Insect Molecular Biology (2004)

13

(5), 481–493

© 2004 The Royal Entomological Society

481

Blackwell Publishing, Ltd.

A functional analysis of

ACP-20

, an adult-specific cuticular protein gene from the beetle

Tenebrio

: role of an intronic sequence in transcriptional activation during the late metamorphic period

A. Lemoine*, J. Mathelin*, C. Braquart-Varnier†, C. Everaerts* and J. Delachambre*

*

UMR CNRS 5548, Développement et Communication Chimique chez les Insectes, Université de Bourgogne, Dijon, France; and

†

UMR CNRS 6556, Génétique et Biologie des Populations de Crustacés, Université de Poitiers, France

Abstract

A gene encoding the adult cuticular protein

ACP-20

wasisolated in

Tenebrio

. It consists of three exons inter-spersed by two introns, intron 1 interrupting the signalpeptide. To understand the regulatory mechanisms of

ACP-20

expression,

ACP-20

promoter–luciferase reportergene constructs were transfected into cultured pha-rate adult wing epidermis. Transfection assays neededthe presence of 20-hydroxyecdysone, confirming that

ACP-20

is up-regulated by ecdysteroids. Analysis of5′′′′

deletion constructs revealed that three regions arenecessary for high levels of transcription. Interactionexperiments between intronic fragments and epidermalnuclear proteins confirmed the importance of intron1 in

ACP-20

transcriptional control, which resultsfrom the combined activity of regulatory

cis

-actingelements of the promoter and those of intron 1.

Keywords: insect cuticular protein gene regulation,intron, metamorphosis, ecdysteroids, cotransfection.

Introduction

Insect integument consists, as in all arthropods, of anepidermis secreting an outer extracellular material, the

cuticle. This exoskeleton serves as a protective coveringin contact with the environment. Its rigidity leads to a dis-continuous growth interrupted by moults. All types of cuticleare mainly composed of long chitin filaments embedded inan heterogeneous protein matrix (Neville, 1975).

The protein composition varies according to the cuticularregion (flexible or rigid cuticle) (Chihara

et al

., 1982; Cox &Willis, 1985; Andersen

et al

., 1986; Lemoine

et al

., 1990) andof developmental stages (larval, pupal and adult) (Roberts& Willis, 1980a,b; Chihara

et al

., 1982; Skelly & Howells, 1988;Souliotis

et al

., 1988; Lemoine

et al

., 1989, 1993). Thesevariations correspond to differential cuticular protein geneexpression regulated by a fine balanced action of 20-hydroxyecdysone (20E) and juvenile hormone (JH) (Apple& Fristrom, 1991; Hiruma

et al

., 1991; Nakato

et al

., 1994;Kramer & Wolbert, 1998). Although the 20E molecularmechanism of action is now well understood (Thummel,1995, 1996; Cherbas & Cherbas, 1996; Segraves, 1998),little is known about the molecular basis of the fine spatio-temporal regulation of cuticular protein genes (Murata

et al

.,1996; Shiomi

et al

., 2000; Togawa

et al

., 2001; Kawasaki

et al

., 2002).In Lepidoptera, Diptera and Coleoptera a number of

cuticular protein genes have been cloned. Most show a similargenomic organization comprising three exons separatedby two introns, the first one interrupting the signal peptidesequence. Another frequent common feature is their organ-ization in clusters (Willis, 1996; Charles

et al

., 1997).In the beetle

Tenebrio

, the same epidermal cell lineagesynthesizes successively larval, pupal and adult cuticle.The main change, in cuticular protein composition, occursduring the pupal–adult transition (Roberts & Willis, 1980a;Lemoine & Delachambre, 1986). Moreover, protein patternvaries during cuticle deposition within a same stage (Roberts& Willis, 1980a; Lemoine

et al

., 1989, 1990, 1993), thussuggesting a sequential activity of coordinately expressedgenes (Bouhin

et al

., 1992; Charles

et al

., 1992; Braquart

et al

., 1996; Rondot

et al

., 1996; Mathelin

et al

., 1998).Therefore, in the present study, a genomic clone wasisolated. This clone contains the transcribed region of

ACP-20

, a cuticular protein gene whose expression is limited

Received 24 February 2004; accepted after revision 8 June 2004.Correspondence: Dr A. Lemoine, UMR CNRS 5548, Développement etCommunication Chimique chez les Insectes, 6 Bd Gabriel, 21000 Dijon,France. Tel.: +33 3 80 39 63 03; fax: +33 3 80 39 62 89; e-mail: aleth.lemoine@u-bourgogne.fr

The nucleotide sequence of

ACP-20

reported in this paper has been de-posited by C. Braquart-Varnier in the EMBL/GenBank database, accessionno. AJ518833.

