The moulting hormone ecdysone is able to recognize target elements composed of direct repeats

ELSEVIER Molecular and Cellular Endocrinology 113 (1995) l-9

ti!iF’., and Mar Endocrhdogy

The moulting hormone ecdysone is able to recognize target elements composed of direct repeats

Pier Paolo D’Avino”, Stefania Crispi”~‘, Lucy Cherbasb, Peter Cherbasb, Maria Furia”*

aDiptntimento di Genetica, Biokqia Genemle e Moiecokue, Univetsit2 di Napoli, uia Mezzocannone 8, I-80134 Naples, Ztafy bDepament of Biotogv, Indiana iJniversi& Bkxmsirgton, IN 47405, USA

Received 3 May 1995; accepted 10 May 1995

Abstract

In Drosophila melatwgaster, three temporally distinct ecdysone-responsive puff sets, the so-called intermoult, early and late puffs, have been described on the salivary gland polytene chromosomes. We have analyzed in detail a DNA segment of the 3C polytene region, from which originates one of the most prominent intermoult puffs, with the aim of identifying ecdysone response elements (E&Es). Here we report that two putative EcREs of identical sequence are located at this puff site. Interestingly, these elements display a novel structural feature, being composed of directly repeated half-sites. Our results show that the EcR/USP heterodimer known to constitute the ecdysone funcrional receptor complex is able to bind to and transactivate through target elements composed of directly repeated half-sites. In addition, we show that these elements are also able to bind efficiently USP alone, suggesting that USP and EcR/USP could compete for their binding to DNA.

Keyworak Ecdysone; E&E; Drosophila; Intermoult puff

1. Introduction

The complex life cycle of holometabolous insects is entirely controlled by variations in the hemolymph titre of a sesquiterpenoid, the juvenile hormone, and a single steroid, the moulting hormone ecdysone (reviewed by Riddford, 1993). Our knowledge about the molecular mechanisms by which the ecdysone acts in its wide pleiotropic role of gene regulation has in- creased substantially in recent years, following the cloning of a gene encoding the ecdysone receptor (EcR) (Koelle et al., 1991). Drosophila EcR binds to DNA as a heterodimer with the USP protein, another member of the nuclear receptor family (Thomas et al., 1993; Yao et al., 1992, 1993). This protein, en- coded by the ultraspiracle (usp) gene, shows both

*Corresponding author, Tel.: + 1-39 81 5526208; Fax: + 1-39 81 5527950.

‘To be considered as joint first author

structural and functional homology with the vertebrate retinoic X receptor (RXR) (Henrich et al., 1990, Oro et al., 1990; Shea et al., 1990). Three isoforms of the ecdysone receptor, showing different tissue dis- tribution, have recently been described (Talbot et al., 1993). However, considering that this single hormone directs a great variety of different tissue-specific re- sponses, and in each tissue a large set of genes is differently regulated at the same developmental time, it is unlikely that this large pleiotropic effect can be explained simply by the presence of different tissue- specific receptor subforms. It seems probable that a large repertoire of E&Es with different properties might exist. Nevertheless, this repertoire still needs to be identified_ Although quite a large number of ecdysone-regulated genes have been characterized, only few functional EcREZ-s have been so far unambiguously defined. A 23-bp sequence present in the hsp27 gene promoter was the first functional EcRE element to be recognized (Riddihough and Pelham,

0303-7207/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0303-7207(95)03584-T

2 P.P. D’Avino et al. / Moiecular and Cellular Endocrinology 1 I3 (1995) l-9

1987). Other EcREs identified at the sequence level came from the Eip28/29 and the Fbpl genes (Cher- bas et al., 1991; Antoniewski et al., 1994). Because these EcREs are all composed of imperfect palin- dromes, the EcR/USP heterodimer is generally thought to bind exclusively to target elements composed of inverted repeats.

