antiserum. (B) Competition analysis of the selected DNA fragment BS.3-6 in the EMSA. The binding reaction contained no protein (probe,lane 1) or in vitro translated T protein (lanes 2-16). The strong binding site BS.3-6 was used as a probe and was competed with unlabelled BS.3-6(lane 3-6), fragments derived from the polylinker region of pKS [pKS(H,K) lanes 7-10, pKS(H,S) lanes 12-14] or the weakly binding fragmentBS.4-35 in the molar ratios indicated above each lane. Unlabelled DNA fragment BS.3-6 successfully competed with the probe for binding to the Tprotein, and abolished the formation of the labelled complex at an excess of 50-fold (1:50). A 100-fold excess of the fragment BS.4-35 or a

1000-fold excess of two unrelated fragments did not influence the formation of the BS.3-6-T protein complex. (C) T protein binds strongly to a

perfect palindrome but not to one half-site alone. Binding reactions included unprogrammed rabbit reticulocyte lysate (RRL) or rabbit reticulocytelysate programmed with T RNA (RRL-T) with (+Ab) or without the antiserum a-TC328-420. Probes were BamHI-EcoRI fragments from pBS.p or

pBS.p/2, respectively, pKS derivatives harbouring the palindromic binding site BS.p or the half-site BS.p/2, respectively, in the SmaI site. T proteindoes not bind to one half-site, but binds strongly to the perfect palindromic fragment as demonstrated by shift and supershift. (D) The proteinsencoded by the Xenopus or zebrafish homologues of T also bind to the palindrome sequence BS.p. (a) EMSA with mouse T , Xenopus T andzebrafish T proteins. Binding reactions included no protein (probe, lane 1), rabbit reticulocyte lysate (RRL, lanes 2 and 6) and mouse (RRL-T, lanes3 and 7), Xenopus (RRL-XeT, lanes 4 and 8) or zebrafish T protein (RRL-ZfT, lanes 5, 9 and 10). All three T proteins produce DNA-proteincomplexes with similar electrophoretic mobility. The presence of T protein in the complex was confirmed by binding to the antiserum ax-TNI-123(Abl, lanes 6-9) or to an antiserum directed against the zebrafish T protein (Ab2, lane 10) (Schulte-Merker et al., 1992) which is only weaklyrecognized by ca-TN-1123. (b) Mouse, Xenopus and zebrafish T proteins are synthesized with similar efficiency in an in vitro translation reaction.Mouse (T), Xenopus (XeT) and zebrafish (ZfT) T RNAs were translated in a rabbit reticulocyte lysate in the presence of [35S]methionine and the invitro translation reactions separated on a 10% SDS-polyacrylamide gel. Autoradiography revealed that a protein of the expected molecular weightwas synthesized in each case whereas in the control (C) with unprogrammed reticulocyte lysate no labelled translation product was found.