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et al.

© 2004 The Royal Entomological Society,

Insect Molecular Biology

,

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, 481–493

to the

Tenebrio

epidermis mainly secreting adult hard pre-ecdysial cuticle (Charles

et al

., 1992). The genomic structureobtained for

ACP-20

is characteristic of cuticular proteingenes with the first of its two short introns (intron 1) locatedin the signal peptide (see Discussion for further details).

A molecular analysis of the transcriptional activity of

ACP-20

promoter sequences was undertaken. Because nocell line and no stable transformation system are avaible inthis species, a transient cotransfection assay of the epider-mis cultured

in vitro

was developed. We report here that,although the upstream regulatory sequences are neces-sary for

ACP-20

expression, a strong transcription activityrequires the presence of intron 1. These transfection ex-periments confirm also that

ACP-20

expression is stage-specific and triggered by 20E.

Results

Structure of the ACP-20 gene

The nucleotide sequence of 2.78 kb, encompassing the com-plete transcribed region (880 nucleotides) and the flankingsequences, is illustrated in Fig. 1. The sequence corre-sponding to the transcribed region matches the previouslydescribed

ACP-20

cDNA (Charles

et al

., 1992), except forsix nucleotide changes at positions +46, +509, +542, +672,+710 and +716. Only one of these changes, at position+672, causes replacement of an amino acid (leucine byvaline) in the

ACP-20

polypeptide sequence. Three exonsinterspersed by two introns were identified. Both introns areshort, 54 and 46 nt, respectively, intron 1 is located after thefourth amino acid in the signal peptide and intron 2 begins175 bp after the end of intron 1. All intron/exon boundariesmatch the consensus for splice sites proposed for

Drosophila

(Mount

et al

., 1992). The cap site (AGTGT) previously deter-mined by primer extension is located at 46 nt upstream ofthe open reading frame and corresponds to the arthropod capsite sequence defined by Cherbas & Cherbas (1993). A typi-cal TATA box [TATA(A/T)A(A/T)] (Breathnach & Chambon,1981) and polyadenylation signal (AATAAA) (Proudfoot &Brownlee, 1976) were, respectively, found at positions

−

31/

−

25 and +862/+867 from the transcription start site.

Localization of

ACP-20

promoter activity

To identify the critical elements required for

ACP-20

pro-moter activity, nine DNA constructs, named Luc-F1 to Luc-F9, containing progressive deletions of 5

′

-flanking

ACP-20

DNA inserted in front of the firefly luciferase reporter gene,pGL3-Basic vector, were prepared (Fig. 2A). As describedin the Experimental procedures, all luciferase constructswere functionally tested by transient cotransfection experi-ments with pRL-CMV control vector in the presence of 10

µ

M

20E, on the upper epidermis of the pharate adult forewingstaken at day 8 after pupation when

ACP-20

mRNA wasstrongly expressed (Charles

et al

., 1992). The results are

summarized in Fig. 2B. No luciferase activity was detectedafter cotransfection of the promoterless pGL3-Basic vectoror Luc-F1 (

−

2900/+1) and Luc-F2 (

−

851/+1) constructs,which contained only sequences located upstream of thetranscription start site. Therefore, new constructs includingsequences located both upstream and downstream of thetranscription start site were tested. Luc-F3, containing the

−

851/+118 sequence, presented the highest luciferaseactivity. The results obtained with Luc-F4 (

−

759/+118) andLuc-F5 (

−

583/+118) were not significantly different fromthat of Luc-F3, so the maximal rate of luciferase activity wasunaffected by 5

′

-deletion from

−

851 up to

−

583 bp. The fullyactive

ACP-20

promoter thus maps between

−

583 and+118, so computer analysis of the putative transcription-binding sites located in this DNA fragment was performed(Table 1). To localize more precisely the promoter activity,progressive deletions in the 5

′

- and 3

′

-flanking regionsof this fragment were made. Deletion of 5

′

-flanking

ACP-20

DNA, between

−

583 and

−

405 (Luc-F6) or between

−

583and

−

227 (Luc-F7), resulted in a decrease in luciferaseactivity (3.7-fold) but there were no significant differences inthe activities of these two constructs. These results suggestthat positive regulatory elements could be located between

−

583 and

−

405. Sequence analysis of this fragment revealedseveral putative binding sites: one ecdysteroid responseelement (EcRE) half-site and one each of GAGA-, GATA-,LEF-1- and zeste-binding sites (Table 1). Additional 5

′

-deletion of DNA between

−

227 and

−

64 (Luc-F8) had adramatic effect on the activity of the

ACP-20

promoter (overeighty-fold compared with the full activity), indicating that thisregion contains important positive regulatory elements in

