Development 112, 351-364 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

351

Gene regulatory factors of the sea urchin embryo

II. Two dissimilar proteins, P3A1 and P3A2, bind to the same target sites that are

required for early territorial gene expression

CHRISTER HOOG*, FRANK J. CALZONEf, ANN E. CUTTING, ROY J. BRITTEN

and ERIC H. DAVIDSON

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

'Present address Department of Molecular Genetics, Karohnska Instituted S-10401, Stockholm, Swedent Present address Department of Developmental and Cell Biology, University of California, Irvine, CA 92717, USA

Summary

Previous work demonstrated that a negative regulatoryinteraction mediated by factor(s) termed 'P3A' isrequired for correct territory-specific gene expression inthe sea urchin embryo. A probe derived from a P3Atarget site in the skeletogenic SM50 gene of Strongylocen-trotus purpuratus was used to isolate a cDNA clonecoding for a factor that binds specifically to this site. Thisfactor, called P3A1, contains two sequence elements thatbelong to the Zn finger class of DNA-binding motifs, andin these regions is most closely similar to the Drosophilahunchback factor. The P3A1 factor also binds to asimilar target sequence in a second gene, Cyllla,expressed in embryonic aboral ectoderm. Another seaurchin embryo protein factor, P3A2, has been isolatedby affinity chromatography and cloned, as described in

Calzone et al. Development 112, 335-350 (1991). P3A2footprints the same target sites in the SM50 and Cylllagenes as does P3A1, but lacks the Zn finger sequencemotifs and in amino acid sequence is almost entirelydissimilar to P3A1. A deletion analysis of P3A2delimited the DNA-binding region, revealing that fivespecific amino acids in the first P3A1 finger region andfour in the second P3A1 finger region are also present inequivalent positions in P3A2. The P3A1 and P3A2factors could function as regulatory antagonists, havingevolved similar target specificities from dissimilar DNA-binding domains.

Key words: DNA-binding protein, embryogenesis

Introduction

As described in the preceding paper (Calzone et al.1991), DNA-protein interactions at cw-regulatorytarget sites called the P3A sites are required for spatialregulation of the Cyllla actin gene. These interactionscause repression of the Cyllla gene in territories otherthan the aboral ectoderm, to which Cyllla expression isnormally confined (Hough-Evans et al. 1990). Calzoneet al. (1991) purified by affinity chromatography aregulatory factor, P3A2, which binds tightly andspecifically to the P3A target sites. In this paper, wedescribe a second DNA-binding factor, P3A1, whichrecognizes the same target sites. P3A1 was isolated bydirect ligand screening of a cDNA library made fromcleavage stage embryo mRNA. On cloning andsequencing P3A1, we discovered, unexpectedly, thatthe early embryo in fact contains two very differentDNA-binding proteins with high target specificity forthe same P3A target site sequences. P3A1 is of the

finger protein family, most closely related to theDrosophila Zn finger factor that is the product of thehunchback gene. P3A2 bears almost no resemblance toP3A1 in sequence, except for two short, adjacentsequence motifs that lie within the DNA-bindingdomain of P3A2 and also appear within the fingerregions of P3A1. Though the P3A2 sequence does notpossess the canonical features of known classes of fingerproteins, these similarities in sequence may indicate thelocus of its target site specificity.

There are several curious and interesting features ofthe P3A regulatory system. First, it appears that whollydissimilar proteins have evolved to recognize the sameregulatory target sequences. This in turn implies afunctional significance, e.g. competitive interaction ofP3A1 and P3A2 at these sites. In addition, the P3Afactors must interact with other regulatory elements toaccount for the fact that they are required forspecification of gene expression in nonoverlapping andfunctionally distinct lineage elements of the early

352 C. Hbog and others

embryo. That is, in the aboral ectoderm the Cyllla geneis on and the SM50 gene is off, while the converse is truein skeletogenic mesenchyme, and yet both genes mayrequire P3A interactions to prevent ectopic expression.

Materials and methods

Construction of cDNA librariesTotal RNA was isolated from unfertilized eggs or embryos, asdescnbed by Posakony et al. (1983) and Lee et al. (1986).Poly(A)+ RNA was partially purified from the total RNA, asdescribed by Posakony et al. (1983). 5/ig of poly(A)+ RNAprepared from embryos of different stages were used tosynthesize cDNA. The BRL cDNA synthesis kjt was usedwith oligo(dT) primers. The cDNA was prepared for cloningas descnbed by Watson and Jackson (1985) and ligated intoAgtll (Promega Biotec) which had been digested with £coRIand dephosphorylated, or into AZAP DNA (Stratagene)Ligated DNA was packaged in vitro using a commercialextract ('Gigapack Gold', Stratagene). The cDNA librarymade in Agtll from poly(A)+ RNA prepared from 4h(3rd-4th cleavage) embryos contained 5.2xlO6 primaryplaques, and was not amplified before use (average insert sizewas 2 3 kb). The second cDNA library was constructed inAZAP from poly(A)+ RNA prepared from 14 h (9th cleavage)embryos. This library contained 8 7X105 primary plaques andwas amplified to a titer of 1x10" pfuml"1.

Isolation of P3A1 cDNA clonesAn aliquot of the 4 h embryo Agtll cDNA library was probedby the direct ligand filter detection method of Vinson et al(1987) 1 3xl(T plaques were screened at a density of 2X104

plaques per filter, after lytic infection of E. coh strain Y1090r~(Young and Davis, 1983) The probes used in screening thefilters were prepared by nick translation (Rigby et al 1987) ofsize-selected, double-stranded concatenated ohgonucleotides25/26 (see Calzone et al. 1991 for sequence) using a^32P-dATPand a^P-dCTP (SOOCimmoF1), according to Vinson et al.(1988) Typical labeling reactions yielded probe with a specificactivity of about SxlC^ctsmin"1 ng~l In the initial screen,the filters were exposed to 2xlOVtsmin~'ml"1 of radio-labeled P3A probe DNA. 45 positive clones were identifiedand one A38 was found to bind the P3A DNA probe in thefollowing rescreens. Lambda 38 phage DNA was purified inlarge scale by standard methods A 1.5 kb cDNA insert wassubcloned into the EcoRl site of pBluescript SK~ (Strata-gene) creating clone p38. A fragment of p38 was nicktranslated and used to screen 6X105 ZAP plaques from the14 h library. 112 positives were found, 50 of which were plaquepurified and converted into plasmids according to themanufacturer's protocol (Stratagene). The longest clone, p43,was isolated for further analysis. An oligonucleotide wassynthesized complementary to the 5' region of p43 (5'-AATGCAGAAACATCTCCAGGGTCATATAGATCGAG,position 127-161 in Fig. 4) and used to rescreen the sameZAP library. The longest clone isolated in this screen, p26,was selected. Another clone, pl3, extended furthest in the 5'direction, and was used to obtain the 5' terminal sequence asindicated in Fig 4.

The mRNA coding for the P3A1 factor was later shown bysingle strand probe excess titration to belong to the rare classof maternal and early embryo mRNAs (Cutting et al. 1990).At 4h postfertilization (the stage from which Agtll cDNAlibrary was made), there are only about 600 copies of thismRNA per embryo Since there is about 30 pg of total mRNA

in the egg, of average length ~2.5 kb (Davidson, 1986, p.71),P3A1 message constitutes only 3xlO~ of the mRNA, and weshould have expected on the average 37 P3A1 clones to havebeen present in the 1.3 x 106 clones initially screened. Of theseabout six should have been in the correct orientation andreading frame. That we found only one such clone could bedue either to failure of many clones to include the 5' half ofthe mRNA, which codes for the necesssary DNA-bindingregions of the protein (see below); to insufficiently vigorousprotein expression, or to a bias against cloning of thisparticular sequence. However, screening by DNA hybridiz-ation of other various embryo A libraries with P3A1 DNAprobes demonstrate that if anything this sequence is slightlyover-represented. A subsequent direct comparison with aclone encoding a different DNA-binding protein isolatedfrom the same library, of the number of clones that testedpositive by direct DNA ligand screening with those positive bynucleic acid homology, yielded a ratio of 1:46 Taking intoaccount the requirement for correct orientation and readingframe, the ratio of clones expected to be recovered by directDNA ligand screening to clones actually detected was 7.7. Wewere thus fortunate to have recovered the single initial P3A1isolate. The moral of this excursion is that for this method tobe routinely useful in cases where the factor is encoded by anmRNA of such low prevalence, very large numbers of cDNAclones must be screened.