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Fig. 4. Characterization of the T DNA binding site. (A) -(D), DNase I protection analysis (A), methylation interference analysis (C) and missingcontact probing analysis (B and D) were performed on a 117 bp XhoI-SacI fragment from the polylinker region of pKS harbouring the perfectpalindrome BS.p in the SmaI site. All reactions were coelectrophoresed on a denaturing polyacrylamide gel and autoradiographs of the labelledfragments are aligned to demonstrate the relationship between the protected region and the important contact points. Lanes A+G and G contain theproducts of a chemical sequencing reaction for A+G and G residues, respectively, on the same fragment and are coelectrophoresed to create asequence marker. Numbers 40 and 87 refer to the position of bases with the respective distance from the labelled end. The sequence and position ofthe perfect palindrome BS.p within the 117 bp fragment used as probe are indicated on the left side of (A). The position of interfering residues isshown on the left side of the A+G sequencing ladder. The degree of interference was judged only qualitatively as strong (large, filled circles),intermediate (small, filled circles) and weak (open circles). -, binding reaction with rabbit reticulocyte lysate alone; +, binding reaction with invitro translated T protein; U, unbound fraction; B, bound fraction. (A) DNase I protection analysis. The bracket indicates the region protected by Tprotein from DNase I digestion. The protected region covers the complete palindrome and includes some flanking bases. (B) Depurination and (C)methylation interference analyses define the G residue at position 5 of the right half-site of BS.p as a major contact point. Base removal andN7-methylation at this residue both interfere strongly with binding of the T protein whereas neighbouring purines exhibit only weak and intermediateinterference. (D) Depyrimidination interference analysis reveals that the T protein contacts the palindromic binding site mainly at pyrimidine residues.Strongest interference is observed at the T residues at positions +4 and +6 of the right half-site suggesting that the TGT triplet is a major contactsite for the T protein. All pyrimidines of the left half-site exhibit interference upon base removal. (E) EMSA of complexes of T protein with perfectpalindromic DNA fragments of various sizes. Binding reactions contained no protein (lane 1), rabbit reticulocyte lysate (lane 2) and in vitro translatedT protein (lanes 3-8). The probes used are indicated on the right, they represent the 24 bp palindrome flanked by two SmiaI half-sites (BS.p) andfragments which were truncated by 3, 4, 5, 7 and 8 bp, respectively, from both ends of BS.p. BS.p-4 is sufficient for binding to T protein asdemonstrated by the shift. The further truncation to BS.p-5 drastically reduces the stability of the complex so that only after prolonged exposurecould a shift be observed (not shown). (F) Summary of the contact points of the T protein with a 20 bp palindromic fragment as gathered bymethylation interference, depurination and depyrimidination interference data. Due to the palindromic nature of the binding site used for these assays,the interference pattern on one strand of one half-site can be considered identical to the pattern on the complementary strand of the other half-site.Large filled circles, strong; small filled circles, intermediate; open circles, weak interference.

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precipitated with the fusion protein. Only the fusion proteinwas recovered after precipitation, strongly suggesting thatunder our experimental conditions the T protein does notdimerize in the presence of target DNA. However, it cannotbe ruled out completely that glutathione-S-transferase in thecontext of the GST-T fusion protein might inhibit thedimerization of T.

DiscussionWe have employed a protocol for the selection of bindingsites from a pool of random 26-mer oligonucleotides todemonstrate that the T gene of mouse encodes a novel DNAbinding protein. Antisera directed against an N-terminal anda C-terminal region of the T protein were used in independentexperiments for the immunoprecipitation of DNA -proteincomplexes. Fragments derived from either experiment werehighly similar in sequence and revealed a nearly palindromicconsensus binding site of 20 bases. The inner five bases ofone half-site (AGGTG) are identical in all selected fragments.The other half-site is more variable. A minimum of six baseson one of the half-sites in combination with a completesecond half-site is required for DNA binding, a singular half-site of 12 bp is not sufficient. Some fragments wererepresented several times among the clones analysed. Theybelong to the class of strong binding sites which comprisesthe majority of clones. A few weak binding sites have alsobeen isolated. Their sequence differs substantially from theconsensus site. A likely explanation for the isolation of weakbinding sites is the observation that binding of the antibodiesto the T protein seems to stabilize the complex. The signalstrength of weak T protein-DNA complexes detected inelectrophoretic mobility shift assays is strongly increasedwhen antiserum is added to the reaction. This shows thatthe DNA binding behaviour of T protein can be modulatedby interaction with other proteins, a property which mightbe important for the action of T protein in vivo.

Interference analyses for the 24 base palindrome revealedthe major contact points of the T protein on the DNA. Thecentral TGT triplet provides the major contact site; flankingpyrimidines seem to represent weaker contact points on thecounterstrand. The importance of the TGT contacts isemphasized by the fact that the triplet is conserved in allselected binding sites except the weakly binding fragmentsBS.3-4 and BS.4-35. Accordingly, the ACA triplet in theleft half-site of the selected binding sites is also highlyconserved. Taken together the data suggest that the T proteincontacts two 10 bp elements one of which has to be verysimilar to the sequence AGGTGTGAAA while the other candiffer from it to some degree. Preliminary evidence obtainedby us recently showed that the T protein can also bind tovariably spaced direct or indirect repeats of the 10 bp targetsequence in genomic mouse DNA (A.Kispert andB.G.Herrmann, unpublished).DNA binding is conferred by the N-terminal half of the