ACP-20

regulation. Sequence analysis of this fragmentrevealed two EcRE half-sites, two Twist-binding sites, oneeach of GATA- and zeste-binding sites and three LEF-1-binding sites (Table 1). The luciferase activity obtained withLuc-F8 is weak and corresponds to the minimal activity ofthe promoter, which may be due to the activity of the TATAbox located at position

−

31 to

−

25 (Table 1). Deletion of 3

′

-flanking

ACP-20

DNA between +47 and +118 (Luc-F9)resulted in a drastic fall in luciferase activity (over eighty-four-fold compared with the full activity). Thus, this 72 bpfragment is required for

ACP-20

expression. Sequenceanalysis revealed two putative EcRE half-sites and oneeach of octamer- and GATA-binding sites (Table 1).

Gel-mobility shift assay for intron 1

In order to confirm the potential role of intron 1, a gel-mobility shift assay was performed (see Experimental pro-cedures) with three double-stranded oligodeoxynucleotidic

32

P-labelled probes (P1–P3) spanning the 72 bp fragment(Fig. 3). The three probes were shifted by nuclear extractsfrom forewings taken when

ACP-20

is expressed, generat-ing specific bands (*), among which the P2 probe–proteincomplex was the major retarded band. Another slightly faster

Functional analysis of an insect cuticular protein gene

483

© 2004 The Royal Entomological Society,

Insect Molecular Biology

,

13

, 481–493

migrating band (NS) was not displaced in competitionexperiments using a 100-fold molar excess of unlabelledprobes P1, P2 or P3, and thus considered as nonspecific.

Effect of 20E on

ACP-20

promoter activity

The continuous presence of 20E was needed for long-term

in vitro

expression of

ACP-20

(Braquart

et al

., 1996). There-

fore, we investigated the effect of 20E on luciferase activityafter Luc-F3 cotransfection in

ACP-20

-secreting epidermis.When no 20E was added, luciferase activity was notdetected. As illustrated in Fig. 4, luciferase activity was veryweak after addition of 0.1–2

µ

M

20E, but increased sharplyto reach the highest level when 10

µ

M

was used. At higherconcentrations, luciferase activity fell drastically.

Figure 1. Nucleotide sequence of the ACP-20 gene from Tenebrio molitor (EMBL/GenBank accession no. AJ518833). Nucleotides are numbered from the transcription initiation site (+1). The line shifts indicate the start and the end of the transcription unit. The deduced amino acid sequence is shown under the nucleotide sequence. The nucleotides in the genomic sequence that differ from those occurring in the cDNA sequence are underlined as is the one amino acid that differs. The putative TATA box and the polyadenylation site are double-underlined.

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,

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, 481–493

Figure 1. (Continued )

Functional analysis of an insect cuticular protein gene 485

© 2004 The Royal Entomological Society, Insect Molecular Biology, 13, 481–493

ACP-20 promoter activity varies during pupal–adult development

Previous Northern blot analysis has shown that ACP-20is mainly expressed during the secretion of the adult pre-ecdysial cuticle (Charles et al., 1992). In order to check thepromoter activity of ACP-20, transient cotransfection exper-iments of Luc-F3 were performed, after addition of 10 µM

20E, on wing epidermis taken before and during the phys-iological expression of ACP-20. The results, illustrated inFig. 5, show that luciferase activity was undetectable intransfected epidermis taken when ACP-20 is not physiolog-ically expressed (newly ecdysed pupae). After transfectionof epidermis secreting adult pre-ecdysial cuticle, an importantincrease in luciferase activity was observed from day 7 today 8 after pupal ecdysis, and this activity then fell drastically.

Discussion

The ACP-20 gene has a 880 nt transcribed region inter-rupted by two short introns, intron 1 spliting the signalpeptide. This structure has been described in many cuticularprotein genes of Lepidoptera (Horodyski & Riddiford, 1989;Binger & Willis, 1994; Rebers et al., 1997; Togawa et al.,

2001) and Tenebrio (Bouhin et al., 1993), but a single intronwas described in Lepidoptera (Nakato et al., 1992; Lampe& Willis, 1994; Shiomi et al., 2000), Diptera (Snyder et al.,1982; Henikoff et al., 1986; Apple & Fristrom, 1991; Qiu &Hardin, 1995; Charles et al., 1997; Dotson et al., 1998) andTenebrio (Mathelin et al., 1998; Rondot et al., 1998), whichwas interpreted as a lack of the intron of an ancestral gene(Charles et al., 1997). Exceptionally, three and four intronshave been found in lepidopteran genes (Rebers & Riddi-ford, 1988; Nakato et al., 1994) and even intronless genesin Drosophila (Charles et al., 1997). Another feature of allcuticular protein genes so far described is the location ofintron 1, interrupting the signal peptide after the first to thefourth codon and more often after the fourth, except for afew genes of the 65A cluster in Drosophila (Charles et al.,1997). Binger & Willis (1994) suggested that such a con-served location of intron 1 may indicate a role in generegulation, although it may also reflect a simple homology.The small size of ACP-20 introns (about 60 bp) is also com-mon to many cuticular protein genes, although it couldexceptionally reach 3.5 kb and 5.8 kb in two lepidopterangenes (Nakato et al., 1992; Lampe & Willis, 1994).