Here we report the identification and the characterization of two putative E&Es of identical sequence representing the first example of naturally occurring target elements composed of directly repeated half- sites. Both the newly identified EcREs map at the 3C polytene region, from which one of the most prominent intermoult puff originates.

In D. melanogaster, three temporally distinct ecdysone-responsive sets of puffs have been described on the salivary gland polytene chromosomes: the intermoult, early and late puffs (reviewed by Ashbumer and Berendes, 1978). For a number of years three of us have studied the molecular organization of the 3C intermoult puff region. Six small genes, named Sgs4, Pig-l (Pre-intermoult gene-l), ng-1, ng-2, ng-3 and ng-4 (nested genes 1, 2, 3 and 4) have been so far identified at this polytene region (Muskavitch and Hogness, 1980; Hofmann and Korge, 1987; Furia et al., 1990; Furia et al., 1993). However, only Sgs-4, ng-1, ng-2 and ng-3 are transcribed at a high level exclusively during the third larval instar (Muskavitch and Hogness, 1980; Furia et al., 1990, 1993), and thus belong to the ‘intermoult’ gene class. The finding that ng-1, ng-2 and ng-3 are specifically transcribed during the puffing stage 1 (PSl), the same developmental stage in which the 3C puff has been reported to be most prominent (Ashbumer and Berendes, 1978), has recently suggested a close relationship between 3C puffing and the expression of these genes (D’Avino et al., 1995a).

We have then searched for ecdysone receptor binding sites in the DNA segment harbouring ng-1, ng-2 and ng-3 by using the DNA blotting assay technique (Cherbas et al., 1991). This analysis enabled us to identify two ecdysone receptor binding sites, each mapping within the homologous coding regions of ng-1 and ng-2, a pair of duplicated genes which are more than 95% homologous at the DNA sequence level (Furia et al., 1993). These two high affinity binding sites have identical sequence and share a novel structural property, being composed of direct repeats. The native ng-Elements, as well as synthetic oligonucleotides containing the directly repeated half-sites, are able to bind specifically not only the EcR/USP heterodimer but also USP alone, revealing a property that might have general functional implica- tions in the mechanisms of ecdysone-mediated gene regulation.

2. Materials and methods

2.1. DNA-blotting assay The DNA-blotting assay was performed as previ-

ously described (Cherbas et al., 1991) by using, as starting DNA, various segments of the 3C region previously subcloned in the pUC18 plasmid (Furia et al., 1993).

2.2. Electrophoretic mobility shift assay (EMSA) Electrophoretic mobility shift assays were per-

formed by using EcR (Bl isoforrn) and USP synthesized by coupled in vitro transcription/translation according to the manufacturer’s instructions (Pro- mega, TNT kit). The EcR-BI cDNA clone pCA1 (generously furnished by C. Antoniewski and J.A. Lepesant) and the ~27-1 usp cDNA clone (generously furnished by V. Henrich) were utilized. To detect the binding of the EcR/USP heterodimer, in vitro translated EcR and USP were preincubated as described by Yao et al. (1993); then 1 pmol of the labeled probe (l-5 x lo* counts/min), prepared through forward reaction by T4 polynucleotide kinase in the presence of [ y32 P] ATP (Amersham), was added and the reaction incubated at 20°C for further 30 min. The reaction was loaded onto a 5% non-denaturing polyacryl- amide gel and run in 0.5 X TBE at 4°C; after electrophoresis, the gel was dried and autoradiographed. In EMSA assays using middle-third instar salivary gland nuclear protein extracts, 3 pg of protein extract were used in each assay. The preparation of the extracts was performed essentially as described by George1 et al. (1991).

2.3. Antibodies EcR monoclonal antibody AD4.4 (Talbot et al.,

1993) was a generous gift of D.S. Hogness; this antibody recognizes the Bl isoform. USP monoclonal antibody AI311 (Khoury Christianson et al., 1992) was a generous gift from Dr. F.C. Kafatos laboratory.

