cooperative binding sites, promoter context, and co-evolution

Transcriptional control ofDrosophila bicoidby Serendipityd:cooperative binding sites, promoter context, and co-evolution

Cecile Ruez, Franc¸ois Payre, Alain Vincent*

Centre de Biologie du De´veloppement, UMR 5547 CNRS/UPS, Bat 4R3,118 route de Narbonne, 31062 Toulouse cedex 4, France

Received 1 July 1998; revised version received 28 August 1998; accepted 28 August 1998

Abstract

Concentration of maternal BICOID (BCD) establishes the anterior pattern in theDrosophilaembryo. Successive deletions in thebcdpromoter allowed us to localize an enhancer sequence in the 5′-UTR and a down-regulating element downstream of the ATG initiatorcodon, and identify a 49 bp region sufficient to drive transcription of a reporter gene specifically in nurse cells. This fragment contains twobinding sites for the Serendipity (Sry)d zinc finger activator, that mediate its cooperative binding. Both sites (sdbs) are essential forbcdexpression. Further analysis showed that thebcdpromoter configuration is decisive for Sryd activating function. Replacement of sdbs bybinding sites for Sryb, the Sryd paralog, restoresbcd transcription insry d mutant ovaries, demonstrating that the functional divergencebetween these two proteins during evolution was mainly driven by changes in their DNA-specific recognition properties, resulting in thecontrol of separate developmental pathways. 1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: bicoidtranscription;Drosophilaoogenesis; Serendipityd homodimers; Co-evolution

1. Introduction

Elaboration of the body plan in the head and thoracicregions of theDrosophilaembryo is largely controlled bythe activity of the maternal morphogen bicoid (bcd) (Frohn-hofer and Nusslein-Volhard, 1986; Nu¨sslein-Volhard et al.,1987; review by Driever, 1993).bcdmRNA transcribed bynurse-cells in the egg chamber is transported to the oocytewhere it remains localized at the anterior pole (St Johnstonet al., 1989). The localizedbcd RNA is translated afterfertilization to give a concentration gradient of proteinalong the antero–posterior axis of the embryo that peaksat the anterior pole (Driever and Nu¨sslein-Volhard,1988a). Bicoid is a transcription regulator that acts as amorphogen by eliciting distinct transcriptional responsesfrom its several targets at different concentrations (Tautz,1988; Driever and Nu¨sslein-Volhard, 1989; Struhl et al.,1989; review by Driever, 1993). In addition, Bicoid is

thought to block translation of a posterior determinantcaudal, even at low concentration (Dubnau and Struhl,1996, Rivero-Pomar et al., 1996). Thus, one key featureof BCD is its concentration dependent action. The observa-tion that the concentration of Bcd protein was roughly pro-portional to the number ofbcdgene copies strongly arguedfor a control of the level ofbcdactivity at the transcriptionallevel (Driever and Nu¨sslein-Volhard, 1988b; Struhl et al.,1989). Yet, whereas genetic screens for maternal-effectmutations identified factors involved inbcd mRNA locali-zation, they failed to uncover mutations affectingbcd tran-scription. One possible explanation was that severaldifferent activators with, at least partly, redundant functionswere involved, whose mutations would only lower but notabolishbcd transcription. One difficulty in identifying such‘hypomorphic’ mutations is the plasticity in the embryonicdevelopment ofDrosophila: embryos laid by mothers witheither a single or up to five, rather than the normal two,copies ofbcd, although showing initial distortions in theexpression of the zygotic segmentation genes and the posi-tion and size of mitotic domains, develop into relativelynormal larvae and adults (Frohnho¨fer and Nusslein-

Mechanisms of Development 78 (1998) 125–134

0925-4773/98/$ - see front matter 1998 Elsevier Science Ireland Ltd. All rights reservedPII S0925-4773(98)00159-2

* Corresponding author. Tel: +33-5-61-55-82-89; fax: +33-5-61-55-65-07; e-mail: [email protected]

Volhard, 1986; Namba et al., 1997). One other possiblereason was that only transcription factors with earlierrequirements during fly development were involved forbcd transcription, precluding their identification in screensfor maternal-effect mutations.