protein, consisting of 229 amino acid residues. This DNAbinding domain is unusually large and does not contain anysignificant similarity to known DNA binding motifs (Buschand Sassone-Corsi, 1990; Harrison, 1991). It is highlyconserved in the T homologues ofXenopus and the zebrafish(Smith et al., 1991; Schulte-Merker et al., 1992). Thisconservation is functionally significant, since, as we haveshown, all three vertebrate T proteins bind to the palindromic

binding site identified in this work. We therefore suggestthat this novel DNA binding domain be called the 'Tdomain'.

Recently a striking similarity between the DNA bindingdomain of the vertebrate T protein and a 224 amino acidsubregion of the Drosophila optomoter-blind (omb) proteinhas been described (Pflugfelder et al., 1992). Forty-fourpercent of the amino acid residues of this subregion of ombare identical to residues in the N-terminal half of the mouseT protein. The conserved domain of omb binds to calfthymus DNA suggesting that omb is also a DNA bindingprotein. omb is structurally different from T and plays a rolein the development of individual neurons and whole brainparts. It is probably not the homologue of T, but rather adifferent protein which shares the DNA binding domain withT. This raises the possibility that omb may bind to a sitethe same as or similar to T. DNA binding motifs often arecommon to a number of genes. Experiments to isolate othermembers of a T gene family are under way.

Pflugfelder and coworkers (1992) reported a clusteringof charged amino acid residues in the N-terminal half of Tand the conserved homologous region in omb, and nearlyan exclusion of SPXX motifs from this region and anincreased occurrence outside this region. SPXX motifs havebeen shown to occur at a high relative frequency intranscription factors in the regions flanking the DNA bindingdomain (Suzuki, 1989). Schulte-Merker and coworkers(1992) demonstrated helical structures in the N-terminal halfof the zebrafish T protein which are conserved in the mouseT protein. Two pairs of these helices are located at eitherend of the T domain and are likely to be involved in DNAbinding. This is consistent with our finding that the removalof amino acid residues from either end of the T domainabolishes DNA binding. Under our experimental conditionsa monomer ofT protein is sufficient to bind to a palindromicsite. It is conceivable that the two helical ends of the Tdomain interact with the two half-sites of the palindromictarget. Of course this does not exclude that the T proteinmight form homo- or heterodimers in vivo.The nuclear location and DNA binding property of T

protein suggest that T may act as a transcription factorrequired for the formation of mesoderm and thedifferentiation and function of chorda mesoderm. Thefunction of T is impaired in mutations in which the C-terminal end of the T protein is truncated. Occurrences ofsuch mutations are represented by the alleles 7Yis and 7t.Heterozygous TWis/+ and 7/+ embryos show a moreanterior arrest of mesoderm and axial development thanembryos carrying a single wild-type gene. The mutantprotein therefore reduces the amount of functional T proteinin these embryos suggesting that they compete or interferewith the wild-type T protein. Since the N-terminal DNAbinding domain is intact in these mutant proteins, they mostlikely can bind to DNA, but may not be able to confertranscriptional activation. This would suggest that the C-terminal region of T protein is required for transcriptionalactivation. However, at the moment it cannot be excludedthat T may function as an auxiliary protein for transcriptionalactivation. Recently it has been shown that the MCM1protein of yeast, which binds to DNA, sets the spacing andorientation of the homeodomains of an a2 dimer therebyraising the target specificity of this transcription factor (Smithand Johnson, 1992).