2.4. Plasmids The expression constructs tested for ecdysone re-

sponsiveness in transient expression assays were obtained by inserting each of the synthetized oligonucleotides in the X-188-cc-cat vector (Cherbas et al., 1991). The WT oligo flanked by Hind111 sites was blunt ended with Klenow enzyme and then inserted within the SnaBI site of the vector, in one (1 X WT) or two copies (2 x WT). The DR3 and IR oligos were flanked by BglII sites and inserted within the vector BglII site. The Mt-EcR expression vector was con- structed by inserting a FspI/HindIII fragment (blunt-ended in with Klenow enzyme), containing the entire coding region from the EcR-Bl cDNA pMK1

P.P. D;luirw et al. /Molecular and Ceftular Enakiinofogv 113 (1995) I-9 3

(Talbot et al., 19931, into the HincII site of pRmHa-1 plasmid (Bunch et al., 1988). Mt-usp contains the entire insert (excised with SmaI and EcoRV) from the usp cDNA clone pZ7-1 (Henrich et al., 19901, inserted into the HincII site of pRmHa-1.

2.5. Cell culture transfection and CAT assay Kc167/M3 cells were grown in M3 medium (Sigma)

supplemented with 5% foetal bovine serum (Sigma). Transfections and ecdysone treatments were carried out as described earlier (Cherbas et al., 1991). Typi- cally, 5 pg of the cat reporter construct and 10 pg of the HZSOPL plasmid (Hiromi and Gehring, 1987), a /?-Gal expression vector used to normalize the CAT assays, were added in each plate; pUC18 DNA was used as carrier to bring the total amount of DNA to 20 pg. In cotransfection experiments, 5 pg of Mt-EcR and/or 5 pg of Mt-usp were also added. 16 h after transfection, the medium was replaced with fresh medium supplemented with cupric sulfate to a final concentration of 0.7 mM, with or without 1 PM 20-OH ecdysone (Sigma). The cells were then incubated for additional 24 h before harvesting.

For CAT assays, the cells were harvested by centrifugation and washed two times in PBS (phosphate- buffered saline). The cell pellet was resuspended in 0.25 M Tris-HCl (pH 7.5) and the cell lysates, prepared by five cycles of freezing/thawing, were clari- fied by centrifugation. After quantification of the protein concentration by the Bio-Rad assay, the P-Gal assay was performed as described (Sambrook et al., 1989). Appropriate aliquots of the cell extracts were then assayed for CAT activity (Gorman et al., 1982) in a range in which the reaction was linear. Quantitation of CAT activity was done using a Molecular Dynamics PhosphorImager and analyzed with the ImageQuaNT software.

3. Results

3.1. Identification of two high afjinity ecdysone receptor- binding sites at the 3C polytene region

We have screened the genomic region harbouring the ng-1, ng-2 and ng-3 genes for high affinity ecdysone receptor-binding sites by using the DNA-blotting assay (Cherbas et al., 1991). This technique consists of the electrophoretic separation of restriction fragments, electroblotting onto a membrane, and incubation of the membrane with crude cell extract in which the ecdysone receptor has been labeled with the hormone analog 26-[‘251]iodoponasterone A. While restriction fragments devoid of specific receptor binding sites yield an autoradiographic signal roughly propor- tional to their length, those containing high affinity binding sites can be recognized by their disproportio nately strong signal.

When a 3.5-kb DNA segment containing the ng-1, ng-2 and ng-3 genes was subjected to this procedure, two high affinity sites for the ecdysone receptor were unambiguously revealed. These binding sites lie within the ng-1 and ng-2 coding sequences, which, as previously described, are more than 95% homologous at the nucleotide sequence level (Furia et al., 1993). As depicted in Fig. 1, strong disproportionate signal was in fact shown by both a 229-bp XhoI/HindIII fragment containing the 3’ end of ng-2 coding region (lane 5) and by a 491-bp &I/Hind111 fragment similarly containing 123 bp of the 3’ end of ng-1 coding sequence and an additional 368 bp of ng-1 3’ flanking region.