Although numerous cuticular protein genes have beenanalysed, knowledge of the fine mechanisms contributing

Figure 2. Analysis of promoter activity of the ACP-20 5′-flanking region after transient cotransfection with pRL-CMV into the upper epidermis of the pharate adult elytra, in the presence of 10 µM 20E. (A) Luc-F1 to Luc-F9 indicate plasmid constructs of promoter-firefly luciferase fusion vectors containing different lengths of 5′-flanking region from −2900 bp up to +118 bp, including the first intron and the promoterless luciferase vector pGL3-Basic. The arrow in each construct denotes the position of the transcription start site and direction of the transcription. The rectangle represents the putative TATA box. The grey, black and striped rectangles indicate, respectively, the position of untranslated DNA, translated DNA and intron. (B) Firefly luciferase activity was normalized with respect to Renilla luciferase activity as described in the Experimental procedures. The data are expressed as means ± SEM with the numbers of transfections (n). nr, Non-responding transfection. Histograms with the same pattern are not significantly different.

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to their transcriptional control is sparse. In three lepidop-teran genes, a sequence located in the 5′ upstream regionand related to the consensus sequence for the vertebratetranscription factors COUP-TF and HNF-4 is able to bindcellular extracts (Togawa et al., 2001). Similar results wereobtained with a putative octamer cis-acting element ofanother lepidopteran cuticular protein gene (Lampe & Wil-lis, 1994). It has been shown also that nuclear receptorsinvolved in 20E-induced regulatory cascade may play a rolein the regulation of cuticular protein genes. This is the casewith Broad proteins and DHR38, which take part, respec-tively, in the formation of pupal (Zhou & Riddiford, 2002)and adult cuticle of Drosophila (Kozlova et al., 1998). Thisis also true for FTZ-F1, because functional FTZ-F1 bindingsites have been described in two pupal Drosophila cuticularprotein genes (Murata et al., 1996; Kawasaki et al., 2002)and in one adult cuticular peptide of Bombyx (Shiomi et al.,2000).

Few cuticular protein genes have been subjected to pro-moter analysis, and therefore it was interesting to study thecontrol of ACP-20 transcription using transfection analysis.In this regard, we have developed a transient cotransfectionassay of Tenebrio wing upper epidermis cultured in vitro.Polyethylenimine (PEI) was chosen as promoter constructsdelivering vector owing to its high efficiency and very lowcytotoxicity (Boussif et al., 1995; Abdallah et al., 1996).

Table 1. Potential cis-regulatory elements, based on the PatSearch V.1 program, in ACP-20 DNA between −583 and +118 bp present into Luc-F5 to Luc-F9 constructs.

Transcription factor Consensus*

Position into −−−−583 to −−−−405†

Position into −405 to −227†

Position into −−−−227 to −−−−64†

Position into −64 to +47†

Position into +47 to +118†

GAGA GAG −−−−567/−−−−563 (–/0)GATA T/A/CGATAA/G −−−−456/−−−−451 (–/1) −364/−359 (–/0) −−−−155/−−−−150 (–/0) −17/−12 (–/1) +111/+116 (+/1)

−286/−281 (+/0) −5/+1 (–/1)−283/−278 (–/1)

EcRE PuGG/TTC/GANTGC/AC/AC/tPy −−−−545/−−−−540 (+/1) −−−−229/−−−−224 (+/1) +52/+57 (+/1)half sites −−−−170/−−−−165 (+/1) +81/+86 (–/0)

LEF-1 A/TA/TCAAAG −−−−454/−−−−448 (+/1) −235/−229 (–/1) −−−−175/−−−−169 (+/2) −42/−36 (+/2)−−−−129/−−−−123 (–/2)−−−−97/−−−−91 (+/1)

Octamer ATGCA/TAAT −365/−358 (–/1) +78/+85 (–/1)−324/−317 (+/1)−287/−280 (+/1)

TATA box TATAA/TAA/T −35/−25 (+/0)Twist CANNTG −−−−173/−−−−168 (+/0)