The P3A1 factor is present in the nuclear extracts fromwhich P3A2 was isolated by affinity chromatography,according to reaction of the anti-P3Al antibody. However,P3A1 did not bind to this column, perhaps because in thenuclear extracts the P3A1 binding site is occluded by anotherprotein bound to it. Thus it was fortuitous that we appliedboth direct ligand screening and affinity chromatography tothe problem of isolating the P3A factor(s)

Sequence of P3A1 cDNA clonesClones p38, p43, p26, pl3 and some additional overlappingcDNA clones were sequenced on both strands using subclonesgenerated by an exonucleaselll/Sl kit (Promega Biotec),using XL1 blue cells as host (Henikoff, 1984). Clones withdeletions of the appropriate size were identified by agarose gelelectrophoresis and used for dideoxy sequencing (Sanger et al.1977) using Sequenase (USB) after alkali denaturation of theDNA (Mierendorf and Pfeffer, 1987). Gaps or ambiguities inthe DNA sequence were resolved by using internal primers.The sequence data were compiled using the IBI program. The5' end of the insert in the original isolate, A38, was sequencedusing a Agtll primer (New England Biolabs).

Preparation of bacterial extract containing P3A1proteinAn Xhol-BamHl fragment was released from p43 (position52-2473) and end filled. A BamHl linker was used to createthe correct frame for ligation into the expression vectorpET3c (Studier and Moffat, 1986) generating clone pET3c-P3A1. A second fragment released by digestion with Hindi(position 332-846) was ligated in a similar way into pET3agenerating clone pET3a-P3Al. BL21(DE3)pLysS cells wereinfected with these constructs or, with pET3c vector alone,grown to mid-log phase, induced with IPTG for 2h andharvested. For electrophoretic sizing and subsequent ligandgel blots, cells were lysed in SDS sample buffer and urea wasadded to a final concentration of 4M. The samples were thenheated for 5min at 90 °C and the solubilized protein wasloaded onto SDS-polyacrylamide gels (Laemmh, 1970). ForDNAase I footprint and gel shift analyses, the cells wereharvested and resuspended in buffer C (20 mM Hepes

Gene regulatory finger protein 353

[pH.7.4], 40mM KC1, 0.1 HIM EDTA, lmM DTT 20%glycerol) with the addition of lysozyme [lmgmF1]). Cellswere incubated on ice for 1 h and then briefly sonicated. Theinsoluble material was spun out and the supernatant wasstored at -70°C For DNAase I footprinting and gelretardation experiments bacterial extracts of P3A1 expressionclones were prepared as described by Calzone et al. (1991).The recombinant protein produced by pET3c-P3Al waspurified by affinity chromatogTaphy using a concatenatedDNA-binding site derived from the Cyllla gene (ohgonucleo-tides 11/12 of Th6z6 et al 1990).

Ligand-specific protein gel blotsBacterial proteins produced by the pET expression constructwere resolved on SDS-polyacrylamide gels and electrophor-etically transferred to nitrocellulose paper, using standardblotting conditions. The filters were then probed according tothe protocol of Miskimins et al (1985) using the same buffersystem as in the ligand clone screening method of Vinson et al(1988). The four double-stranded DNA probes used rep-resented binding sites found in the upstream region of theCyllla gene (Th6z6 et al 1990) and were constructed fromcomplementary ohgonucleotides with offset termini to gener-ate 'sticky ends' that would allow concatenation. Thesequences of some of these probes are given in Table 1 ofTh6z6 et al. (1990). Those utilized in this study wereohgonucleotides 23/24 (the PI site); 5/6 (the P6 site); 19/20(the P7I site), all given by Th6z6 et al. (1990) Ohgonucleo-tides 25/26 are given in the accompanying paper (Calzone etal 1991), and the relevant native P3A site of the SM50 gene isshown in Fig. 3C of this paper (see Table 2 of accompanyingpaper for other P3A target sites in these genes)

Gel shift reactions and DNAase I footprintsThe quantitative gel shift reactions used for estimation of K^,were carried out as described by Calzone et al. (1988) and inlegend to Fig. 2. DNAase I footprints were carried out bystandard methods as described in detail by Calzone et al.(1991).

Locating the DNA-binding region of P3A2A Bluescnpt cDNA clone containing the complete codingsequence of the P3A2 protein (Calzone et al. 1991) was usedas the starting material. The 3' series of deletions wasgenerated from a slightly shortened version of this clone, inwhich 30 nt were removed from the 5' end by exonucleasedigestion (to eliminate a foldback that caused premature stopsin transcription). The shortened clone, pP3A2A, was linear-ized by digestion with different restriction enzymes, at thefollowing positions in the sequence (Calzone et al. 1991):

P3A2A 1 1335 Espl2 1070 Sphl3 1014 Hpal4 854 Stul5 745 Xmnl6 695 Ncol1 623 Seal8 571 Hindi9 480 Styl

To construct 5' deletions PCR was used to generate copies ofselected regions of the P3A2 coding sequence (Sarkar andSommer, 1989). The template was a Hindlll-Htndlllfragment from the P3A2 clone [position 1-2883 in the P3A2DNA sequence (Calzone et al. 1991)]. The ohgonucleotidesthat were used contained, in addition to a sequencecomplementary to the desired locations in the P3A2 cDNA, a23-base T7 promoter sequence followed by a 9-base trans-

lation initiation signal that fused a methionine in frame withthe P3A2 protein sequence. These oligonucleotide primerswere as follows (the digits in the name for each oligonucleo-tide indicate the start of the region of the cDNA clone towhich the oligonucleotide is complementary):561 5'-TAATAO^CTCACTATAC«WCACCGCCATCGCTACATCGGTCAACAAGATG

505 5'-TAATA«^CTCACTATACKX^GAC«KXATGCCGTCTAATGAGAATTCAGAG

4 5 3

351 5'-TAATAC<^CTCACTATAGGGAGACC«X^TGCGGCAGCAA<XNTRRCGTTCTA

153 5'-TAATACX^CTT:ACTATAGGGACACCGCCATGCCAAGTATGACGGATGATGTC

1 1 6 3 5'-CAATGTGGTTACTGTTTGCCC (includes no T7 promoter sequence)

PCR was performed with oligonucleotide 1163 (3'-end), andone of five additional ohgonucleotides (5'-end) using aPerkin-Elmer 'Gene-Amp' kit according to the manufac-turer's recommendation in a 100pi\ reaction. After lO min at94 °C, 35 cycles of PCR were performed with the Perkin-Elmer Cetus automated thermal cycler. 1 jA (—0.5 fig) of theamplified DNA were added to 43 jA of RNA transcriptionmixture and was transcribed using T7 RNA polymerase(Stratagene)

The linear DNA obtained from the PCR reactions was usedas template for T7 RNA polymerase to transcribe uncappedRNA suitable for in vitro translation (Promega Biotec). TheRNA templates (about 2fig) were added to a rabbitreticulocyte lysate (Promega Biotec) containing [35S]methio-nine according to the manufacturer's specifications, and theprotein products were confirmed by analysis of the reactionproducts on SDS-polyacrylamide gels.