In order to map these high affinity binding sites more precisely, the 491-bp X&I/find111 fragment was gel-purified and then digested further. Note that, given that the non-specific signal decreases with the fragment size, the smallest fragments of the molecular weight marker (lane M) are not detected in the autoradiography. Conversely, fragments of compara- ble sizes containing high-affinity binding sites are detectable as a consequence of their disproportionately strong signal. A 93-bp xhoI/DdeI fragment (Fig. 1, lane 2) was the smallest fragment to test positive in the assay. Sequence analysis revealed that this fragment contains two directly repeated copies of the heptamer 5’-AAGGTCA-3’ spaced by 11 bp. This sequence includes a perfect match to the hexamer 5’-RGT/GTCA-3’, generally accepted as consensus for the EcRE half-site (Cherbas et al., 1991). A Copy of the putative EcRE identified by the DNA blotting assay is present in both the ng-1 and ng-2 coding regions. Interestingly, the coding regions of these pair of highly homologous genes diverge only 46 bp down- stream (Furia et al., 1993).

3.2. The q-Elements are able to bind the EcR / USP heterodimer as well as USP alone

The 93-bp xhoI/DdeI fragment containing the putative ng-EcRE was then tested for its ability to bind specifically the in vitro-translated EcR/USP heterodimer in an Electra Mobility Shift Assay (EMSA). When this fragment was used as a probe, a low mobility complex corresponding to the binding of the EcR/USP heterodimer is clearly detected (Fig. 2a, lane 8). It has been reported that the binding of the EcR/USP heterodimer to the palindromic hsp27- EcRE is enhanced by the addition of 20-OH ecdysone (Yao et al., 1993). We confirmed this effect also on the ng-Element (Fig. 2a, lane 9; 2b, lane 2). As expected for such a heterodimeric species, both EcR and USP antibodies can supershift the complex (Fig. 2a, lanes 10-11). Surprisingly, we noticed that the addition of antibodies to USP causes the supershifting of an additional complex of higher mobility (Fig. 2a,

603

310 281 271 234

194

118

72 t

Ml23456 --_-_--

P.P. DAvino et al. /Molecular and Cellular Endocrinology 113 (1995) l-9

MM123456 -------

ng-2 ng-3 ng-1

x H X P P X H

I I I I I I

e 1

lane S

XD HeH

id ‘j

lane I

lane 2

lane 3

lane 3

Xhol * * Ddel CmmmAT*T-mGG CBTA__ ccnlTAr?lwlmmccccAGAGTc

Fig. 1. Identification and mapping of the ng-Elements by DNA blotting assay. The DNA fragments were separated by electrophoresis in duplicate panels; the first panel was stained with ethidium bromide (a) and the second incubated with a ‘251-labeled receptor ligand mixture lb). The 491-bp XhoI/HindIII DNA fragment containing the 3’ end of ng-1 and its 3’-flanking sequence was isolated and digested with SauIIIA (lane 1); DdeI/HincII (lane 2); HincII (lane 3) and AuuII (lane 4) restriction enzymes. A 659-bp %I fragment derived from the corresponding 3’ region of the ng-2 gene was also analysed after digestion with Hind111 (lane 5). A DNA fragment containing the Upstream E&E of the Eip28/29 gene was loaded as positive control (lane 6) Hue11 digest of 0X174 DNA was used as molecular weight marker (lane M). In (c), the map outlines the organization of the genomic region; the relative position of the fragments that bind the labeled receptor disproportionately is shown below. Enzyme abbreviations are H, HindIII; X, BoI; P, &I; A, AuaII; D, DdeI; Hc, HincII; S, SauIIL4.