−−−−130/−−−−125 (+/0)Zeste T/CGAGT/CG −−−−525/−−−−520 (–/0) −−−−177/−−−−172 (–/1)

*References for consensus binding sites. GAGA: Wilkins & Lis (1998); GATA: Merika & Orkin (1993), Ravagnani et al. (1997); EcRE: Cherbas & Cherbas (1996), Antonieski et al. (1993); LEF-1: van de Wetering & Clevers (1992); Octamer: Verrijzer & Van der Vliet (1993); TATA box: Breathnach & Chambon (1981); Twist: Ephrussi et al. (1985); Zeste: Benson & Pirrotta (1988).†In parentheses are the orientation of the putative binding sites (+ on the strand, – on the other strand) and the number of mismatches compared with the consensus. DNA fragments containing potential positive regulatory elements are in bold type.

Figure 3. Gel-mobility shift assays with nuclear extract from upper epidermis of pharate adult elytra. Assays were performed as described in the Experimental procedures. Ten micrograms of nuclear extracts were incubated with double-stranded labelled probes P1 (located from +47 to +76), P2 (+74 to +90) and P3 (+90 to +118) (lanes 2–7). Lane 1 displays the probe P1 alone. For the competition experiment (lanes 5–7), a 100-fold molar excess of unlabelled probes P1, P2 and P3 was added, respectively. Positions of specific DNA–protein complexes are indicated with asterisks; NS, position of the nonspecific; Free, the free DNA probe.

Functional analysis of an insect cuticular protein gene 487

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Numerous preliminary experiments allowed us to obtainoptimal PEI/DNA and recombinant pGL3-Basic vector/pRL-CMV vector ratios, and to optimize the duration of incuba-tion in transfection medium and of the whole experiment.

Cuticle deposition is known to be triggered directly orindirectly by 20E via a regulatory cascade of gene expression(Ashburner et al., 1974). Two types of ecdysone-responsivecuticular protein genes have been reported, most of whichare expressed after a 20E pulse followed by a decrease inthe hormone (Apple & Fristrom, 1991; Hiruma et al., 1991;Noji et al., 2003), whereas others are 20E up-regulated(Horodyski & Riddiford, 1989; Noji et al., 2003). ACP-20belongs to this second group; in previous in vitro studies,the continuous presence of 20E was necessary for thesecretion of a typical adult cuticle and for the stimulationof ACP-20 expression (Braquart et al., 1996). In our trans-fection assays, the presence of 20E in the culture mediumduring the entire duration of the experiment was necessaryto activate the promoter, confirming that ACP-20 is up-regulated by 20E. After transfection of Luc-F3 (−851/+118),the highest luciferase activity was obtained in the presenceof 10 µM 20E, which corresponds to the pupal physiological20E peak (Delbecque et al., 1978).

Cuticular protein gene expression is tissue- and gener-ally stage-specific. After transfection of Luc-F3 in wingepidermis taken at different stages, luciferase activity wasdetected only in pharate adult epidermis. This indicates thatthe sequence (−851/+118) contains the regulatory elementsof the stage-specificity of ACP-20 expression. However, the

unexpected weak luciferase activity, observed in vitro beforeday 7 and after day 8 following pupation, was unexplainedbecause ACP-20 mRNA was still detected at theses periodsin vivo (Charles et al., 1992).

These in vitro results indicate that this sequence seemsto control practically as well as in vivo the ecdysone respon-siveness and the temporal specificity of ACP-20 geneexpression; therefore, a functional analysis of 5′ and 3′DNA deletions was undertaken. First, a basal promoter ofACP-20 that contains a TATA box was identified between−64 and +1. The correct transcription of ACP-20 is notensured by sequences located upstream of the tran-scription start site but needs a cooperative control throughregulatory sequences located upstream and downstreamof the transcriptional start site. At least three distinctiveregions were found to be responsible for high levels of ACP-20 transcription, two located in the upstream region, atpositions −583/−405 and −227/−64, and one located down-stream of the transcription start site, at position +47/+118.The two last sequences are essential because the deletionof each one reduced the luciferase activity over eighty-fold,and removal of the first sequence resulted in a transcrip-tional decrease less than four-fold.

The nucleotide sequence of these three regulatoryregions was analysed for cis-acting elements assumingthat those lying within could be important positive media-tors in the transcriptional acivity of ACP-20 because theirremoval resulted in a strong decrease in promoter activity.Several potential developmental and tissue-specific cis

Figure 4. Effect of 20E on the promoter Luc-F3 activity after transient cotransfection with pRL-CMV into the upper epidermis of the pharate adult elytra in the presence of 0.1–100 µM 20E during all experiments. Firefly luciferase activity was normalized with respect to Renilla luciferase activity as described in the Experimental procedures. The data are expressed as means ± SEM with the numbers of transfections (n). nr, Non-responding transfection. Histograms with the same pattern are not significantly different.