Supporting this second possibility, we previouslyreported that the Serendipity delta (Sryd) C2H2 zinc fingerprotein is a direct activator ofbcdtranscription (Payre et al.,1994).sry d is a zygotic lethal gene whose mutation resultsin pleiotropic somatic and germ-line defects, the relativeseverity of which depends upon the allele considered (Cro-zatier et al., 1992; and unpublished data).sry d and its closeparalog,sryb, provide an interesting paradigm for studies offunctional aspects of the modular structure of C2H2 zincfinger proteins (Ferrer et al., 1994). While a single copy ofwild-type sry d fully suppresses thesry d mutant lethalityand sterility, increasing the copy number ofsry b does notrescue any of these phenotypes including the lack ofbcdexpression (Crozatier et al., 1992; Payre et al., 1994).

The specific requirement of Sryd for bcd expression, anunexpected link between a zygotic factor with pleiotropicfunctions and the establishment of coordinates of theDro-sophila egg, led us to investigate in detail the Sryd/bcdinteraction. In this paper, we show that a 49 bpbcd DNAfragment including two Sryd binding sites and the 5′-mostbcdtranscription starts is sufficient to mimicbcdexpressionin ovaries. We then show that each of the two Sryd bindingsites is essential forbcd transcription and that thebcd pro-moter configuration potentiates the ability of Sryd to acti-vatebcdtranscription specifically in nurse cells. Finally, weshow that abcd transgene where the two Sryd binding siteshave been replaced by consensus recognition sites for Sryb

(Payre and Vincent, 1991), is transcribed in Sryd mutantovaries. These results strongly suggest that the main deter-minant of the functional difference between the Sryb andSry d paralog zinc finger proteins resides in their DNArecognition properties, a co-evolution of these proteinsand their target binding sites having resulted in the regula-tion of distinct sets of genes.

2. Results

2.1. A 49 bp bcd DNA fragment drives nurse cell-specifictranscription of a reporter gene

In order to define the 5′ and 3′ limits of the functionalbcd

promoter, we made a series of constructs fusingbcd geno-mic fragments to thelacZ reporter gene and analyzed theirexpression in transformant lines (Fig. 1). Several indepen-dent lines were assayed for each construct and LacZ expres-sion examined in embryos, third-instar larvae, whole maleand female adults, and dissected ovaries and testis. Thereference construct, Kex2-Z (Fig. 1a), included 2732 bp ofDNA upstream of the 5′-most bcd transcription start site(Seeger and Kaufman, 1990). It also included 923 bp oftranscribed DNA corresponding to the entire first exonand intron plus 13 bp of the second exon. With this con-struct, LacZ expression was only observed in ovaries, andexclusively in the germ-line, starting at around stage 5–6 ofoogenesis, similar to the pattern of accumulation ofbcdmRNA (Fig. 1a, St Johnston et al., 1989). Analysis of aseries of progressive deletions, using various restrictionsites, positions -1790, -1229, -635, -361 and -37, showedthat the same pattern and similar level of transcription of thelacZ reporter gene was retained when all DNA upstream ofa Sryd binding site, previously shown to be essential forbcdtranscription, (Payre et al., 1994), was removed (Mex2-Zconstruct, Fig. 1b and data not shown). Position of this Sryd

binding site (designated below as sdbsA) thus defines the 5′border of the functionalbcd promoter. We then tested theeffect of a 3′ truncation moving the position of thebcd/lacZfusion from exon 2 to exon 1, 15 bp downstream of thebcdtranslation initiation codon (position+202). With this con-struct (Kex1-Z, Fig. 1c) expression of LacZ was still onlyobserved in the female germ-line. However, this ovarian-specific expression was much stronger than observed withthe reference construct, assayed in parallel (Fig. 1a), sug-gesting the existence of cis-regulatory elements in the introndown-regulating the level ofbcd expression. The sameincrease in LacZ expression was observed whether or notDNA upstream of sdbs A was present in the construct (com-pare Kex1-Z and Mex1-Z, Fig. 1c,d). The main conclusionat this point was that a 239 bpbcdDNA fragment, betweenpositions−37 and+202, is able to drive expression of thelacZ reporter gene specifically in the nurse cells. Furthertruncation from the 3′ side would result in deletion of the5′ bcd untranslated region (UTR). In order to test for thepossible existence of cis-regulatory elements within thisUTR, we replaced it by the Xenopus beta-globin UTR, pre-viously shown to functionally substitute for thebcdsequence in transgenic rescue experiments (McDonald andStruhl, 1988), leaving only 49 bp ofbcdDNA (position−37to +12, 49bg-Z construct). Thisbcd/Xenopus globin 5′-