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Fig. 5. DNA binding is conferred by the N-terminal half of the T protein. (A) Full-length and truncated T proteins are synthesized with similarefficiency in vitro. T proteins of various lengths (as indicated by the numbers in brackets) were translated in the presence of [35S]methionine and invitro translation reactions were separated on a 15% SDS -polyacrylamide gel. Autoradiography shows that proteins of the expected sizes were

synthesized in similar amounts. (B) Polypeptide regions of the T protein, synthesized in rabbit reticulocyte lysates, were tested for binding to thepalindromic fragment BS.p in a gel-shift assay. Only full-length T protein and a polypeptide consisting of the N-terminal 229 amino acid residuesshowed strong binding to the probe, truncation of this polypeptide resulted in very weak [T(18-229)] or complete loss of binding to the targetDNA. The numbers in brackets refer to the amino acid residues of the T protein synthesized in vitro and contained in the binding reaction. RRLbinding reaction with rabbit reticulocyte lysate, lane 2; probe binding reaction without protein, lane 1. (C) Schematic representation of the resultsshown in (B). (D) An excess of antibodies to the N-terminal half of the T protein (ai-TNI-123) inhibits the formation of a complex with the targetBS.3-6. However, complex formation is enhanced by excess amounts of the antibodies ca-TC328-420. Binding reactions included no protein (probe,lane 1), or an aliquot of an in vitro translation reaction with full-length T protein (lanes 2-9). ca-TNI-123 (lanes 3-5) and a-TC328-420 (lanes 7-9)antisera, respectively, were added to the binding reaction as indicated above each lane. BS.3-6-T protein complex formation was analysed withEMSA.

Previously sequences very similar to genomic binding siteshave been isolated by random target site selection (Oliphantet al., 1989; Pollock and Treisman, 1990). Thisdemonstrates that the in vitro DNA binding specificity ofa protein can be identical to that in vivo, and suggests thatnatural target sites can be isolated from genomic DNA invitro. Therefore, the approach used here will be applied tothe isolation of genomic fragments recognized by T protein.This might allow the identification and isolation of genescontrolled by the Tgene product in vivo. Similar procedureshave already been used successfully for the isolation of targetgenes of Ubx (Gould et al., 1990; Graba et al., 1992). Weexpect that the cloning of target genes of T will give insightinto the formation of mesoderm in general, and in particular

in the formation and differentiation of the notochord. The

latter plays an important role in the organization and

differentiation of the embryonic structures (Yamada et al.,1991; Rong et al., 1992), and it is conceivable that the T

protein might regulate genes expressed in the notochord and

involved in axial organization.

Materials and methodsBinding site selectionThe procedure was carried out essentially as described previously for the

isolation of target DNA fragments bound by serum response factor (Pollockand Treisman, 1990). An aliquot of an in vitro translation reaction containingfull-length T protein was mixed with 1 ng of the double-stranded DNA

fragment R76 which contains a 26 random-mer flanked by two

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Fig. 6. A monomer of T protein binds to DNA. (A) Full-length T protein (T) and the N-terminal half (amino acid residues 1-229; TN),synthesized in rabbit reticulocyte lysates, were mixed in various ratios and tested for binding activity in a gel-shift assay. No DNA-protein complexof intermediate mobility between T and TN was generated indicating that dimerization does not occur between the N-terminal domains of 229 aminoacid residues. The concentration of full-length T protein was reduced in steps of 0.25 td, whereas the concentration of the N-terminal half wasincreased by 0.25 1l per step (lane 2, 2 pd T, 0 ,ul TN; lane 3, 1.75 I1 T, 0.25 41 TN; to lane 10, 0 1l T, 2 1l TN); lane 1, probe Bs.p alone. Gel-shift analysis was performed using BS.p as probe. (B) A glutathione-S-transferase-T fusion protein (GST-T) and full-length T protein werecotranslated in vitro in the presence of [35S]methionine and incubated with the target fragment Bs.p. DNA-protein complexes were precipitated bybinding of glutathione-S-transferase to glutathione-sepharose beads and separated on an SDS-polyacrylamide gel. Since T protein was notcoprecipitated with GST-T, we conclude that T protein does not dimerize in this experiment. Lanes 1-3 contain in vitro translation products;lane 1, T protein; lane 2, T and GST-T fusion protein; lane 3, GST-T; lanes 4-6, protein recovered after precipitation with glutathione-sepharosebeads; lane 4, T protein only was incubated with Bs.p; lane 5, GST-T and T protein were incubated without Bs.p; lane 6, GST-T and T incubatedwith Bs.p; lane 7, GST-T protein only.