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regulatory elements as GATA-, octamer-, Twist- and LEF-1-binding sites that are known to be developmentaland tissue-specific regulatory factors (reviewed in Jan &Jan, 1993; Verrijzer & Van der Vliet, 1993; Wegner et al.,1993; Patient & McGhee, 2002; van Genderen et al., 1994;Castanon et al., 2001; Roel et al., 2002) were identified inthese three regions. Interestingly, a similar putative octamer-binding site and a Pit-like sequence, both recognized byPOU-domain proteins, were found in the 5′-flanking regionof stage- or region-specific cuticular protein genes (Nakatoet al., 1992; Bouhin et al., 1993; Lampe & Willis, 1994). Thesequence analysis revealed also putative cis-actingelements, as two zeste sequences and a GAGA-bindingsite, implicated in chromatin spatial structure (reviewed inBenson & Pirrotta, 1988; O’Brien et al., 1995; Tsukiyamaet al., 1994; Wilkins & Lis, 1997).

Considering the important role of 20E in ACP-20 expres-sion, we were particularly attentive towards searching bind-ing sites involved in hormonal regulation. No perfect EcREconsensus sequences but five half-site EcRE consensussequences were found in the three regulatory regions.However, in the 5′-flanking region extending from −603 to

−587, a pseudopalindromic EcRE was found. Severalcuticular protein genes were reported to contain imperfectEcRE and half-sites that might mediate their response toecdysteroids but none of them has been demonstrated asbeing functional (Bouhin et al., 1993; Binger & Willis, 1994;Lampe & Willis, 1994; Rebers et al., 1997; Dotson et al., 1998).Nevertheless, in other noncuticular 20E-induced genes,single motifs have been shown to act as enhancing elementscooperatively with the palindromic sequence (Antoniewskiet al., 1996; Christophides et al., 2000) or to confer alonethe ecdysone responsiveness (Bruhat et al., 1993; Tourmenteet al., 1993). These results indicate that a perfect EcREis not needed for a direct 20E gene regulation. However,cycloheximide experiments strongly suggest that ACP-20is not directly regulated by 20E but requires the synthesisof intermediary regulatory proteins (Braquart et al., 1996).Such proteins involved in the 20E-induced regulatorycascade (TmGRF, TmßFTZ-F1) were found to be syn-thesized by epidermal cells during ACP-20 expression(Mouillet et al., 1999). GRF and FTZ-F1 recognize thesame FTZ-F1 response element (FTZ-F1 RE) (Ueda &Hirose, 1991; Charles et al., 1999). A putative FTZ-F1 RE

Figure 5. Developmental expression profiles of the promoter Luc-F3 activity after transient cotransfection with pRL-CMV into the elytral upper epidermis taken at different times, in the presence of 10 µM 20E. Firefly luciferase activity was normalized with respect to Renilla luciferase activity as described in the Experimental procedures. The data are expressed as means ± SEM with the numbers of transfections (n). nr, Non-responding transfection. Histograms with the same pattern are not significantly different. Timescale is indicated in days. A representation of cuticular events during metamorphosis is shown below. AE, adult ecdysis; AP, adult apolysis; PE, pupal ecdysis; postAC, postecdysial adult cuticle secretion; postPC, postecdysial pupal cuticle secretion; preAC, pre-ecdysial adult cuticle secretion; prePC, pre-ecdysial pupal cuticle secretion; ACP-20 mRNA levels are after Charles et al. (1992).

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is present at position −591/−584 embedded in a putativepseudopalindromic EcRE site. Unexpectedly, its deletiondid not significantly alter promoter activity, suggestingthat the putative FTZ-F1 RE is not functional in transfec-tion experiments. The role of these potential cis-actingelements, located in the three regulatory regions, is stillquestionable and the involvement of unidentified factorscannot be excluded. Nevertheless, the present resultsprovide the basis for a functional characterization of activebinding sites involved in the spatiotemporal regulationof ACP-20.