Fig. 1. A 49 bpbcdDNA fragment drives nurse cell-specific transcription. Top: schematic representation of the 7.5 kbbcdgenomic region. Restriction sitesused to generatelacZ reporter genes and their position relative to thebcd transcription start (+1, broken arrow) are indicated. B, BamHI; E, EcoRI; H,HindIII; K, KpnI; M, MunI; N, NruI; S, SalI. Positions of the initiator codon (ATG,+186), and Sryd binding sites (AB) are indicated. The introns are drawnas broken lines. Bottom: the names of thebcd/lacZ fusion genes and their diagrammatic representation are shown on the left. The 5′ positions of Kex andMex/49 bg-Z constructs are the KpnI site and MunI site, respectively. Thebcdtranscribed regions are shown in yellow, thelacZcoding region in blue and theXenopusb-globin5′-UTR in black. X-gal staining of dissected ovaries (a to e) or in situ hybridization tobcdRNA (d′,e′) is shown on the right. *indicates thatthe X-Gal staining in (e) was five times longer that in panels a–d, emphasizing the low level of transcription of the 49bg-Z reporter gene. A conspicuousantero-posterior gradient of LacZ staining was observed with this construct (e) but a uniform signal was detected by in situ hybridization (e′).

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UTR/lacZ reporter gene showed an expression restricted toovaries although this expression was weak and displayed apeculiar antero-posterior gradient in the nurse-cell chambernot observed with the reference construct (Fig. 1d,e). In situhybridization on dissected ovaries from transformant linesusing a probe complementary to the 3′ lacZ tag showed thatthe RNAs transcribed from the 49bg-Z transgene were uni-formly distributed in the nurse cell chamber (Fig. 1d′,e′, anddata not shown). This indicates that the observed gradient ofLacZ activity specifically observed with this construct islikely due to translation-regulatory signals in the Xenopus5′ globin UTR. We therefore can conclude that the 49bp(−37 to+12) fragment ofbcdDNA is the primary determi-nant ofbcdspecific expression in ovaries. The much weakerlevel of accumulation of transcripts observed with 49bg-Z,compared to the full-length construct, confirms the presenceof an up-regulating cis-element in thebcd 5′-UTR, as pre-viously suggested by McDonald and Struhl (1988).

2.2. Cooperative binding of Sryd homodimers to two sitesin the bcd promoter is required for bcd transcription

In our initial study of the control ofbcd transcription, weshowed the essential role of a single Sryd binding site(sdbs1, renamed below as sdbsA) located between positions−33 and−21 upstream of thebcdtranscription start (Payre etal., 1994). Analysis of the 49 bp proximalbcd promoterregion revealed another potential Sryd binding site, denotedbelow as sdbs B, located between positions−14 and−1, i.e.separated from sdbsA by 7 bp (Fig. 2A). In vitro footprint-ing experiments indicated that sdbsB was a high affinitybinding site responsible, together with sdbsA, for the coop-erative binding of Sryd to thebcd promoter (Payre et al.,1997). EMSA analysis (Fig. 2B) confirmed that either muta-tion of sdbsA or sdbsB results in a similar decrease of Sryd

binding in vitro, typical of the loss of binding cooperationobserved when going from two to one Sryd binding sites(sdcs) (Payre et al., 1997). We therefore tested whether, likesdbsA, sdbsB was required forbcd transcription. Each ofthese two sites was separately mutated in the context of abcdtransgene (p2107) previously shown to be able to rescuebcdmutations (McDonald et al., 1993, Fig. 2A). This func-tional transgene contains a 7.5 kbbcd genomic fragment(Driever and Nu¨sslein-Volhard, 1988b), modified by theinsertion of a 1.3 kb lacZ RNA tag in thebcd3′-UTR region.The unmodified and mutated transgenes, p2107, p2107A−