Results

P3A1 cDNA clonesTarget binding sites for the P3A factors in the SM50 andCyllla genes show a core sequence element, C / T XC / T G C G C A / T (Calzone et al. 1988, and precedingpaper; Th6z6 et al. 1990; Thiebaud et al. 1990). TheP3A1 factor described herein was isolated by screeningan expression cDNA clone library with concatenatedoligonucleotides, each monomer of which contains twoof these core sequence elements. This probe representsa naturally occurring, high affinity double P3A sitelocated at position -128 to -102 of the SM50 gene. Theexact sequence of this site is shown in Table 2 of theaccompanying paper of Calzone et al. (1991). Oligonuc-leotides 25/26, which were used to produce the probe,consist of precisely this sequence with a few additionalflanking nucleotides from the SM50 gene, as also givenin the accompanying paper. The concatenated probewas labeled and used to screen a set of early embryocDNA libraries by the method of Vinson et al. (1988).The initial P3A1 isolate was obtained from a Agtlllibrary containing inserts of 4h embryo cDNA. Onereproducibly positive isolate, called A38, was obtainedfrom 1.3x10 plaques produced by a portion of theunamplified packaging mixture. After purification theinsert of this recombinant, which was about 1.5 kb inlength, was converted into a subclone (p38) and afragment of p38 was used to screen a second cDNAlibrary, produced from 14 h embryo cDNA, andconstructed in AZAP. The longest of the positive clonescontained a 2.6 kb insert (p43), and this clone was usedfor much of the following study.

354 C. Hdog and others

Interactions in vitro between P3A1 protein and DNAtarget sitesAn insert from clone p43 containing sequence codingfor amino acids 9-387 (the -COOH terminus) wastransferred to the pET3c expression vector (Studier andMoffat, 1986). This clone is denoted pET3c-P3Al.After IPTG induction the proteins were extracted (seeMaterials and methods, and legend to Fig. 1), andloaded onto an SDS-polyacrylamide gel. Followingelectrophoresis they were transferred to nitrocelluloseand reacted with the probe containing the P3A site, andwith three other probes that bind tightly to other seaurchin embryo nuclear factors involved in the regu-lation of the Cyllla gene, viz the PI, P6 and P7I sites(Calzone et al. 1988; Th6z6 et al. 1990). Fig. 1 showsthat only the P3A probe is bound by the bacterialproducts of the expression clone pET3c-P3Al. Thisresult is dependent on IPTG induction, and is notobtained with extracts from bacteria containing thevector alone (data not shown). The apparent relativemolecular mass of the active fusion protein is about60X103 (in this vector the fusion product includes only12 amino acids encoded by the host sequence).

The fusion protein produced by the pET3c-P3Alexpression construct was recovered and used forquantitative gel shift reactions in order to measure itsrelative affinity for the SM50 and Cyllla target sites.The protein product of pET3c-P3Al was purified usinga site-specific DNA affinity column (Calzone et al.1991). We utilized quantitative gel shift competitionseries (cf. Calzone et al. 1988) to determine values forthe equilibrium dissociation constant (KD) by least

P3A P1 P6

x10-3

200 -

92 -

69 -

46 -

30 —

squares analysis. Representative gel shift competitionsare shown in Fig. 2A, for reaction of the recombinantP3A1 protein with the Cyllla target sequence; and inFig. 2B for its reaction with the SM50 target sequence.Beneath each panel the data are plotted in a form whichallows KD to be calculated directly from the slope(which, as given in the legend, is —ATp"1). Fig. 2 showsthat the bacterial protein produced by pET3c-P3Alindeed reacts specifically with both the SM50 P3A andthe Cyllla P3A sites. The /CD values obtained in theseparticular reactions are about the same, i.e. 6.Ox 10~8 Mfor the Cyllla target fragment and 3.5X10~8M for theSM50 target fragment. These values are not signifi-cantly different, and experiments in which the SM50oligonucleotide competitor was used with the Cylllaprobe, show that the recombinant P3A1 proteinpartitions equally between these target sites. It is usefulto compare these values with the />CD measurementsobtained in similar experiments using the P3A2 factor,and presented in the accompanying paper of Calzone etal. In contrast to the equivalent binding affinities for theSM50 and Cyllla sites displayed by P3A1, P3A2 bindsto the SM50 probe about 50-fold better than it does to aprobe containing a single Cyllla site, though it binds tothe Cyllla site with about the same affinity as doesP3A1. Thus the apparent KD values extracted from theexperiments of Calzone et al. were KD=7Ax 10~8 M forthe reaction of P3A2 with the Cyllla site, butATD=1.7X10~9M for its reaction with the SM50 probe.However, there are two reasons why interpretation ofthis comparison in itself is not straightforward, (i) TheCyllla fragment used for the gel shifts as probe contains

Fig. 1. Ligand gel blot of fusionpj] proteins generated from pET3c-

P3A1 expression clone. Thisclone lacks only the initial 12

' amino acids of the complete. • open reading frame (see

1 Materials and methods). Afterinduction with IPTG for 2 h the

^ cells were collected andresuspended in SDS-lysis bufferwith 4 M urea An identicalamount of protein was loaded infour separate wells of anSDS-PAGE gel. Followingelectrophoresis the proteins were

, transferred electrophoretically toa nitrocellulose filter, which wascut into four strips. Each strip

I was reacted with a uniquedouble-stranded concatenatedDNA probe; P3A (A), PI (B),P6 (C), and P7I (D) (for probessee Materials and methods).Molecular weight standards(Amersham) are shown on theleft. Minor bands represennngnonspecific DNA-binding

^ ^ proteins can be observed" between 30-40 xlO3 and at

>90xl03. Two probableproteolytic P3A2 derivatives

• ' migrate at -38X103.

(A) (B)Gene regulatory finger protein 355

1 2 3 4 5 6 7

1 2 3 4 5 6 7

CO

O

16

12

a 8

a

8 r

O

C

S

PDs(x101u)

Fig. 2. Specific gel shift reactions mediated byrecombinant P3A1 protein. The fusion protein encodedby pET3c-P3Al was recovered and purified as describedin Materials and methods, and utilized for gel shiftcompetition series with (A) Cyllla and (B) SM50 targetsites as probes. In A, the probe was the 86 nt Zfragment of the Cyllla regulatory domain, whichcontains a single P3A target site (see map in Th6z6 etal. 1990), and the competitor was constructed byconcatenating the 25 nt complementary ohgonucleotides11/12 (see Materials and methods; the sequenceincluded in ohgonucleotides 11/12 is indicated in Fig. 4Cof this paper). Lanes 2-7 contain increasing amounts ofspecific competitor. Probe was present at 2.5X10~LOM.The significance of the two complexes detected whenP3A1 was reacted with the Cyllla Z probe is unknownand, as seen in B, only a single complex was observedwhen the probe contained the tandem P3A target sitesThe graphs in the lower portion of the figure showdeterminations of the equilibrium dissociation constant

PDs(x10a)

(KD) from this reaction, determined by least squares analysisusing the function

where PDj/D, is the ratio of complex to free specific probe(measured from the gel shift reaction), and Po is the concentrationof active factor in the reaction KD was 6.0X1CT8M for thisreaction, Po was about 9X10~10M, and poly(dI)-poly(dC) waspresent at about 8.0X10~4M. In B, the SM50 target sequenceprobe was a monomenc ohgonucleotide containing the tandemP3A sites, and the competitor was concatenated ohgonucleotide25/26. Lane assignments are as in A. The KD value extractedfrom this experiment was 3 5X1CT8M, PO

w a s about 4.1X10~9Mand poly(dI)-poly(dC) was present at 8.0X1(T5M. Probe waspresent at about 2.0X10~9M. KD for the nonspecific reaction ofP3A1 with poly(dI)poly(dC) should be about 1 8X10"3M (cf.Calzone et al 1988). Thus, in A -40% of the protein isexpected to be bound to the nonspecific DNA, and in B lessthan 5 % of the protein was in nonspecific complex.

a single target site while the SM50 probe fragmentcontains two nearby but nonidentical sites (see below),and (ii) the P3A1 protein was a recombinant product ofa bacterial expression system that was affinity purified,while the P3A2 protein utilized for these studies was

purified directly by affinity chromatography from seaurchin embryo nuclear extract, and this factor is subjectto modifications that may affect its DNA-bindingactivity. Nevertheless, the low molar values for Ko

measured in Fig. 2 of this paper, which fall well within

356 C. Hddg and others

the range reported for known DNA-binding regulatoryproteins, demonstrate that P3A1 is indeed a specificDNA-binding protein that recognizes the P3A targetsites of both SM50 and Cyllla genes. Furthermore,although proof of the point must await direct purifi-cation of the native P3A1 protein, the comparison withP3A2 raises the possibility that P3A1 does notdiscriminate between the Cyllla and SM50 sites, whileP3A2 does. The same distinction obtains in the case ofclosely spaced P3A sites in a third gene, Sped. Hereagain P3A2 binds these sites relatively tightly, but P3A1responds to them the same as it does to the singleCyllla site utilized for the experiment of Fig. 2A (datanot shown). Were this difference to obtain in vivo.differential functional significance of the two factorswould be implied, with respect to interactions with theCyllla, SM50 and other genes that bear regulatory P3Atarget sites.