oligonucleotides containing a BamHI (primer R) and an EcoRI (primer F)restriction site, respectively, in 250 A1 BBE binding buffer (20mM HEPESpH 7.4, 100 mM KCI, 20% glycerol, 0.25 mM EDTA, 1 mM DTT, 0.1%Nonidet P-40, 1 1tg/ml BSA, 1 AM pepstatin, 1 ytM leupeptin, 1 mM PMSF)and 2 Ag poly(dI-dC).poly(dI-dC) as non-specific competitor. DNA-Tprotein complexes were precipitated overnight at 4°C with an antiserumdirected against an N-terminal (ai-TNI - 123) or a C-terminal (oa-TC328 -420)portion of the T protein, respectively, and 10 11 protein A agarose beads(Boehringer). Protein A beads were washed four times with 500 d1 bindingbuffer at 4°C. The remaining DNA fragments were eluted from the agarosebeads in 5 mM EDTA, 0.5% SDS, 100 mM sodium acetate, 50 mM TrispH 8.0 at 37°C, and an aliquot was used for PCR amplification (94°C-1min, 62°C-1 min, 72°C-1 min, 15 cycles). The reaction products werepurified on 8% polyacrylamide gels. An aliquot of the pool of the selectedDNA fragments (0.4 ng) was subjected to another round of binding siteselection.

Oligonucleotides and probesPrimer R (5'-CAGGTCAGTTCAGCGGATCCTGTCG-3'), primer F(5'-GCTGCAGTT GCACTGAATTCGCCTC-3') and R76 (5'- CAGGT-CAGTTCAGCGGATCCTGTCGN26 GAGGCGAATTCAGTGCAACT-GCAGC-3') were purified on preparative polyacrylamide gels before use.R76 was rendered double-stranded using primer F and the Klenow fragmentof Escherichia coli DNA polymerase.DNA fragments BS.3-1, BS.3-4, BS.3-5, BS.3-6, BS.3-8, BS.3-10,

BS.3-13, BS.3-14, BS.4-1, BS.4-5 and BS.4-35 were released from theplasmid vector by a BamHI-EcoRI double digest, isolated from preparativeagarose gels after separation and dephosphorylated with calf intestinal alkalinephosphatase. BS.p/2 was prepared by annealing the oligonucleotide5'-GGGAGGTGTGAAATTCCC-3' to its complementary DNA, creatinga double-stranded DNA fragment which represents a half-site of thepalindromic sequence flanked by SmnaI half-sites. BS.p was made by self-annealing of the oligonucleotide 5'-GGGAATTTCACACCTAGGTGTG-AAATTCCC-3' which represents a 24 bp palindromic fragment flankedby SmaI half-sites. The base positions in this fragment were numbered asfollows:5'-GGGAATTTCACACCT AGGTGTGAAATTCCC-3'

-12 -1 +1 +12Similarly the palindromic DNA fragments BS.p-3, BS.p-4, BS.p-5, BS.p-7and BS.p-8 were made by self-annealing of the respective oligonucleotides.All these DNA fragments were end-labelled with T4-PNK in the presenceof [-y-32P]ATP. Unrelated DNA fragments for competition studies[pKS(H,K) or pKS(H,S)], are HindHI-KpnI or HindIII-SacI fragments

from the polylinker region of the plasmid vector pBluescript KS, respectively.The plasmids pBS.p and pBS.p/2 were created by inserting the double-stranded fragments Bs.p and Bs.p/2, respectively, in the SmaI site ofpBluescript KS. BamHI-EcoRl fragments from these plasmids were gel-purified, dephosphorylated and used as probes in experiment 3C afterlabelling with T4-PNK. The context of the vector sequence ensures thatthe half-site and the palindromic site can be used as probes in a gel-shiftassay in a comparable manner.