and p2107B−, respectively, were introduced into the flygenome, and transgenic lines established. Using standardgenetic crosses, each transgene was then introduced inbcd mutant backgrounds and the cuticles of embryos laidby the bcd transgenic females were examined for anteriorpatterning defects. For these experiments, we used thebcdE3

allele because of its intermediate phenotype likely to besensitive to even small changes in the level of expressionof the bcd gene transgene. While one copy of p2107 com-pletely rescued thebcd phenotype, neither p2107A− nor

p2107B− did significantly improve this phenotype, indicat-ing that both sdbs sites are required for normal levels ofbcdactivity. Only with two copies of p2107B−, did we observea slight rescue (Fig. 2C). To further confirm that the absenceof rescue by p2107A− or p2107B− resulted from a lack ofbcd transcription, we looked at the accumulation of thecorrespondingbcd-lacZ transcript in ovaries of otherwisewild-type, transgenic females. Endogenousbcd mRNAlevel were used as internal standard for quantification ofmRNA. Fig. 2D shows that mutation of either sdbsA orsdbsB results in the reduction ofbcd transcript accumula-tion to undetectable levels, demonstrating that both Sryd

binding sites present in the proximalbcd promoter regionare essential forbcd transcription.

Together with the previous report that Sryd makeshomodimers in solution (Payre et al., 1997), these in vivoresults indicated that activation ofbcdtranscription requirescooperative binding of Sryd homodimers mediated by thetwo sites present in the proximal promoter region. Thisrequirement could explain by itself the high sensitivity ofbcd transcription to sryd mutations that affect either itsaffinity for DNA or its homodimerization properties(Payre et al., 1997). Yet, Sryd is expressed and requiredin both various types of somatic cells and male and femalegerm line cells (Payre et al., 1989; Crozatier et al., 1992)whereasbcd transcription only occurs in the female germ-line. This suggested the possible existence of other transact-ing factor(s) involved in the regulation ofbcd transcription,that had escaped genetic screens. We therefore undertookexperiments to (i) identify putative tissue-specific cis-regu-latory elements other than sdbs in the 49 bpbcd promoterregion and (ii) test whether Sryd binding sites are by them-selves able to mediate transcriptional activation of a down-stream gene and, if so, whether thebcdpromoter context isdeterminant for the restriction ofbcd activation to nursecells.

2.3. The bcd promoter configuration potentiates the abilityof Sryd to activate bcd

In addition to sdbsA and sdbsB, the 49bpbcd fragmentdriving lacZ expression specifically in ovaries contains 12bp of DNA, downstream of sdbsB, that include two (out ofthe three) majorbcd transcription start sites (Fig. 3A andSeeger and Kaufman, 1990). We determined whether these12 bp contained a cis-regulatory element, by mutating ninenucleotide positions, leaving intact the two transcriptionstart sites (Fig. 3A). The effect of these mutations was testedin the context of a strongbcdpromoter configuration, i.e. theMex1-Z construct, (construct Mex1-Zm12). We could notdetect any difference in the pattern oflacZ expression dri-ven by Mex1-Zm12 compared to Mex1-Z transgenes (Fig.3a,b), suggesting that the 12 bp nucleotide sequence is notmeaningful for the nurse-cell specific activation ofbcdtran-scription. Sryd consensus binding sites are able by them-selves to promote a strong transcription of a downstream


Fig. 2. Each of two Sryd binding sites in thebcdpromoter is essential forbcd transcription. (A) Schematic representation of thebcdgenomic region presentin the p[2107] construct, indicating the position of thelacZRNA tag in thebcd3′-UTR. The proximal promoter region is enlarged to show the position of theSry d binding sites sdbsA and sdbsB (boxed) relative to thebcd transcription start (+ 1). Upwards pointing arrowheads indicate the positions of mutationsintroduced separately in these two sites. (B) A 112 bpbcdDNA fragment (− 72, + 40) was used for EMSA analysis with increasing amounts of Sryd; fromleft to right Sryd concentrations were 0, 12.5, 25, 50 and 100 ng. Mutation of either sdbsA (panel A−B) or sdbsB (panel AB−) abolished cooperative bindingof Sry d observed with the intactbcd promoter (panel AB). (C) Cuticle preparations of embryos laid bybcdE3 females carrying a copy of p[2107], eitherunmodified or with mutated sdbsA (p[2107A-]) or sdbsB ([p2107B-]). Only [p2107] does rescue thebcdphenotype while a minor rescue is observed with twocopies of [p2107B−]. (D) Northern analysis ofbcd/lacZ mRNA in ovaries of wild type females carrying a copy of either p[2107], p[2107A−] or p[2107B−].Two separate lines are shown for p[2107A−] and p[2107B−], revealing the absence ofbcd/lacZ reporter gene transcription when either sdbs A or B has beenmutated. Endogenousbcd and rp49 mRNAs were used as internal standards for quantitation of deposited mRNA.