The footprints and hypersensitive sites shown inFig. 3A,B demonstrate explicitly the sequences en-gaged by the DNA-binding domains of the P3A1 factorin the SM50 and Cyllla target sites. For theseexperiments, a second P3A1 expression clone was used(pET3a-P3AlD), which contains a shorter insert codingfor amino acids 104-272. The protein encoded by thisconstruct includes the complete DNA-binding domainof the P3A1 protein. The truncated pET3a-P3AlAprotein was employed for the experiments of Fig. 3because its accumulation in the bacterial expressionsystem and stability in extracts greatly exceeds that ofthe nearly complete protein encoded by pET3c-P3Al.

In Fig. 3C the target regulatory sequences of theCyllla and SM50 genes that are protected by P3A1 arecompared with those protected by P3A2. P3A2footprints are shown in Calzone et al. (1991) and in B ofFig. 3. Both proteins clearly protect the core T/cXT/CGCGCA/T elements of these sites, which aremarked by boxes in Fig. 3C (their relative orientationsare indicated by arrows). Methylation interferenceexperiments (not shown) also indicate that bothproteins make contacts with G residues on both strandsof the central GCGC element. These data, the footprintstudies summarized in Fig. 3C and the oligonucleotidecompetition results shown in Fig. 2 confirm that P3A1and P3A2 react with the same DNA target sites. On theCyllla site the DNAase I footprint patterns overlapexactly on the 5'—>3' (top) strand, and within one ortwo nucleotides at each end on the 3'<—5' (bottom)strand. However, the P3A1 protein binds very differ-ently from the P3A2 protein on the double SM50 site.P3A1 reacts strongly with only the proximal site, whereit covers 13 nucleotides just as on the lower strand of theCyllla site, while P3A2 equally protects both sites (thisalmost certainly requires two P3A2 molecules). Athigher concentrations P3A1 weakly interacts with theupstream SM50 site as well. This impressive differencebetween the P3A1 and P3A2 footprints obviously couldaccount for the striking difference in the measured KDvalues for the two proteins. Thus the footprints providea reasonable qualitative interpretation for the obser-vation that P3A2 prefers the SM50 probe about 50

times more than it does the Cyllla probe, while P3A1regards them more or less indiscriminately, and binds toboth with about the same affinity as P3A2 does to theCyllla site. However, the P3A1 protein may respondspecifically to a different feature of the proximal SM50site, v/z that if considered by itself, this site issymmetrical (vertical arrow). Thus the P3A1 factorpositions itself directly over its axis of symmetry, so thatboth termini of the protected region lie in equivalentpositions, within the sequence elements CA/GCCCC.This feature of the SM50 site could be functionallyimportant, since this symmetrical sequence motif, towhich P3A1 is sensitive, is not found in the Cyllla site.

P3A1 is a finger proteinFig. 4 displays the sequence of the P3A1 cDNA and theprotein for which it codes. The actual mRNA probablybegins a short distance upstream of the 5' terminusshown. However, it is unlikely that this mRNA has avery large 5' untranslated leader because, though theclone includes only 25 nucleotides prior to thetranslation initiation site, the size of the sequencedtranscript, 2473 nt, is indistinguishable from the size ofthe transcript revealed by RNA gel blots (Cutting et al.1990) measured by reference to standards as about2500±50nt. Use of the first ATG is supported by theadjacent sequence CAAATCATGG, which at all exceptthe starred positions is identical with the 10 nt consensustranslation start site of Kozak (1987). There follows an1161 nt open reading frame, a stop codon at position1186 in the sequence, and a 1287 nt long 3' untranslatedregion, which in the recovered clones lacks a poly (A)tail, though the mRNA is indeed polyadenylated in theembryo (Cutting et al. 1990). The open reading frame ofthe P3A1 sequence specifies a protein with a relativemolecular mass of 42X103 (387 amino acids), although,as noted above, the recombinant expressed proteinmigrates on SDS-polyacrylamide gels as if its masswere 60X103 (Fig. 1, lane 1). Similar overestimates ofsize have been noted for other transcription factors,including GCN4 (Hope and Struhl, 1986), RAP1(Shore and Nasmyth, 1987) and HSF (Sorger andPelham, 1988). Since in the present case the recombi-nant protein was expressed in bacteria, its anomalousmigration is unlikely to be due to significant post-translational modification, and its cause might besought in its unusual amino acid composition. TheP3A1 protein is predicted to have a high content ofserine, threonine and proline, together ~29%, as alsonoted for some other DNA-binding proteins, e.g. GF1(Tsai etal. 1989), Spl (Kadonaga et al. 1987) and SRF(Norman etal. 1988). The amino acid terminal region ofthe P3A1 protein (positions 1-53) is strongly acidic,with a net charge of —11 and perhaps could function asa transcriptional activator region (reviewed by Mitchelland Tjian, 1989). A short strongly basic region (7/11amino acids) appears after position 237, similar to thenuclear localization signals described in other proteins(Dingwall and Laskey, 1986). No significant similaritiesare detected, comparing the derived P3A1 proteinsequence with those in the available sequence data

Gene regulatory finger protein 357

(A)1 2 3 4 1 2 3 4

(B)

(Hind III) -244-, r-114 (Ddel)(BstNI) -49-i

1 2 3 4 5 6 1 2 3 4 5 6r-200 r-200(HpaII)

-174

(Ddel) -114-

•115

-102

(Hpall)-200" -200-P3A2 P3A1

- 4 9P3A2

-49 (BstNI)P3A1

Coding Noncoding- -244 (Hindlll)

Noncoding Coding

(C)

CyHIa

-197 •170

5-TTGTTTTTAAAAAGAATAAATGA^GCGCAAAAACAAAAATTTTTCTTATTTACT rCGCGTTT

•1741I

i:AAACTTTATTAAGCAAAAAAGCSTTTGAAATAATTCGTTTTTTCC

SM50 •115 102!