AntibodiesAn N-terminal and a C-terminal portion of the T protein were expressedin bacteria as fusions to glutathione-S-transferase (GST): forpGEX2T.T(1-123) the coding region of pme75 (Herrmann et al., 1990)was amplified by PCR using the primers 5'-TAGAATTCGCATGAGCT-CGCCGGGCACAGAG-3' (sense, introducing an EcoRI site close to thestart codon) and 5'-AGATCTGAATTCCAGGATTTCAAAGT-3' (STO13,position 1767 on pme75) and an EcoRI-AccI fragment of 369 bp (encodingamino acid residues 1-123) was cloned into the SniaI site ofpGEX2T (Smithand Johnson, 1988) after filling recessive ends with the Klenow fragmentof DNA polymerase. pGEX2T.T(327-420) was constructed by cloninga HaeHI fragment of pme75 (nucleotides from position 1088-1366,corresponding to amino acid residues 327-420) into the SmiaI site ofpGEX2T. Plasmids were transformed in E.coli JM101 and fusion proteinspurified essentially as described by Smith and Johnson (1988) except thatwe used buffer A (50 mM Tris-HCl pH 7.9, 200 mM NaCl, 2 mM EDTA,2 mM 2-mercaptoethanol, 1 mM PMSF, 1 itM pepstatin, 1 itM leupeptin,0.2 mg/ml lysozyme) as lysis buffer. For the fusion of the N-terminal Tpolypeptide an inclusion body preparation (Rio et al., 1986) had to be carriedout due to the insolubility of the fusion protein. The eluates of the GSHagarose column were separated on a preparative 12% SDS-polyacrylamidegel. Bands containing the fusion proteins were excised, homogenized andsuspended in Freund's complete adjuvant for the first injection and inincomplete adjuvant for the subsequent injections. Two rabbits wereimmuniized with the N-terminal fusion protein, one rabbit with the C-terminalGST-T fusion at intervals of 4-6 weeks.

Polyclonal antisera from the rabbits were affinity purified. For that purpose(His)6-tagged polypeptides were made using the Qiaexpress system(Diagen). The vector pQE12 confers a C-terminal (His)6 tag to expressedproteins. For construction of pQE12.T(I -157) a part of the pme75 cDNAwas amplified by PCR using the primers 5'-AGGATCCATGAGCTCGC-CGGGCACA-3' (sense, position 109) and 5'- AGGGATCCCTGTCCC-CCTCCATTGAG-3' (antisense, position 582), digested with Sau3A andcloned in the BamHI site of pQE12. For pQE12.T(328-435) primers

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5'-GAGGATCCCACACCAGCATGCTGCCTGT-3' (sense, position 1090)and 5'-CTAGATCTCATAGATGGGGGTGACAC-3' (antisense, position1413) were employed to amplify the part of pme75 corresponding to aminoacid residues 328-435. The product was cut with BamHI and Bgll andcloned in the BamHI site of pQE12. pQE12 constructs were transformedin E. coli Sure cells. Recombinant proteins were induced and purifiedessentially as recommended by the manufacturer. (His)6-tagged proteinswere purified under denaturing conditions (6 M guanidinium chloride, 0.1M sodium phosphate, 0.01 M Tris pH 8.0) on a nickel-chelate affinitycolumn. Proteins were eluted in 8 M urea, 0.1 M sodium phosphate, 0.01M Tris pH 4.5. To renature proteins the eluate was dialysed against severalchanges of buffer C (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3). Proteins werecoupled to CnBr-activated Sepharose 4B beads according to the manufacturer(Pharmacia). T(l - 157)His6 protein or T(328-435)His6 protein coupledto beads (2 ml), was incubated with 12 ml of antiserum derived from therabbit immunized with GST-T(1-123) or GST-T(327-420) fusionprotein, respectively. Incubation was carried out overnight in 15 ml falcontubes on a rocking platform in the coldroom. The beads were washed with5 x 15 ml of PBST [0.1% Tween in phosphate buffered saline (PBS)] andfilled in a column. 10 ml PBST containing 0.9 M NaCl were applied tothe column to wash low-affinity antibodies off before high affinity antibodieswere eluted with 0.1 M glycine pH 2.5. Eluates were immediately neutralizedby addition of 1/10 volume of 1 M Tris pH 8.0. Peak fractions (OD280)were dialysed against PBS, glycerol was added to final 10% and aliquotsstored at -70'C. Antisera against the N- or C-terminal domain of the Tprotein were designated a-TNI-123 or a-TC328-420, respectively.