gene in culturedDrosophilaSL2 cells (Payre et al., 1997).The restriction ofbcd activation to nurse cells, contrastingwith the ubiquitous expression of Sryd raised the possibi-lities that either the structure of sdbsA and sdbsB, or thelength or sequence of the spacer in between was determinantfor this tissue-restriction. We tested the first possibility byplacing upstream of a TATA box in the hsp70 minimalpromoter, either two sdcs in the same configuration thanpreviously used in cell culture, or the 37 bpbcdDNA frag-ment containing sdbs A and B (AB-HZ50 construct, Fig. 3Band data not shown). Both constructs gave the same result:the complete absence of expression of thelacZreporter genein ovaries. This contrasted with results obtained with the 49

bpbcdDNA fragment, whereas expression was observed inother tissues, the testis and larval CNS in all independentAB-HZ50 lines examined, and additional sites specific toeach line (Fig. 3c,d and data not shown). These results led usto conclude that, whereas functional in cultured cells, Sryd

binding sites are not sufficient to activate the expression of areporter gene in all tissues where Sryd is expressed duringfly development, and that other specific requirement must bemet for the activation ofbcd transcription. We then testedwhether the length and/or nucleotide sequence of the 7 bpspacer separating sdbsA and sdbsB was determinant, bysubstituting for a 11 bp spacer of unrelated sequence. Themodifiedbcd transgene remained efficiently transcribed, as

Fig. 3. Thebcdpromoter configuration is decisive forbcdactivation by Sryd. The names of thebcd/lacZ fusion genes and their diagrammatic representationare shown on the left according to Fig. 1. (A) Nucleotide positions mutated in the Mex1-Zm12 construct are indicated by arrowheads (in red) and thebcdtranscription start sites (+1 and+9) by broken arrows. (B) Full sequence of the 49 bpbcd DNA fragment inserted directly upstream of theXenopus beta-globin/lacZ transcribed region (49bg-Z construct) and the 37 bp fragment inserted upstream of the TATA box of the hsp70 minimal promoter in the HZ50reporter construct (AB-HZ50 construct) is given. X-gal staining of dissected ovaries (a–d) is shown on the right.


assayed by both phenotypic rescue and Northern blot ana-lysis (data not shown), indicating that the structure and exactlength of the 7 bp spacer present in wild typebcd is notdeterminant. The only cis-regulatory elements identifiedwithin the 49 bpbcd DNA fragment were therefore sdbsA and B, indicating that thebcdpromoter configuration, i.e.the position of sdbs immediately upstream of thebcd tran-scription start with no intervening TATA box, was decisivefor bcd activation in ovarian nurse cells. Although thepossibility that sdbs could bind another factor than Sryd

cannot be formally excluded, it further suggested therecruitment by Sryd of a co-activator specifically presentin these cells.