P.P. DXvino et al. /Molecukuand Cellu&r&bcrinoiogv 113 (1995) 1-9 5

a) 2OHF. - - - - + - - - + + + EcR -.-mm-++ +++ USP - - - + + + - + + + + Ab-EcR _ _ _ _ _ _ _ a _ + - Ah-USP _ _ + _ _ + _ _ _ _ +

b)

20HE - + + +

Ab-EcR _ _ + -

Ab-USP _ _ - +

t

I 2 3 4 5 6 7 X 9 IO II

Fig. 2. The ng-Elements bind both to the EcR/USP heterodimer and to USP alone. (a) The 93-bp XhoI/DdeI fragment which was positive in the DNA blotting assay was tested in EMSA experiments for its ability to bind to in vitro translated USP alone (lanes 4-61, EcR alone (lane 7) or the EcR/USP heterodimer (lanes 8-11). Monoclonal antibody against EcR or USP was added as indicated on the top. As control, lane 1 shows the probe alone; lanes 2-3 the probe after incubation with the unprogrammed rabbit reticulocyte lysate, in the absence (lane 2) or in the presence (lane 3) of monoclonal antibodies against USP. As expected, antibodies to USP do not recognize the non-specific complexes formed after incubation with the unprogrammed lysate (lane 3). The probe is able to bind efficiently to USP alone (lane 4); the complex - marked by the lower arrow on the left - does not increase after addition of ecdysone (lane 5) and, as expected, is supershifted by the addition of anti-USP antibody (lane 6). The probe does not bind EcR alone (lane 7), while it efficiently binds the EcR/USP heterodimer (lane 8); the addition of 20 PM 20-OH ecdysone enhances the formation of the heterodiieric complex (lanes 9-ll), which is supershifted by both anti-&R (lane 10) and anti-USP monoclonal antibodies (lane 11). In (b), the same probe was incubated with third instar salivary glands nuclear protein extracta in the absence (lane 1) or in the presence of 20 /.LM 20-OH ecdysone (lanes 2-4). The major complex, marked by the arrow on the left, is enhanced by the addition of ecdysone and is supershifted by both anti&R (lane 3) and anti-USP antibodies (lane 4).

lane 11). This additional supershifted complex corre- sponds to the binding of USP alone. In fact, USP alone is able to bind the probe efficiently (Fig. 2a, lanes 4-5), while incubation with EcR alone does not result in the formation of any retarded complex (Fig. 2a, lane 7). Although the USP-complex often comi- grates with additional complexes which do not contain USP, USP-specific binding can be unambiguously detected by the supershifting caused by the addition of USP-specific antibodies (Fig. 2a, lanes 6 and 11).

USP binding is not affected at all by ligand addition (Fig. 2a, lane 5); this is to be expected, since USP is incapable of binding ecdysone (Yao et al., 1993). The property of binding USP in the absence of EcR has so far not been reported for any other ecdysone target element. This ability is likely to depend on the se-

quence and arrangement of the element’s half-sites. Interesting to note, the 5’-AGGTCA-3’ hexamer identically repeated within the ng-Element half-sites is also included in the USP binding site described at the chorion ~15 gene promoter (Khoury Cristianson et al., 1992).

To check whether the ng-Element could be in vivo a potential direct target of the EcR/USP heterodimer, we have tested this element as a probe in an EMSA assay after incubation with protein nuclear extracts of middle-third instar salivary glands, that is the same tissue in which the ng-genes are specifically expressed. As shown in Fig. 2b, several nucleoprotein complexes can be detected in this assay (lane 1). However, the major retarded complex clearly derives from the binding of EcR/USP, since it is enhanced

6 P.P. D’Avino et al. /Molecular and Cellular Endocrinology 113 (1995) l-9

by the addition of 20-OH ecdysone (Fig. 2b, lane 2) and is specifically supershifted by the addition of anti-EcR or anti-USP antibodies (Fig. 2b, lanes 3-4). In contrast, the binding of USP alone has not been detected in third instar salivary gland nuclear extracts. Whether the binding of USP to the ng-Elements in vivo might be restricted to other tissues or at developmental times in which the ng-genes are not expressed remains to be established by future experiments.