S ACGCCCCGTTTTCGGCTrCTGCGCATGCGGGGCAAAAGCCGAAGACGCGT

I-197-184

182

CACCCiTGGG

t

loo

ACGCGCArGGGGCGTGCACGGATGCGCGUCCCCGCACGTGCCT

138

11/12 2S12S

Fig. 3. DNAase I footprint and sequences protected by recombinant P3A1, compared to sequences protected by P3A2.The recombinant P3A1 used in these experiments was the 24X103 fragment produced by the pET3a-P3Al construct (seetext) P3A2 was affinity-purified from nuclear extract as described in the accompanying paper. The sequences of the Cylllaand SM50 regulatory DNA present in each probe are marked in the maps aligned vertically with the reactions. The labeledstrand is noted below each set of reactions. The boxes represent the regions of each probe that were protected fromnuclease attack by P3A1 or P3A2 as indicated The filled in boxes locate the P3A target site noted in the text. (A) Cylllafootprints. The reactions shown in lanes 1 and 4 for each probe did not contain recombinant protein. In lanes 2 and 3, theprobe was incubated with 2[A and 4/il of bactenal extract containing recombinant P3A1 before treatment with DNAase IControl extract generated a DNAase I profile identical to that shown in lanes 1 and 2. Marker lanes are not shown. HSindicates sites which became hypersensitive to DNAase I when probe was incubated with P3A1 (darker bands than in noprotein control) P3A2 footprints obtained with the same probes are shown in the first paper of this series. (B) SM50footprints. Control reactions that did not contain P3A1 or P3A2 are shown in lanes 1 and 6. The footprint obtained withP3A2 is shown in lane 2. The reactions shown in lanes 3, 4, and 5 were incubated with 2, 4, and 6/il of P3A1 bactenalextract before DNAase I treatment. (C) Sequences protected by P3A1 and P3A2 proteins. Only strong footpnnts areshown. Solid brackets represent nucleotides protected by P3A2, dashed lines represent nucleotides protected by P3A1DNA-binding fragment produced by pET3a-P3Al (or by the nearly complete protein produced by pET3c-P3Al; not shownhere). Left, Cyllla target site; nght, SM50 target site. The boxes indicate the canonical core of the P3A target siterecognized by both proteins (Th6z6 et al 1990; Calzone et al 1988), viz 5'-T/cXT/cGCGCA/T. Arrows (5'-»3') indicatethe onentation of this site with respect to the sequences shown (transcnption starts downstream to the right). Thesequences included in oligonucleotides 11/12 and 25/26 referred to in text are indicated The vertical arrow shows a dyadaxis of symmetry in the SM50 site covered by the P3A1 protein.

358 C. Hdog and others

||3 * U * * * * |43n i l T T I T T C CACTCCCTAC AATC ATC CCC CAT CAA CAC AAA CAA CTC CAC ACC CCT CCC ACC CAT CCC TTT TCA CCT CAC AAT CAC CAA AAT CAC CAC TCC ACT 105

M A D E Q K R L D T P C 5 D C F I A Q N Q Q H D D S 5 27

« + * * * * * * * *

ATA CCA AAC CAT CCA CCA CAA AAT CCA CAA ACA TCT CCA CCC TCA TAT ACA TCC ACA CAT CAC CCA ACT CAC ATC CAA CCT TCA ACC CTC CCA TCT ACA CCA 207

I C W D C C E H A E T I P C S Y B 5 B D D C 3 E M E A 5 3 V P 5 T P 61

* * * * # • * * # #

CCA CCA ACC TTT CTC CCT CTA CCA CCA ATT TTA ACA CCT ACC CAA CCC ATT CCC ACC CCC CCC TCA TCA TCC ACT TCA CAC TCC CTA ACA CAA CCA ACC CAT 3 M

P P T F V R L A P I L T P S E A I C T C C S 3 S S S Q 5 V T E C T D 95

tr * * * * * * * * * +

CAA TCA ATA TTC TTT CAA CAC TCA ACT CAA CCT ACC TCT CCT TCC CCC CAT ATC CAT TAC AAC TTT CTC CAT CCC AAA TAC CTT CAC TAC ACA CCT ACC CCA 4 1 1

Q 3 1 L F E Q S T E C T S C S C H 1 D Y K P V D C K Y V Q Y T P T C 129

* * * * * * * * * *

CAA CAC CCC CAT CCA ACC CCA CAC TTC ATC CAT TTC AAC CCA AAC TCA TCC ACT CTC ACC TCA CTA CAT CCC CCC CAC CCC TTC ATA TCT AAA CTC TCT CCT 5 1 3

Q E C D C S P Q L M D P H C K S 3 S V T 3 V D /?//6//p///c//4//////////////// 163

CCT TCC TAT AAC CTT CCC CAC TCC CTC AAC TCC CAC ATA CAC AAC CAA CAT CAC CCC AAC CCA TTC AAC TCC TCC AAC TCT CCC TAC ACC ACA AAC ACA CCC 6 1 5

CAT CAT CTC TAT CCT CAT CAT CCC CAC CTA CAC ACC CCT ACC CCC AAC CAA ACC CTC CCT CTC CTC TCT CCC ATA CCC AAA CTC ACA CCC AAA CCC AAC ACC

CTA CCA CCA CCT CTC AAC AAC CCT CAC CAC ACC ACA ATA CTC AAC AAC CCA CTC ACC ATT ACC TCA CCC ACC TTT ACC CCC CCC ACC ATA ACC CCA CCT ACC 8 1 9

V A A A L K K R D E R I I L K K P L T I S S P 3 F T P A T I S P A S 265

* * * * * * A u * * *

TTC ACT CCT CCC AAT CCC ACA TCC CTC AAC TTC ACC TCT CCC ACA ATT ACC TTA CAA AAC AAC TCC CTC AAC CAA CTC CTC AAT CTA CCA CAA CCT CCT AAC 9 2 1

L S A A H P T S V H L S S P T I S L E H K S V K Q L L H L C E C A H 2 9 9

u * * * * t t * * tr *

TCC CAC CAC TTC CTC CTC CTC ACT CAC CCC ATC CAC CCC CCT CTC TAT TCC CAA CCC CCC TCT CCT CAC CAC AAT CAC AAC ACA TCC CCA TCC CTC CAA CAC 1023

S Q Q L V V V S D C M Q C A V Y S E P P S P Q Q H E H B 3 C U L E E 3 3 3* + * * * * * * * *

AAT TCA ACC AAC AAC ACC ACC ACC CCC ACC AAC CCC CAC ACC CAC ACC CAA TCC TTT CAC CAC CAC CTA CCA ACC CCT CCC ATC TTC ACA CCC ATC ACA CAA 1125

H S S W H S S S C S K P H R . Q T E S F E H Q L P S C A I P K R I T E 367

» * * * * * * * * * *

ATC ACA TTC TTC CCT CAT CCA CCC ATT CCA CAC ACT CCT ACA CAC CAA TAT CAC ATC CCA TCA TT TCATACACAA ATCACCACCT CTTTCACTTA AACTCTACTT 1 2 3 0

I T F F P D C I I R E T R T E E Y E H P 0 387

* * * * * * * *

TACACTTTCC TAAAATCCAC TCTTCTCATC TCACAACTTC TAAATCCCTT TTTCCCAATC TCCCATTTAC TTTAAACCAA AATTCTTCTT CAATCAAATT CAACTTCCCA ATTCATCCTC 1 3 5 0

TATTTCACTA ATCCTACTAT ' CCATCCACAA TCTTCAACAT CCCTCACTAA CTTCAAACAA CATTTCCACA ACATTATCAT CCTTCCAAAT TTACATACTT ATATAAACAA

TCTTCCACAT ACAATCATCT AATTATTTCT TTCCCTCATA ACTCACCATC AACACCTACA TCCATTAATT ACACAAACAT AAAATACTTT CTTTTCAAAC ACCCTTCCCT TCTCCCCACT 1590

* * * * * * * * * * * *

TTCTCAACTC AACTAATTAT TTATAACAAC AACTTTCATA AACTTATTTA TCACACCTCA ATCATCATCA ATAAATTCTA TTTATTTTTC TTTTCTTAAT TCACACTTCT AATTAAATAT 1 7 1 0

* * * * * * * * * * * *

CACTCCACTC TACTTCAAAA TTCCAATACA TCATTTTCCA ATCCTTCCAT TTCATCAAAA TTTCTCAAAA CTTTAACTTA CATCATACCA TATCCCATAT ACTATCTACA CCATACTTCA 1 8 3 0* * * * * * * * * * * *

CTAAACTTCT 1 1 1 1 1 1 1 ICT CTTCAAACAC AACCAACTTC TATATCACAC ATCTTCTTAC CACACTCCCC AAAAACTTCA TCTCCATATT TACACAATCT ACCTTAATTT TCTTCTACAC 1 9 5 0

* * * * * * * * * * * *

AACTTTTACA TAACAACTCC TCAAACAACC TATCCCACCT TCTAATTTAC ACACCACATT ATATTTTTAT ACATCACATC CTCTATAACT ATTACACTCC TTCAATCATC ATTTAAACAA 2 0 7 0

* * * * * * * * * * * *

ACTATCAACA CTTTATACAC AATCAAAATA TTATATATCT CAATTTTCAT CTAAATATAA CTTATTCTAT AATATCCTTA TCCACCTCCA TTA1III1 .I1 CTTATCACAT TCAACCTACA 2 1 9 0