Either antiserum was employed in the immunoprecipitation step of thebinding site selection protocol (3 Al) and in antibody shift analyses inelectrophoretic mobility shift assays (0.2-2 Al).In vitro transcriptionPlasmids for the synthesis of mRNA encoding full-length (pBP4.T) orterminally deleted T protein were constructed by amplification of therespective coding region contained in the cDNA pme75 by polymerase chainreaction using primers allowing cloning as NcoI-EcoRI fragments into thevector pBP4 (a gift from D.Stein), a derivative of pSP64T (Krieg and Melton,1984). It contains the Xenopus globin leader sequence downstream of theSP6 promotor which ensures a high translation efficiency. Plasmids for thesynthesis of mRNA encoding the Xenopus and zebrafish T protein(pBP4.XeT and pBP4.Zff) were constructed correspondingly employingthe cDNAs xt6 (Smith et al., 1991) and pBSCT-ZFc1 (Schulte-Merker et al.,1992) as templates for amplification. For the plasmid encoding a fusionprotein of Schistosoma japonicum glutathione-S-transferase (Smith andJohnson, 1988) and T (pBP4.GST-T) the coding region of GST wasamplified from the plasmid pGEX3X using the oligonucleotides AK083(5'-AGCTCCATGGCCCCTATACTAGGTTATTGG-3') and AK084(5'-AGCTCCATGGGGATCCCACGATCCTTC-3') as primers. The PCRproduct was digested with NcoI and cloned into the NcoI site of pBP4.Tand the orientation of the insert was checked by restriction digests usinginternal sites. The expression of fusions of GST with other proteins in vitroshould be applicable to most other proteins and might be particularly usefulin those cases where full-length foreign proteins cannot be expressed inbacteria. Plasmids were linearized with EcoRI, and mRNA was synthesizedusing SP6 RNA polymerase.

In vitro translationIn vitro transcribed mRNA was translated in a rabbit reticulocyte lysateaccording to the supplier (Amersham). Separation of translation reactionscontaining [35S]methionine by SDS-PAGE (Lammli, 1970) andautoradiography confirmed that proteins of the expected size were produced.For testing whether T binds as a monomer or dimer, T and GST -T proteinswere cotranslated in rabbit reticulocyte lysate containing [35S]methionineand an aliquot was incubated with glutathione-sepharose beads (Pharmacia)in BBO (see below) for 1 hat RT with or without BS.p (3 tg). Beads werewashed three times with 500 Al BBO and proteins bound to the beads werereleased by boiling in Lammli buffer. Proteins were separated on a 10%SDS-polyacrylamide gel. For comparison the in vitro translation reactionswere coelectrophoresed. Gels were dried unfixed on Whatman 3MM paperand exposed on Kodak X-AR5 for autoradiography.

Electrophoretic mobilty shift assayBinding reactions contained binding buffer BBE or the optimized bufferBBO (25 mM HEPES pH 7.4, 10% glycerol, 75 mM NaCl, 0.25 mMEDTA, 10 ,zg/ml bovine serum albumin, 1 mM DTT, 0.1% Nonidet P-40,mM MgC12, 1 mM PMSF, 1 /tM leupeptin, 1 ,tM pepstatin), 10 000

c.p.m. of labelled DNA fragments, 1 11 of the in vitro translation reaction,1 Ag of the non-specific competitor poly(dI-dC).poly(dI-dC) in a final volume

of 10 /d. The reaction was preincubated for 10 min at RT before the labelledoligonucleotide was added. Complexes were allowed to form for 20 minat RT. For competition analysis increasing amounts of unlabelled DNAfragments were included in the binding reaction. For antibody shift analysis0.2-2 Al of the N- or C-terminal polypeptide specific antiserum were addedand the samples incubated for an additional 15 min. The samples were loadedon 4%-TAE polyacrylamide (40:1) gels prerun at 4°C for I h and complexeswere separated at 10 V/cm at 4'C. Gels were dried unfixed on Whatman3MM and exposed for autoradiography.