3.3. Further molecular characterization of the ng- Elements

The DNA blotting and EMSA assays permitted us to identify a 93-bp fragment capable of binding EcR/USP and USP alone. This fragment contains a pair of direct repeats of the EcRE consensus half-site. In order to test whether these repeats are actually responsible for the binding properties, we synthetized a 33-bp oligonucleotide centered about the directly arranged half-sites (see Table 1). The wild type oligonucleotide (WT) and a mutated version (Mut 1) carrying specific sequence alterations within the half- sites were tested for their ability to bind the EcR/USP heterodimer (Fig. 3). The wild type oligonucleotide, encompassing the direct repeated half-sites and 4 flanking bp on each side, is sufficient to form specific complex with both the FcR/USP heterodimer (Fig.

a)

w1’_ Mutl

b)

3a, lanes l-3) and USP alone (Fig. 3~). Mut 1, carrying two substitutions within the right half site and a single substitution in the left half site failed to bind both the FcR/USP heterodimer and USP alone (Fig. 3a, lanes 4-6). As expected, the WT oligonucleotide, when added as cold competitor, is able to compete efficiently the binding of the EcR/USP heterodimer and that of USP alone to the entire 93-bp DNA fragment, whereas Mut 1 fails to compete (data not shown). Note that two out the three nucleotide substitutions introduced in Mut 1 involve bases which have been previously shown by methylation and hydroxyla- tion interference analyses to be important for USP binding [23], specifically the G - A transition introduced in the right half-site and the A + G transition introduced in the left half-site (see Table 1).3tbl 1

An additional oligonucleotide, named DR3, was designed to test the effect of the spacer length on the efficiency of EcR/USP binding. In DR3, two wild type half-sites were separated by 3 bp, rather than I1 bp as in the native ng-Elements. This reduction in the spacer length does not change significantly the efficiency of EcR/USP binding (Fig. 3b, lane 4). The EcR/USP heterodimer is also able to bind an additional oligonucleotide (IR) in which the ng-half-sites were arranged as inverted repeats with a single nucleotide spacing. This oligonucleotide is essentially

IR DR3 -- 20HE + - - -

Fig. 3. Molecular characterization of the ng-Elements. Synthetic oligonucleotides (see Table 1 for structures) were tested for their ability to form specific complexes with in vitro translated EcR/USP. Monoclonal antibodies against EcR or USP were added as indicated on the top. In the figure, the position of the specific complexes is marked by the arrows on the left, while the position of the same complexes after supershifting is marked by the arrows on the right. The WT oligonucleotide is shown to form both heterodimeric (a, lane 1) and USP complexes Cc; in lane 3 USP binding can be followed by the supershifting caused by the addition of USP antibodies). In Cd), EcR atone is shown to be able to bind the IR oligonucleotide (lane 1); the complex is supershifted by the anti-EcR antibody (lane 2).

P.P. D’Avino et al. /Mokc&rand Cellular Edwinoh 113 (1995) l-9 7

Table 1 Nucleotide sequences of the synthetic oligonucleotides used in the EMSA and the transient expression experiments described in the text

Name Sequence

WT GCG.4MGGTCAAGAGGCCAAAGAAGGTCAGGAA CGCITI’CCAG’IT~CCGUGTCCIT

Mut 1 GCGAAAGGTCsAGAGGCCAA4GA~GTCAGGAA CGCITTCCAGCITCTCCGGTIT~*CAGTC~

DR3 GCGAAAGGTCAAGGMGGTCAGGAA CGCTTI’CCAGTTCCITCCAGTCCTT

IR GCGA AAGGTCAGTGACCITGGAA CGC?ITECAGTCACTGGAACCll-’

identical to the EcRE consensus sequence (Cherbas et al., 1991). As expected, IR had high afkity for EcR/USP (Fig. 3b, lane 1). Interestingly, we noticed that EcR alone is able to bind to this palindromic structure at a low but detectable level (Fig. 3d). The low mobility of the EcR complex suggests that it binds to the IR probe as homodimer. Since EcR alone is unable to bind to the hsp27 EcRF (Yao et al., 1992; Thomas et al., 1993) we assume that the perfect palindrome included in the IR oligonucleotide matches more satisfactorily the EcR optimal binding site. Taken together, the data obtained indicate that subsets of suitable E&Es can bind USP or EcR either alone or in combination.