2.4. Converting bcd from a Sryd into a Sryb target

The close paralog of Sryd, Sryb, does not substitute forSry d in activatingbcd (Crozatier et al., 1992). The Sryband d DNA consensus recognition sites, sbcs: 5′-TGCGCATCTCTGR-3’ and sdcs: 5′-TTYCCATCTC-TAR-3’, respectively, differ from each other at 4 out of 13positions, and these differences are sufficient for discrimi-natory in vitro binding of Sryb and Sryd to their respectiverecognition sites (Payre and Vincent, 1991, and data notshown). We could then ask whether this difference inDNA recognition properties was sufficient to account forthe failure of Sryb to activatebcd transcription. We thusreplaced both sdbsA and sdbsB by sbcs in the p2107bcdtransgene and assayed for expression of this modified trans-gene, p2107sbcs (Fig. 4A), in a wild type orsry d mutantbackground. Northern blot analysis of RNA from p2107sbcstransgenic females showed the accumulation ofbcd-lacZtranscripts in ovaries, to levels similar to those observedwith p2107, taking into account variations between indivi-dual transformant lines (Fig. 4B and data not shown). Tofurther verify that the transcription of p2107sbcs was inde-pendent of Sryd activity, the p2107 and p2107sbcs trans-genes were introduced into asry dSF2 mutant background.sry dSF2homozygous escaper females lay eggs, a fraction ofwhich develop to the point of making cuticles (Crozatier etal., 1992). These cuticles typically show a strongbcd-likephenotype with a deletion of anterior structures and thoracicsegments and duplication of the posterior telson (Fig. 4C).While p2107 even present at two copies did not significantlyimprove this phenotype, one copy of p2107sbcs restored thedevelopment of thoracic and some anterior structures. Withtwo copies of the transgene, a complete rescue wasobserved, with the cuticles of rescued embryos being indis-tinguishable from wild type cuticles. We therefore can con-clude p2107sbcs is efficiently transcribed in ovaries,independently of Sryd activity. In situ hybridization ondissected ovaries of p[2107sbcs] transgenic females, orembryos laid by such females, indicated that transcriptionof this modifiedbcd transgene was restricted to nurse cells(data not shown). Altogether these results indicate thatreplacing the two binding sites of Sryd by binding sites

for Sry b is sufficient to turnbcd from a Sryd target intoa Sryb target, still expressed specifically in nurse cells.

3. Discussion

3.1. Cooperative binding of Sryd homodimers to the bcdpromoter is required for bcd transcription

Since the initial report of the structure of TFIIIA, a vastnumber of C2H2 zinc finger transcription factors have beencharacterized in all eucaryotes (Klug and Schwabe, 1995;for review). Characteristic of this class of transcription fac-tors is the variable number of fingers per protein, each fingerfolding into an independent domain capable to specificallyinteract with DNA. Contrary to other families of zinc fingertranscription factors, however, C2H2 proteins display nonpalindromic recognition sites and are generally thought tobind DNA as monomers (Mackay and Crossley, 1998; forreview). The involvement of a subset of C2H2 fingers inmediating specific protein-protein interactions has beenrecently reported as, for example, the related proteins Ikarosand Aiolo, two transcriptional regulators of lymphocyte dif-ferentiation that form homo- and heterodimeric complexes(Sun et al., 1996; Morgan et al., 1997). We have alsorecently shown that Sryd forms homodimers; in this case,homodimerization requires the sixth C2H2 finger plus anevolutionary conserved non canonical C2C2 zinc fingerlocated at the N-terminus of the protein (Payre et al.,1997). Homodimer formation is required for Sryd coopera-tive binding to multiple tandem binding sites and synergisticactivation of a reporter gene in transfected cells (Payre et al.,1997). Variation in thebcdgene dosage has a strong effecton the positioning of morphological and molecular land-marks in the early gastrulating embryo (Driever andNusslein-Volhard, 1988b; Struhl et al., 1989; review byDriever, 1993; Namba et al., 1997). The level ofbcd tran-scription reached when either sdbs A or B is mutated, is wellbelow the minimal threshold required for embryo survival.This demonstration that each of the two sdbs in thebcdproximal promoter is required forbcd transcription con-firmed, in vivo, the crucial role of Sryd homodimerization.

3.2. The bcd promoter context is decisive for nurse-cellspecific activation of bcd by Sryd

Successive deletions in thebcd promoter allowed us tolocalize an enhancer sequence in the 5′-UTR and a down-regulating element located downstream of the ATG initiatorcodon probably in the first intron, and identified a 49 bpminimal promoter region sufficient to drive expression ofa reporter gene specifically in nurse cells. Detailed analysisof this 49 bp DNA fragment which includes the two sdbsand bcd transcription starts with no TATA box, failed toreveal any additional cis-regulatory element. We have pre-viously shown that the orientation of scds relative to the