* * * * * * * * * * * *

AAATCATTCA TCTTATACAT CTACCTCCTC TTCACACTTC CATTTAATAA ATATACTTTT TCTTCTCAAT CTAATTTATT CTACATACTC CCTTACCACC TCAACCTACC ATTCACCCCA 2 3 1 0

* * * * * * * * * * * *

AAAACAAAAA CTAACTAAAT CTATCACTTT 111111 .1111 TACATAATCT CTTTATTTCA 11111.11111 TATCCCCATT 11IL. 11 ILIA TACTTTAAAA CCAAAAACCT TTCTATCATA 2 4 3 0

* * * *

TTCTCCCCCC AAACATCCCT TCTATCAAAT TCAAACTCAA TTC 2 4 7 3

Fig. 4. DNA and deduced amino acid sequence for P3A1. The complete P3A1 cDNA sequence was denved fromfragments of cDNA clones as indicated in Materials and methods. 5' endpoints of inserts of cDNA clones p43, p26 and pl3are indicated by arrows The two proposed finger sequence elements are cross hatched The circled asterisk indicates theTGA stop codon. The sequence has been entered in the Genbank/EMBL database, accession no. X57013.

banks, except for two regions that are obviously similarto the Zn finger sequences of many known DNA-binding proteins. These regions are indicated byshading in Fig. 4. They conform to the general form ofZn finger motifs, Phe-X-Cys-X(2_4)-Cys-X3-Phe-X5-Leu-X2-His-X(3_5-)-His [as described by Miller et al.(1985) and Berg (1988)]. We showed in the experiment

of Fig. 3 that a truncated version of the P3A1 proteinincluding only amino acids 104-274 and thus these twofinger sequence elements, is capable of binding the P3Atarget sites specifically.

In Fig. 5 the P3A1 finger region is shown aligned witha number of other finger proteins, all of which areknown directly to serve as DNA-binding regulatory

ADR1

SW15

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H ] E C L F P G C TY S C D H P G C D l

c TFR]A|T |AFRIo EIc N L«J c LFJ T LEJ R o

FKI TIT1 KFR]R YI[KJA[FJV[RJN H

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N I[R[S HCDO©-D[JJI[RJH K[R]5 -

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H i c H ® Q G c G[K1VF]M C T W s Y c U f

C ® E - - c

1 (7]K C D E - - C O[K|M Y S T S M G[L1S[F1H R O F2 H S C E E - - C G K L 1 T T I G A L p H M f f i

3 C K C®X)- - C G KUms[R]P W L LEG H(D(BiS

4 [F]o c ® o - - c PLBJSLEJA o R s K|IJR]A H O Q(D

NGF1-A 1 Y A C ® V E 5 C ol

Spl

Sna

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Hb

P3A-1

H O N K ;

- - H- - H

0T C K ® - - C S [ R | S [ F | G Y K H V[T1Q N H E JR®E C ® E - - C O K f t j F T[R]D H(HJLnrjT H M B I

Y H C S H - - C D|_R[Q F V O V A 1 I R p L |Rj VY T C E ® - - C O G ® l f J S D S N Q I L I K I S H M L V

Y K C K T - - C G V V A I T K V O F W A H T | R ® - - H M K P D IL 0 C ® K - - C P F V T E F K H ® f T | E Y H ( n R K - - H K N 0 K[P]

[ f ] Q C D K - - C S Y T C V N K S M M N S H R K S - - H S 5 V Y 0Y R C A D - - C D Y A T K Y C H S F 0 L H L [fiJK Y G H K P G M VY E C K Y - - C D I F(T |K D A V L Y T I H M G Y - - H S C D D V

|T]K C N M - - C G E ® C 0 G P V G[T)F V H M A R N A H S

0JCKC

K V - - C G A W Y N V R O SfTlK 5 H ® 0 N Q - H 6 [ G 1 K [ R ] -S K - - C P Y S T N 0 R H O U J Y H H D 6 0 V H R[GJT A IC

® [ [ ]D 6 0 V - H R[GJT A IC

Fig. 5. Comparison of selected finger sequences with thoseof P3A1. References for the sequences shown are asfollows: ADR1; Hartshorne et al. 1986; SWI5, Stillman etal. 1988; NGF1-A, Milbrandt, 1987; Spl, Kadonaga et al.1987, snail, Boulay et al 1987; Kruppel, Rosenberg et al.1986; hunchback, Tautz et al. 1987). At two locations,dashes have been introduced to produce the maximumalignments The horizontal space separates the hunchback-P3A1 group of 8 fingers from the remaining 18 fingerelements. The canonical C-C and H-H residues shared bythe known Zn finger structures and with P3A1 are shownin bold face. Residues other than these that occur in givenpositions in 5=9 of the 18 fingers of the top compansongroup are boxed In this companson, arginine (R) andlysine (K) are considered equivalent. Residues that occurcommonly but in less than half of the 18 finger elements inthe top comparison group are circled (arbitranlyconsidered as >5/l8 occurrences). Residues that occur ingiven positions in the P3A1 fingers and also in any of theother finger elements are shaded.

factors, so as to display the subfamilial relationshipsamong the examples included (see legend). Theinvariant C-C and H-H residues (bold face in Fig. 5), aswell as several other residues that frequently (i.e. inover half the examples) also occur in specific locationsin Zn finger domains (boxed residues) are also presentin the P3A1 finger sequences. There is no evidence forP3A1 that Zn2 + or other metals are required for DNAbinding, however. Fig. 5 displays a specially closerelationship between the sequence of the P3A1 fingersand those of the Drosophila hunchback factor. Aminoacids shared (in register) between either of the P3A1finger sequences and any of the other finger sequencesin Fig. 5 are shaded (excluding those that are commonto more than half of all the Zn fingers shown, i.e. theinvariant C and H residues and the boxed residues). Itcan be seen that of the six hunchback fingers two sharesix such residues and two five with the P3A1 fingersequences. One finger sequence of the four snail fingers

Gene regulatory finger protein 359

also shares five such residues, while all other fingers inFig. 5 are less similar. Furthermore, other amino acidsthat are common (circled) in the 18 finger regionsexcluding the hunchback and P3A1 fingers are rarelyseen in the latter two sets of fingers. For example, fiveof the six hunchback fingers lack the aromatictryptophan or phenylalanine residue that occurs fouramino acids beyond the second conserved cysteine andthat is present in all 18 of the other fingers shown inFig. 5. Berg (1988) suggested that this aromatic aminoacid is part of a hydrophobic core important for thefolding of zinc fingers and that this amino acid can bereplaced by another aromatic residue positioned twoamino acids following the second cysteine; indeed 50%of both the hunchback fingers and the P3A1 fingersdisplay tryptophan or phenylalanine at this position,while these amino acids are found in none of the other18 finger structures included in Fig. 5.