3.4. Functional definition of the ng-Elements by transient expression experiments

To establish the functional role of the ng-Elements identified in the experiments described above, we tested the role of the WI, DR3 and IR oligonucleotides in driving ecdysone-dependent transcriptional activation of the reporter CAT gene. Each construct was then tested by transient expression into Drosophila ecdysone-responsive Kc cells treated or untreated with the hormone. Surprisingly, the wild type ng-Element was unable to confer ecdysone responsiveness in this first set of transfection assays (Fig. 4a). In contrast, a significant hormonal induction was shown by both DR3 and IR constructs, whose hormonal responsiveness was of about 4- and g-fold, respectively. Hence, the reduction of the spacer length from 11 to 3 bp is sufficient to convert the native, unresponsive ng-Element into an active element able to confer ecdysone induction to a minimal promoter in Kc cells. The IR element is very slightly more active than the hsj27-element, indicating that a consensus EcRE sequence supports an induction higher than that given by the hsp27-E&E.

Thus, the native ng-Elements fail to mediate the ecdysone response in Kc cells, whereas the DR3 element is able to confer hormonal-induced response. Given that the only difference between these two

a) 3 25%-

7.7Y 7.1x

4.2x

X-188 hsp IXWT 2xWT DR3 IR

W 4.4x

- ,_ EcR USP EcRlUSP

Fig. 4. The ability of the oligonucleotides derived from the ng-Ele- ment sequence to mediate the ecdysone response in Kc167 responsive cells without (a) or after cotransfection of expression vectors for either EcR and USP (b). The same vector containing a copy of the bsp-27 FcRE was used as internal control. CAT activity was followed by transient expression experiments; grey columns indicate the percentage of acetylated chloramphenicol in the absence of ecdysone, black columns that in the presence of the hormone. Results shown are the average of at least three independent experiments, after normalization for the /3-Gal activity derived from a cotransfected HZ5OPL plasmid (Hiromi and Gehring, 1987); the standard deviation was always s 10%. In (b), EcR and usp down- stream of the inducible metallothionein promoter were separately or simultaneously cotransfected along with the 2 X WT-CAT reporter construct in the presence of 0.7 mM CuSO,.

elements is the length of the spacer, we considered the reasons for their different transactivation ability. A possibility is that the ng-Elements may be struc- turally inactive as E&Es, essentially because of the unusual length of their spacer. Alternatively, we considered the hypothesis that the long spacer of the native ng-Elements may contain an additional binding site for factor(s) abundant in Kc cells. According to this hypothesis, a putative factor(s) present in Kc cells might specifically compete with the EcR/USP heterodimer for its binding to the WT ng-Elements, but not to DR3. To test this possibility, we assayed the ecdysone-responsiveness of the WT oligonucleotide in Kc cells which were expressing elevated levels of EcR, USP, or both proteins. When the reporter plasmid was co-transfected with plasmids expressing EcR and

8 P.P. DYvino et al. /Molecular and Cellular Endocrinology 113 (1995) l-9

USP, ecdysone caused a 4-fold induction of CAT activity (Fig. 4b). Interestingly, EcR alone caused a similar level of induction, while USP alone gave a lower but significant induction. Since USP alone is incapable of binding ecdysone (Yao et al., 1993), the ecdysone induction seen after USP overexpression must be mediated by EcR/USP. We assume that the induction seen after EcR overexpression is also mediated by EcR/USP, since we have been unable to detect binding of EcR alone to the ng-WT element in vitro. The fact that overexpression of either compo- nent of the heterodimer can apparently lead to an increase in the titre of the heterodimer may suggest that the reaction EcR + USP + EcR/USP has an equilibrium favouring the monomers. Alternatively, the hypothesis that USP may heterodimerize with other(s) member(s) of the nuclear receptor family might well explain this result.