Regulatory elements of the +47/+118 fragment, containingexon 1 and intron 1, are essential for high transcriptionalexpression of ACP-20. Because the predominant retardedband of gel-mobility shift experiments was obtained using theintronic probe P2 containing one putative octamer-bindingsite and EcRE half-site, the short intron 1, characteristic ofcuticular protein genes, could be considered as an importantelement in the transcriptional regulation of ACP-20. Increasingnumbers of studies emphasize the importance of regulatoryelements located within introns, and most often in intron 1, ofhousekeeping as well as tissue-specifically expressed genes,and such sequences are characterized as positive or negativeregulatory elements, enhancers or silencers and as alter-native intronic promoters (reviewed in Magin et al., 1992;Fatyol et al., 1999; Wang et al., 2002; Kolb, 2003). It hasalso been reported that the mere presence of an intron canincrease gene expression levels and that an exogenousintron could be recognized by the splicing machinery,leading to an increase in transgene expression levels(Palmiter et al., 1991; Choi et al., 1991; Duncker et al.,1997). In Tenebrio, the interactions between intronicsequences and nuclear proteins, observed in gel-mobilityshift experiments, confirm that the intronic up-regulation ofACP-20 is at least due to cis-acting elements. A search forfactors able to bind these cis-acting elements within intron1 and detailed functional studies are now required to definefully the key role played by intron 1 in controlling cuticularprotein gene expression.

Experimental procedures

Animals

Mealworms, Tenebrio molitor, were mass reared at 25 °C onchicken food containing 1% yeast. Dating of larvae and pupae wascarried out by the determination of physiological criteria based onthe observation of eye migration (Delbecque et al., 1978) and theirage represented a mean time scale in days.

Genomic library screening

The genomic library of Tenebrio was screened as previously des-cribed (Bouhin et al., 1993). Briefly, about three genome equivalentswere plated, transferred on to nylon membranes and hydridized at65 °C for 3 h in 6× NaCl/Cit (150 mM NaCl, 15 mM trisodium citrate,

pH 7.0), 5× Denhardt’s solution (Denhardt, 1966), 0.5% SDS and0.1 mg/ml salmon sperm DNA (Sigma). The filters were thenhybridized to the random primed [α32P] cDNA probe (106 cpm/ml)encoding ACP-20 in the same solution (without Denhardt) at 65 °Covernight, followed by three 20 min washes in 1× NaCl/Cit, 0.1%SDS at room temperature, one 30 min wash in 2× NaCl/Cit, 0.1%SDS and two 1 h washes in 0.1× NaCl/Cit, 0.1% SDS at 68 °C. Thefilters were then dried and exposed to X-ray film. One hybridizingclone was isolated after three cycles of screening.

Nucleic acid sequencing

For sequence analysis, a 3.5 kb EcoRI fragment was subcloned inpBluescript SK(+/–) (Stratagene). Insert deletions were generatedusing Exonuclease III according to Henikoff (1984), and sequencingwas performed on both strands using the dideoxy chain terminationmethod (Sanger et al., 1977) until unambiguous data were obtained.Sequences were obtained using either primers that bind to thecloning vector or internal primers. Another restriction DNA fragment,located upstream of the 3.5 EcoRI fragment, was also sequenced.

Computer-assisted analysis of sequence data

The search for putative binding sites for known transcription factorswas performed using the PatSearch V 1.0 program available at theGene Regulation website (http://www.gene-regulation.com) (Win-gender et al., 2001).

Reporter gene constructs

Different fragments of the 5′-flanking region of ACP-20, namedLuc-F1 to Luc-F9 (Fig. 2A), were obtained by PCR and ligated intothe Bgl II–NcoI sites of the pGL3-Basic vector (Promega, Madison,WI, USA) expressing firefly luciferase. All recombinant plasmidswere checked by sequencing. Plasmid DNA used for transfec-tion was purified with QIAGEN Plasmid Midi Kit (QIAGEN Gmbh,Germany). A second plasmid, pRL-CMV vector (Promega), contain-ing the cytomegalovirus enhancer and early promoter elementsto provide high-level expression of Renilla luciferase, was co-transfected to verify transfection efficiency and to normalizemeasurements of luciferase activity.

Tissue culture and co-transfection assay

Forewings from pupae taken at different ages were dissected, thepupal cuticle removed, the upper wing epidermis isolated andplaced into a ninety-six-well plate containing Landureau’s S20medium (Landureau & Grellet, 1972), modified according to theNa+/K+ ratio (65 : 42) and osmolality (380 mOsm) of Tenebriohaemolymph (Quennedey et al., 1983), supplemented with 5%fetal bovine serum (FBS) (Seromed) and, according to the exper-iments, 0.1–100 µM 20E (Sigma) dissolved in ethanol. Plates wereincubated at 25 °C 1 day before transfection. A 0.1 M stock solutionof 25 kDa polyethylenimine (PEI) (Aldrich) was prepared accord-ing to Abdallah et al. (1996). Plasmid DNA (2 µg Luc-F1 to F9 and0.5 µg pRL-CMV vector/epidermis) was diluted in 5% glucose.Fifty microlitres of PEI–DNA complexes, at a ratio of PEI nitrogenper DNA phosphate of 9 (Boussif et al., 1995), was added, then anadditional 100 µl of culture medium (containing 20E). To increasethe efficiency of transfection, the epidermis was centrifuged for10 min at 300 g in a tabletop centrifuge using flatplate centrifuga-tion adaptors (Verma et al., 1998). After 6 h incubation at 25 °C,

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the transfection medium was replaced by fresh culture mediumsupplemented with 5% FBS and 20E.