Sequence similarities in the DNA-binding domains ofP3A1 and P3A2Standard comparison of the P3A1 and P3A2 sequencesindicated that the proteins are ahnost entirely dissimilar(only about 11% identical amino acids). This resultposes something of a paradox, since as shown in Fig. 3the two factors bind to essentially the same target DNAsequences. To obtain a more focussed insight, weproduced N- and C-terminal deletions of the P3A2protein, and determined their effect on its DNA-binding activity, since the nature of the P3A2 DNA-binding domain was not obvious a priori from anyfeatures of its primary sequence. A cDNA clonecontaining a complete coding sequence, for P3A2(Calzone et al. 1991) was truncated at its 3' end atconveniently located restriction sites, or at its 5' end byoligonucleotide primed PCR mutagenesis, as describedin Materials and methods. The truncated clones werethen transcribed using the vector T7 polymerase startsite, and the RNAs were translated in vitro. The seriesof truncated products resulting from the N-terminal andC-terminal deletions are shown respectively in Fig. 6aland bl. Gel shift reactions with the SM50 target sitewere then carried out with these polypeptides as shownin parts a2 and b2 of Fig. 6. At the N terminus, theP3A2 region required for DNA binding evidentlyincludes a sequence that lies between amino acids 25and 90, since of the N-terminal deletions, only thatterminating at position 25 still recognizes the P3A probe[Fig. 6a2]. Deletions to amino acid 90 and all furtherdeletions completely abolish binding. Truncation fromthe C-terminal end showed that a P3A2 polypeptideincluding residues 1-258 exhibits normal binding [Fig.6b2], while further deletion to position 222 abolishesspecific DNA binding. The DNA-binding domain ofP3A2 is thus <233 and >130 amino acids in length andlies between positions 25-90 and 222-258. Though oflow resolution, this delimitation of the P3A2 DNA-binding region afforded an interesting observation,shown in Fig. 7A. When carefully examined forsequence similarities with P3A1, five amino acids thatlie within or adjacent to the first finger region of P3A1

360 C. Hdog and others

N TER [L

ro CD ro -t». CDcn o co o o

a2 ro co ro -f̂ - cr>cn o co o o

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PCR c o o

] NTER

] CTER

1 2 3 4 5 7 8 9 10 11 12 13 14

fo1 NTER b2 TER

:TER [ -t^ -£»• coc n -«• coCD oo o

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]N

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lift1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Fig. 6. Delimitation of DNA-binding domain of P3A2 using recombmant deletion mutants. 5' and 3' deletions of the P3A2cDNA were constructed as described in Materials and methods, and transcribed in vitro using the vector T7 polymerasesite. These transcripts were then translated in a cell-free reticulocyte system, and labeled with [35S]methionine. In al andbl, the translation products are displayed on an SDS-polyacrylamide gel by autoradiography The respective N termini andC termini of the various products are indicated at the top of each panel In al is shown products of the N-terminal deletionseries. Lane 1 shows the translation products of a clone that extends from position 1 to 418. The remainder of theN-terminal deletions were created from a starting clone truncated at the 3' end at position 360 In bl, illustrating productsof the C-terminal deletion series, lane 1 shows the product of a clone that includes the complete coding sequence, i.e.positions 1—459. In a2 and b2 are shown gel shift assays that demonstrate retention or loss of specific DNA-binding activityof the polypeptides shown respectively in al and bl The probe was the SM50 binding site shown in Fig 3C Lanes 7 in a2and 9 in b2 marked P contain only probe, and the adjacent lanes, marked CR contain probe and reticulocyte extract butno synthetic P3A2 polypeptide. Free probe migrates to the bottom of the gels and the specific complexes, formed in verylarge excess of poly(dI)-poly(dC), migrate more slowly. As expected, their mobility is observed to increase as polypeptidesize decreases

Gene regulatory finger protein 361

(A)

P3A1

G GQ G Fl QGKRFK

(VSWT)(B)

P3A1

P3A2

P3A1

P3A2

P3A1

P3A2

P3A1

P3A2

P3A1

P3A2

P3A1

P3A2

P3A1

P3A2

P3A1

P3A2

10 20 30 40KADEQKELDTPGSDGFSAQNQQNDDSSIGNDGGENAETSP-

MMISEDISEPSSPDTPFDDSDLLNSSMTDDVSAQLAASGPIGVRAAAAIATGKKRKRPHS10 20 30 40 50 60

50 60 70 80 90SEMEASSVPSTPPPTFVRLAPILTP-SEAIGTGGSSSSSQSVTEGTD-

FETNPSIRRRQQTRLIRKLKATIDEYATRVGQQAWLTCTPGKHDGNFKVFGAAPLENIM70 80 90 100 110 120

100 110 120 130 140QSILFEQSTEGTSGSGHMDYKFVDGKY VQYTPTGQEGDGSPQLHDF

RNLKGIVLQDLDNALAQRAPQPSNENSDLYELPPLVIDGIPTSVNKMTQAQLRAFIPLML130 140 150 160 170 180

KYSTGRGKPGWGKESCRPVWWPSDLPWANVRSDVRSEDEKRKVSWTHALVTIVINC-YKHH190 200 210 220 230 240

GRDDLLPEFIEDKCKEIEASQNQVASLPTATLLPSHAWHTINNPDGTVSLIQVDTGATVA250 260 270 280 290 300

260 270 280 290 300SFTPAT ISPASLSAANPTSVNLSSPTISLENKSVKQLLNLGEGA NS

TLADVTQVQQLTNLQTLQQVRLQPLQIQHALGNQQAEATQAVQTLAEVAAAQGGDGELTEG310 320 330 340 350 360

310 320 330 340 350QQLVWSDGMQGAVYSEPPSPQQNENRSGWLEENSSNN SSSGSKPHRQTESFEHQLP

QTVTTLPEGTQLVLASDGSLQAINDGTAQGIVIPASVYQTWAGDGQPIQIANVNIAQQSG370 380 390 400 410 420

360 370 380SGAIFRRITEITFFPDGRIRETRTEEYEHP

GGTTMAAIKNAVHQSQPIPSQVATLWNAASHIX1HT430 440 450

Fig. 7. Comparison of P3A1 and P3A2 sequences. (A) Thefinger regions of P3A1 are shown conventionally aligned astwo Zn-coordinated finger structures (note that we show noevidence for the actual involvement of Zn in this paper).Circled arruno acids occur, in the same registration withinthe DNA-binding domain of P3A2 as well, as shown in BTo obtain the registration shown by the circled amino acidsin the downstream (right) P3A1 finger, four amino acids inthe region between the fingers must be inserted in theP3A1 sequence where indicated by the parenthesis, whichincludes the P3A2 residues at this point. (B) Maximizedalignment of P3A1 and P3A2 sequences (ALIGN program:Myers and Miller, 1988). This alignment yields only about11 % identity, even with free insertion of gaps in theshorter (P3A1) sequence. The alignment shown was chosenby registering the two clusters of identical amino acidresidues that occur in the finger regions of P3A1, as shownin A.

are also found, in registration, within the DNA-bindingregion of P3A2. Furthermore, if a gap of four aminoacids is allowed between the fingers of P3A1, a similarfeature is revealed in the second finger of P3A1, wherethere are four amino acids in the DNA-binding regionof P3A2 that are identical, in register, with respect tothis P3A1 finger. In both fingers, the identical aminoacids begin with the aromatic amino acid following thesecond cysteine. However, as Fig. 7B shows, theoverall sequences of P3A1 and P3A2 show no othersignificant similarities whatsoever, even when thealignment is chosen on the basis of these nine identicalfinger region residues. Since the two factors recognizealmost the same target sequences, these observationsinvite a direct test by mutagenesis of the significance ofthese nine amino acids for the target specificity of theP3A2 protein.

Discussion

The P3A1 target site appears in the regulatory domainsof at least three territory-specific genes that areactivated regionally in the early sea urchin embryo and,in one of these genes, interactions at this site are knownto be required for the correct spatial pattern ofexpression. In this paper, we describe the P3A1 DNA-binding factor, which specifically interacts with thesetarget sites. Unexpectedly, a second factor, P3A2, thatrecognizes the same target sites, has also beendiscovered, as described in the first paper of this series.As we demonstrate in Fig. 7, except for small sequenceelements within the DNA-binding regions of these twoproteins, the P3A1 and P3A2 mRNAs code for whollydissimilar polypeptides. P3A1 displays canonical fea-tures of the Zn-finger class of DNA-binding protein,while P3A2 resembles no known DNA-binding pro-teins, and thus appears to represent a novel class ofregulatory factors (Calzone et al. 1991).