direction of transcription strongly influences the level oftransactivation in transfected cells: this level is severalfold higher in the orientation found in thebcd promoter,compared to the inverse. However, neither two sdcs in theappropriate orientation, nor a 38 bpbcd DNA fragmentcontaining sdbs A and sdbsB, were able by themselves topromote transcription in nurse cells when placed upstreamof the TATA-box containing minimal hsp70 promoter. Thisobservation suggested the imposition of topological con-straints on the ability of Sryd to activatebcd transcriptionby the bcd promoter configuration itself. All our data areconsistent with a model in whichbcd transcription requires

the cooperative binding of Sryd homodimers to the tandemsdbs sites and subsequent recruitment of a nurse cell-speci-fic coactivator. What could be the nature of this co-activa-tor? One possibility is a nurse cell-specific TATA bindingprotein (TBP) associated factor (TAF). TAFS function asrequisite ‘coactivators’ mediating the interaction betweensequence-specific-activators and the general transcriptionmachinery, although not generally required, per se, for regu-lated transcription (Verrijzer and Tjian, 1996; for review). Arole of some TAFs in the specific recognition of promotersthat lack a canonical TATA box (and this is the case ofbcd)has nevertheless recently been proposed (Hampsey and

Fig. 4.bcdtranscription independent of Sryd activity. (A) Schematic representation of the p[2107sbcs] construct as in Fig. 1, showing the nucleotide changesconverting sdbsA and sdbsB into sbcs (arrowheads). (B) Northern analysis ofbcd/lacZ mRNA in ovaries of wild type females carrying a copy of eitherp[2107] or p[2107sbcs] (two independent lines) showing efficient transcription of thebcd/lacZ reporter gene when both sdbs sites have been converted tosbcs; the asterisk denotes the line used for rescue experiments, see panel C. Endogenousbcd and rp49 were used as internal standards for quantitation ofdeposited mRNA. (C) Cuticle preparations of embryos laid bysry dSF2 mutant escaper females carrying one or two copies of either p[2107] or p[2107sbcs].Only p[2107sbcs] does rescue thebcd-like phenotype of embryos laid bysry d mutant females, either partially (one copy) or completely (two copies).


Reinberg, 1997). Another possibility is the existence of analternative form of TPB (TATA binding protein), such asTRF expressed in the CNS and male reproductive organs(Crowley et al., 1993; Hansen et al., 1997), that could func-tion to transduce the effects of activators in establishing thenurse-cell specific transcription program. Further experi-ments are now needed to distinguish between these possibi-lities.

3.3. Turning bcd from a Sryd into a Sryb target. DNAspecific recognition makes it all

The modular structure of the DNA binding domain ofC2H2 proteins allows extensive variations in length andsequence of the DNA sites that they specifically recognizeand is prone to the emergence of new specificities duringevolution. The identification ofbcd as a Sryd target pro-vided us with a direct in vivo assay of the role of DNAspecific recognition in the functional distinction betweenthe two close paralogs, Sryb and Sryd, which both functionas transcription activators in cultured cells (Payre et al.,1997 and data not shown) and are present at high levels innurse cell nuclei. Replacing Sryd by Sry b binding sites(p2107sbcs construct) restored transcription of abcd trans-gene insry d mutant ovaries, even though the level of tran-scription of p2107sbcs was lower than that of p2107 in wild-type embryos. Whereas the absence, so far, ofsryb specificmutations precludes the unambiguous demonstration thatSry b is the protein which activates transcription ofp[2107sbcs], this is likely for several reasons. First, Sryb

is the only nuclear protein which specifically binds to sbcs inan EMSA assay (Payre and Vincent, 1991). Second, like Sryd, it binds cooperatively to multiple sites, leading to syner-gistic transactivation of a reporter gene (P. Buono, unpub-lished data). Third, a chimaeric protein in which the entireN-terminal region of Sryd has been replaced by the analo-gous region of Sryb binds cooperatively to sdcs and is ableto substitute for Sryd in activatingbcdexpression (Noselliet al., 1992; Crozatier et al., in preparation), suggesting thatthe structural divergence between the two proteins in theirN-terminus does not prevent interaction with (a) ternaryfactor(s) at thebcd promoter. We conclude from the sdcsinto sbcs substitution experiment that the main driver offunctional diversification of Sryb and Sryd was the diver-gence in their DNA-specific recognition properties. Thisdivergence resulted in the regulation of transcription ofseparate genes and, as shown here withbcd, the control ofdifferent developmental programs.