DNase I footprinting analysis, DNA methylation and missingcontact interference analysesFor DNase I footprinting analysis the binding reaction was performed in80 ,ud BBO containing 4 yg non-specific competitor poly(dI-dC).poly(dI-dC), 8 Al in vitro translation reaction with full-length T protein and 20 000c.p.m. of end-labelled probe. The control reaction contained unprogrammedreticulocyte lysate. Binding reactions were incubated for 30 min at RT before20 Al Ca,Mg-buffer (10 mM MgCl2, 10 mM CaCl2 in BBO) were addedand the reaction placed on ice. After addition of 5 14 DNase I (0.1 mg/ml,Pharmacia) and incubation for 1 min on ice the digestion was terminatedby mixing with 100 til DNase I stop-buffer (1% SDS, 20mM EDTA, 200mM KCl), phenol/chloroform and chloroform extraction. DNA fragmentswere precipitated in the presence of tRNA (10 ug) and dissolved in a suitablevolume of loading buffer. DNA methylation and missing contact interferenceanalyses were performed essentially as described by Ausubel et al. (1989).Briefly, 10 A1 in vitro translation reaction with full-length T protein wereincubated with 105 c.p.m. of premodified probe [N-7 methylation withdimethylsulfate, depurination with formic acid and depyrimidination withhydrazine (Maxam and Gilbert, 1980)] in the presence of 5 tg poly(dI-dC).poly(dI-dC) in a final volume of 100 Al BBO for 30 min at RT. Afterelectrophoresis the wet gel was exposed for autoradiography and the partsof the gel harbouring free and bound probe were excised. Gel slices weretransferred into a 1 % TAE agarose gel and DNA fragments wereelectrophoretically transferred on to DEAE-cellulose paper (NA45,Schleicher & Schiill). The DNA fragments were recovered from the DEAEpaper as recommended by the manufacturer and precipitated. DNA cleavagereactions at modified bases were carried out by incubation in 10% piperidinefor 30 min at 90'C. Chemical cleavage reaction and DNase I digestionproducts were separated on a 10% polyacrylamide (19:1)-8 M ureasequencing gel. Approximately the same activities of the bound and thefree fraction products were loaded. The autoradiographs of the separatedDNA fragments were judged by optical inspection for differences in bandintensities between the free and bound fraction. A sequence ladder forreference was generated by chemical sequencing of A +G and G residues.The DNA fragment used in these assays was derived from pBS.p. Theplasmid was cut with XhoI and the recessive end filled with [a-32P]dCTPand cold dGTP, dATP and dTTP using the Klenow fragment of E.coli DNApolymerase. The 117 bp fragment released by digestion with SacI was gel-purified and isolated using the Mermaid-Kit (Bio 101).

Sequence analysis of selected sitesDNA fragments eluted after four rounds of binding site selection of eachexperiment (selection using a-TNI - 123 or a-TC328_420) were amplified,digested with BamHl and EcoRI and ligated in pBluescript KS (Stratagene).Ligation products were transformed in Ecoli JM1O1. Plasmid DNA ofindividual transformants was prepared and the inserts were sequenced usingT7 DNA Polymerase according to the supplier (Pharmacia).

AcknowledgementsWe thank U.Schwarz for support and critical comments on the manuscript,R.Schuster for technical help, D.Stein for the plasmid pBP4, S.Schulte-Merker for the cDNA of the zebrafish T gene and for antiserum againstits protein product, and the staff of the Photographic Department forphotographs. This work was supported by the Deutsche Forschungsgemein-schaft.

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Received on January 15, 1993; revised on May 3, 1993

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The Brachyury gene encodes a novel DNA binding protein

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