DNA-binding structures of P3A1 and P3A2 factorsP3A1 evidently contains two adjacent finger sequenceelements of the C-C/H-H class, as shown explicitly inFig. 5. This structure was initially recognized in therepetitive DNA-binding domain of the TFIIIA tran-scription factor (Miller et al 1985), in which each of therepeat elements is organized into a finger-like structureby a tetrahedraUy coordinated Zn ion at its base. ManyDNA-binding proteins utilizing this canonical structureare now known, and a variety of structural andmutational evidence demonstrates that the fingerstructure is indeed that which mediates the sequence-specific interactions with DNA target sequences inwhich these proteins engage functionally (e.g. Diakunet al. 1986; Parraga et al. 1988; Nagai et al. 1988;Kadonaga et al. 1987; Berg, 1988; Redemann et al.1988). Thus it is not surprising that a truncated 24X103

version of the P3A1 protein that includes its two fingersequence elements displays the same site speaficity asdoes the complete 43xl(r protein.

An interesting subdivision of the class of finger

362 C. Hoog and others

proteins is shown in Fig. 5, where it can be seen that theP3A1 fingers share a number of sequence features withthe fingers of the hunchback factor and, like thehunchback factor, lack certain residues that areotherwise common amongst DNA-binding Zn fingerprotein sequences. However, the target site for thehunchback factor, G/C

A/CATAAAAAA (Stanojevid etal. 1989) displays no similarity whatsoever with thetarget site for the P3A1 factor, C / T X C / T G C G C A / T .Thus the specific sequences that are responsible fortarget site recognition within the finger region mustinclude amino acids besides those that are sharedspecifically between the hunchback and P3A1 fingers.This result is consistent with the observation that noneof the hunchback fingers displays the sequence of eitherof the P3A1 fingers, including the five and four aminoacids respectively present in both the P3A1 fingers andthe P3A2 DNA-binding domains. Many though not allof these amino acids are located near the tip of theP3A1 fingers, and between the tip and the histidineresidues, i.e. in the region believed to be configuredinto an a'-helix that lies in the major groove of the targetsite DNA, where sequence-specific contacts probablyoccur (Lee et al. 1989; Berg, 1988; Gibson et al. 1988;Brown and Argos, 1986). These local sequences mightindeed contribute to the target site recognition el-ements of P3A2 as well as of P3A1, accounting for theiralmost identical site specificity. Since P3A2 lacks all thestandard features of Zn fingers, it would then followthat there must be alternative ways to organize a three-dimensional structure in which almost the sameDNA-protein contacts are formed, that do not requirethe recognition elements to be mounted on a canonicalC-C/H-H Zn finger. However, it is important to keep inmind that a much larger domain of the P3A2 factor isapparently required for DNA binding than is includedin the two finger structures of P3A1. The deletionexperiments shown in Fig. 6 indicate the minimumnecessary DNA-binding domain of P3A2 to encompass^140 amino acids. Of course this could includesequences required for other functions interspersedamongst the elements that immediately contact theDNA target site. Since no DNA-binding proteinsdisplaying significant sequence similarities to P3A2have been reported, much more information will berequired to develop an image of structure-functionrelationships within this large region of the P3A2 factor.The interesting fact remains that a finger protein and anon-finger protein recognize the same regulatory siteswith nearly the same affinity.

Possible biological significanceSpecification of both the aboral ectoderm and theskeletogenic mesenchyme, the tern tones of the embryoin which the Cyllla and Sped, and the SM50 genes arerespectively expressed, occurs during cleavage (David-son, 1986, 1989). Both P3A1 and P3A2 are encoded bylow abundance maternal mRNAs; there are about 300copies of P3A1 mRNA per egg, rising to about 2000 perembryo at 24h (at that stage ~4molecules/cell), andabout 1100 copies of P3A2 mRNA per egg, a level that

remains approximately constant (Cutting et al. 1990).Calculations show that the levels of these mRNAspresent in the very early embryo would just suffice toprovide sufficient levels of newly synthesized P3Afactors in the nuclei of the mid-cleavage stage foundercells, when active P3A factors should first be required(Cutting et al. 1990). Since the amounts of P3A1 andP3A2 mRNA are approximately the same by mid-cleavage the amounts of the two factors are probablycomparable. However, we do not yet know where in thevery early embryo the P3A1 and P3A2 factors arelocated. By late, even mid-embryogenesis, aboralectoderm is a wholly committed, terminally differen-tiated cell type, in contrast to oral ectoderm and thevegetal plate derivatives, which continue to differen-tiate in new directions (Davidson, 1986,1989; Cameronet al. 1987). We suspect that in so far as it pertains toaboral ectoderm gene expression the P3A regulatorysystem is a specification system that functions very earlyin development. Thus the factor is present even beforethe Cyllla gene is activated (Calzone et al. 1988), andcompetitive interference with P3A2 interactions in vivoresults in ectopic specification. These factors could,however, have other functions in the still differentiatingembryonic cell types of the oral ectoderm and gut-mesenchyme territories. But it seems most unlikely thatthese maternally encoded factors are physically absentfrom the aboral ectoderm founder cell lineages in earlycleavage. Initially the egg is radially symmetrical interms of potential to generate oral versus aboralectoderm. That is, any quadrant of the 4-cell embryocan give rise to a complete larva (Davidson, 1989). Anhypothesis (Davidson, 1989) is that the cytologicalpolarization that is the initial indication of this axiscauses a modification of radially distributed factors suchas P3A1 and P3A2 that would affect their activity alongthis dimension of the embryonic space.

In considering the P3A regulatory system, we beginwith the argument that two different factors thatinteract with the same or overlapping sites are likely toperform opposite functions, i.e. to act competitively.Since we know that at least one of these factors behavesnegatively the other might be required for transcrip-tional activation. The literature contains severalexamples of negative factors that probably function bycompetitively interfering or modifying positive interac-tions, in which both factors utilize the same oroverlapping binding sites. Among such examples arethe positive and negative interactions in the interferongene (Keller and Maniatis, 1988); the CCAAT andCCAAT-displacement factor interactions in a seaurchin histone gene (Barberis et al. 1987); and theoverlapping and oppositely acting Kr and bed interac-tions, and bed and giant interactions identified in the'stripe 2' regulatory element of the Drosophila eve gene(Small et al. 1990). For our case an additional clue isprovided by the in vitro interaction studies we present inFigs 2 and 3 of this paper. The most striking differencein DNA-binding activities between P3A1 and P3A2that we have detected is their response to the targetsites in the SM50 regulatory domain. Table 2 of Calzone

Gene regulatory finger protein 363

et al. (1991) shows that P3A2 binds about 50 x moretightly to the double site in this gene than to the singleCyllla site, and about 10x more tightly to a third targetsite of the SM50 gene located proximally. In contrast, asshown in this paper, P3A1 recognizes all of the targetsites with about the same affinity (Fig. 2), which is alsoabout the same affinity that P3A2 displays for theCyllla site. Thus, at relatively low factor concentration,the SM50 target site would be expected to be occupiedby P3A2, while the Cyllla site would not. The twofactors could both be involved in regulating both aboralectoderm and skeletogenic genes if they functioncompetitively. Thus, for example, we may suppose thatP3A2 is the factor that functions negatively, as arepressor of transcriptional expression, and that P3A1competitively antagonizes this repression. Then, werethe ratio of P3A1 to P3A2 to increase duringdevelopment in favor of P3A1 by a modest factor, sayover a 10-fold range about equivalence, the transcrip-tion of genes such as Cyllla would be allowed,assuming the remainder of the required transcriptionfactors were available (The'ze' et al. 1990; Calzone et al.1988). However, at such ratios repression of SM50expression by P3A2 would everywhere be dominant.To account for SM50 activation exclusively in theskeletogenic mesenchyme lineage it is likely, aspostulated earlier (Davidson, 1989), that other positiveskeletogenic gene activators should be present ab initioin this autonomously specified lineage. A testableprediction is that there should be a much smallerconcentration of the P3A2 repressor relative to P3A1 inthe confined cellular domain from which the skeleto-genic founder cells descend, i.e. the 5th cleavagemicromeres, than in any other lineage elements of thecleavage stage embryo.

We thank Mariann Brunkener for construction of the Agtlllibrary. This work was supported by an NIH grant HD-05753.Support for the initial phases were provided by a Develop-mental Biology grant from the Lucille P. Markey CharitableTrust C.H. was supported by a postdoctoral fellowship fromEMBO.

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(Accepted 6 February 1991)