4. Experimental procedures

4.1. Fly strains

The sry d EMS induced allelesry dSF2 was described inCrozatier et al., 1992. ThebcdE3 mutant strain was obtained

from the Tubingen stock Center (Tearle and Nu¨sslein-Volhard, 1987). Thery506 strain was used as wild type strainfor injection of the p[AB-HZ50 ry+] construct. All the otherconstructs were injected into aw− strain which was also usedfor control RNA preparations. Flies were grown under stan-dard conditions at 22°C and eggs processed for the determi-nation of the cuticular phenotypes as described (Wieschausand Nusslein-Volhard, 1986).

4.2. RNA and EMSA analysis

RNA preparation from ovaries, Northern blot analysis,whole-mount in situ hybridization and X-Gal stainingwere as in Payre et al. (1989), Payre et al. (1994). Expres-sion in E. coli and purification of the recombinant Srydprotein and electrophoretic mobility shift assays (EMSA)were as described in Payre et al. (1997).

4.3. Reporter gene constructs and transgenic lines

AB-HZ50 was constructed by inserting a 37 bpbcdDNAfragment upstream of the hsp70 TATA box in the HZ50PLplasmid (Hiromi and Gehring, 1987). The p2107 constructis a w+P-vector which contains 7.5 kbp ofbcd genomicfrom a Kpn I site, position−2725, to an EcoRI site, position+4594, i.e. the entirebcdcoding region and 3′-UTR, with a1.3 kb lacZ RNA tag inserted in the 3′-UTR (McDonald etal., 1993). The Kex2-Z construct contains 3648 bp ofbcdDNA, from the Kpn I site to an EcoRI site, position+923,introduced in the second exon by site directed mutagenesis.This DNA fragment was fused in frame to theE coli beta-galactosidase coding region. Mex2-Z was generated fromKex2-Z by removing upstream DNA, from the Kpn I site toa Mun I site, position−37. Kex1-Z and Mex1-Z are Kex2-Zand Mex2-Z derivates, respectively, with thebcd-lacZfusion in exon 1, five amino-acids downstream of thebcdATG, at a EcoRI site, position+202. Mex1-Z-m12 wasgenerated from Mex1-Z by mutating 8 out of the 12 bp ofbcd DNA downstream of sdbsB that are included in thisconstruct. The 49bgZ construct contains 49 bp ofbcdDNA (Positions−37;+12) fused to the 5′-UTR of the Xeno-pus beta globin gene placed upstream of the lacZ codingsequence. For site specific mutagenesis of sdbsA and sdbsB,we subcloned the 1.8 kb BamHI/PstI (−1790,+12)bcdfrag-ment in pTZ18R (Pharmacia) and used the procedure ofKunkel (1985). Sequences of the oligonucleotides used tomutate sdbsA, sdbsB and replace sdbs by sbcs are, respec-tively:

A− 5′-CAATTGTGCCATGGGTACATCTCTTCGC-3′B− 5′-CATCTCTTCGCCCATGGGAAAATAACGGC-3′sbcs 5′-CGCCAATTTGCGCATCTCTGAATCTCTTTG

CGCATCTCTGAATAACGGC-3′

The A− and B− oligonucleotides incorporated a NcoIrestriction site (underlined) to facilitate mutant identifica-


tion. The specific nature of the introduced mutations wasthen confirmed by sequencing. After introduction of thesemutations, the BamHI/PstI fragment was placed back intop[2107] for fly transformation (Rubin and Spradling, 1982).

Acknowledgements

We are grateful to the Tu¨bingen Drosophila Stock Centerfor sending us mutant strains and Paul McDonald for thep2107 construct and transgenic flies. We acknowledge theexcellent technical help of Claude Ardourel and thankMichele Crozatier and Anaı¨d Chahinian for their commentson the manuscript and our colleagues at Toulouse for sug-gestions and discussions throughout the course of this work.The work was supported by Centre National de laRecherche Scientifique and Human Frontier Science Pro-gram. C. Ruez was recipient of fellowship from Associationpour la Recherche sur le Cancer.

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cooperative binding sites, promoter context, and co-evolution

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