4. Discussion

The ability of the EcR/USP functional ecdysone receptor to bind in vitro synthesized oligonucleotides composed of directly repeated half-sites has recently been noticed by our group (D’Avino et al., 1995b) and, independently, by Homer et al. (1995). Here we show that, in addition to the previously described palindromic E&Es, naturally occurring ecdysone binding elements might be composed of direct repeats. Our results also show that directly repeated elements, such as DR 3 or the WT oligonucleotides, are able to confer ecdysone responsiveness to a minimal promoter in transfection assays. This finding suggests that the repertoire of EcREs mediating the ecdysone response in vivo may be much larger and more degenerate than previously suspected. More- over, it adds further relationship between invertebrate and vertebrate hormone response elements, since the property of recognizing HREs formed by either inverted or directly repeated half-sites is typical of the type II vertebrate receptors (reviewed by Stunnen- berg, 1993).

An interesting property displayed by the native ng-Elements and the synthetic oligonucleotides derived from their sequence is the ability to bind USP alone. The fact that both EcR/USP and USP can bind to the same response elements clearly implies that they may compete for DNA binding, a finding that may be relevant for the transcriptional regulation of ng-1,2,3 as well as for other ecdysone-regulated genes. The formation of the heterodimer will depend on the relative level of the two proteins and therefore may be cell type dependent; moreover, given that ecdysone stabilizes the heterodimer, competition between EcR/USP and USP might be modulated by the variations in the hemolymph titre of the hormone.

The observation that the binding of USP occurs preferentially if the 5’-AGGTCA-3’ hexamer forms a direct repeat (this paper; Khoury Christianson et al., 1992) suggests that competition between EcR/USP and USP may be a regulatory mechanism operating preferentially for the E&Es composed of directly repeated half-sites.

The identification and the characterization of high affinity binding sites for the ecdysone receptor within the ng-1 and ng-2 coding regions strongly suggests that these sites may play an active role in triggering the 3C puffing. Although the role of the ng-Elements in vivo remains to be firmly established and deserves further investigation, their functional role on the regulation of the ng-genes in vivo is supported by the observation that, after incubation with third instar salivar gland nuclear protein extracts, the major retarded complex detected in an EMSA assay is due to the binding of the EcR/USP heterodimer. The fact that the ng-Elements are very weak EcREs when tested in Kc cells is likely to reflect differences in the conditions of the in viva/in vitro experiments and reveals aspects of cell type specificity in the Kc cells. We suggest that further conditions, such as, for example, specific interaction of the EcR/USP heterodimer with additional transcription factors, or post-transcriptional modifications of the receptor, must be required to confer to the ng-EcREs in vivo a more effective transactivation response to the hormone than that observed in vitro.

Acknowledgements

We are grateful to C. Antoniewski and J.A. Lepe- sant for having generously furnished the Ecr-Bl cDNA clone pCA1 and to V. Henrich for his generous gift of the usp cDNA clone pZ7-1. We are also grateful to D. Hogness, M. Arbeitman and T. Watanabe for the generous gift of the anti-EcR monoclonal antibodies and to F. Kafatos, D.L. Ring and J. Sutherland for having generously supplied anti-USP monoclonal antibody. We thank J.A. Lepesant, M. Lava1 and C. Antoniewski for sharing useful and helpful informa- tion during the course of this work.

This work was supported by grants of the CNR Target Project on Biotechnology and Bioinstrumenta- tion and MURST 40% and 60% to M.F, and by a NIH award to P. Cherbas. P.P. D’Avino was supported by a Ph.D. CEINGE Fellowship through Napoli Ricerche.

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The moulting hormone ecdysone is able to recognize target elements composed of direct repeats