Measurement of ACP-20 promoter activity

Luciferase activity was measured using the Dual-LuciferaseReporter Assay System (Promega, Madison, WI, USA) 76 h aftertransfection according to the manufacter’s instructions, and aMicrolite TLX1 photon counter (Dynatech Laboratory, Inc.). TheACP-20 promoter activity in transfected epidermis was assessedby the ratio of firefly to Renilla luciferase activity, after subtractingblank and control values for both activities, in order to remove allthe nonrelevant luciferase activity. Blank value was measured inepidermis transfected without the ACP-20 promoter and withoutpGL3, and pGL3-induced luciferase activities were assessed bymeasuring both firefly and Renilla luciferase activities on epider-mis transfected with pGL3 but without the ACP-20 promoter.Samples in which luciferase activity differed from both blank and con-trol experiments were considered responsive. The frequencies ofresponsive transfection are indicated in the figures. For unknownreasons, a few values were not relevant (up to five orders of mag-nitude higher or lower than the remainder). In order to homogenizeour data objectively, for each sample we only took into accountvalues included in the 90% confidence limits. Data were com-pared using Kruskall–Wallis test, eventually complemented with apost-hoc multiple step-by-step comparison test, according to themethod of Nemenyi, Wilcoxon and Wilcox (same sized samples) orthat of Noether (different sized samples) (Scherrer, 1984). Datawere graphically shown (samples with the same pattern are notsignificantly different at P = 0.05).

Epidermal nuclear protein extracts and electrophoretic mobility shift assay

Forewings from pharate adults taken from day 7 to day 10 afterpupation were dissected and upper epidermis samples werestored in liquid nitrogen until extraction. All steps were performedat 4 °C. Wings (around 5 g) were ground in liquid nitrogen using amortar. The resulting powder was vortexed in 20 ml homogeniza-tion buffer A [1 M sucrose, 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES, pH 7.9), 10 mM KCl, 3 mM MgCl2,500 mM dithiothreitol (DTT), 500 mM PMSF, 5 µg/ml bestatin, 5 µg/ml pepstatin], filtered on 100 µm Blutex, then rinsed with 20 ml ofthe same buffer. The homogenate was centrifuged (5 min at 400 g)to remove debris. The supernatant was centrifuged for 5 min at4500 g. The pelleted nuclei were resuspended in 500 µl of bufferB [10 mM HEPES (pH 7.9), 500 mM NaCl, 3 mM MgCl2, 500 mM

DTT, 5% glycerol, 500 mM PMSF, 5 µg/ml bestatin, 5 µg/mlpepstatin] and incubated for 20 min at 4 °C under gentle agitation.The lysed nuclei were centrifuged for 30 min at 20 000 g. Thesupernatant was aliquoted and stored at −80 °C. The proteinconcentration was determined by Bradford assay (Bio-Rad). Double-stranded DNA probes, derived from the +47 to +118 genomicregion, were used for electrophoretic mobility shift assay, probe P1(5′-ATGCTAGTACAAGTGAGTACCACCACCACA-3′) spanning+47 to +76, probe P2 (5′-ACAAATCTGCATTTCCA-3′) spanning+74 to +90 and probe P3 (5′-AGTACCACATTTTCTTCTTGCA-GATTACC-3′) spanning +90 to +118. These probes were labelledwith [α32P]-dCTP using DNA polymerase I Klenow fragment.Nuclear extract (2 µg) was preincubated for 20 min on ice in 20 µlbinding buffer (100 mM HEPES, 250 mM KCl, 12.5 mM MgCl2, 50%glycerol, 10 mM DTT) containing 2 µg poly(dI-dC) with or without a

100-fold molar excess unlabelled probes. The labelled probes(approximately 104 cpm/fmol) were then added and incubated onice for an additional 30 min. All reaction mixtures were analysed byelectrophoresis through a 5% polyacrylamide gel containing25 mM Tris base, 180 mM glycine, 1 mM EDTA and 2.5% glycerol,which had been pre-run for 1 h at 4 °C. Finally, the gel was driedand exposed to a phosphor screen and scanned with a Phos-phoImager SI scanning instrument (Molecular Dynamics).

Acknowledgements

This research was supported by the Centre National dela Recherche Scientifique. C. Braquart-Varnier wassupported by a doctoral fellowship from the Ministère del’Enseignement Supérieur et de la Recherche.

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