Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway

Florence Janody1,*, Rachel Sturny1, Valérie Schaeffer2,{ddagger}, Yannick Azou1 and Nathalie Dostatni1,§

1 Developmental Biology Institute of Marseille, Laboratoire de Génétique et Physiologie du Développement, Université de la Méditerrannée, Luminy, Case 907, 13288 Marseille Cedex 09, France
2 Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA
* Present address: Skirball Institute of Biomolecular Medicine, 540 First Avenue, New York, NY 10016, USA
{ddagger} Present address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
§ Present address: UMR218, Curie Institute-Section of Research, Pavillon Pasteur, 26 rue d’Ulm, 75 248 Paris Cedex 05, France

¶Author for correspondence (e-mail: nathalie.dostatni{at}curie.fr)

Accepted March 27, 2001


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcriptional activity of the Bicoid morphogen is directly downregulated by the Torso signal transduction cascade at the anterior pole of the Drosophila embryo. This regulation does not involve the homeodomain or direct phosphorylation of Bicoid. We analyse the transcriptional regulation of Bicoid in response to the Torso pathway, using Bicoid variants and fusion proteins between the Bicoid domains and the Gal4 DNA-binding domain. We show that Bicoid possesses three autonomous activation domains. Two of these domains, the serine/threonine-rich and the acidic domains, are downregulated by Torso, whereas the third activation domain, which is rich in glutamine, is not. The alanine-rich domain, previously described as an activation domain in vitro, has a repressive activity that is independent of Torso. Thus, Bicoid downregulation by Torso results from a competition between the glutamine-rich domain that is insensitive to Torso and the serine/threonine-rich and acidic activation domains downregulated by Torso. The alanine-rich domain contributes to this process indirectly by reducing the global activity of the protein and in particular the activity of the glutamine-rich domain that might otherwise prevent downregulation by Torso.

Key words: Bicoid morphogen, Homeodomain, Downregulation, Torso receptor tyrosine kinase, Drosophila


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Head and thorax development in the Drosophila embryo requires the maternal determinant of the anterior patterning system bicoid (bcd) (Frohnhofer and Nusslein-Volhard, 1986). The Bcd protein is expressed as a maternal anteroposterior concentration gradient in the early developing embryo (Driever and Nusslein-Volhard, 1988b; Struhl et al., 1989) and it is necessary for the expression of numerous zygotic genes in distinct anterior domains. Among them, the gap gene hunchback (hb), which is required for the formation of the segmented part of the head and of the thorax, is activated by low levels of Bcd and expands from 54% to 100% of egg length (EL). In contrast to hb, the expression of another Bcd target gene, the head gap gene orthodenticle (otd), which is involved in the development of antennal and pre-antennal segments, is restricted to 100-70% of EL in the domain where high levels of Bcd concentration are found. Bcd is a homeodomain (HD)-containing transcription factor that probably activates directly transcription in the blastoderm embryo, by binding to its DNA targets (Driever and Nusslein-Volhard, 1989; Bellaïche et al., 1996; Gao and Finkelstein, 1998). As it specifies distinct developmental fates by specifically activating zygotic genes in distinct domains that respond to a series of Bcd concentration thresholds, the Bcd protein behaves as a typical morphogen.

The terminal group genes mediate the development of the terminal structures called the acron at the anterior and the telson at the posterior (St Johnston and Nusslein-Volhard, 1992). Interestingly, in addition to the lack of anterior development, embryos from bcd females develop a telson instead of an acron at the anterior (Frohnhofer and Nusslein-Volhard, 1986). This indicates that the combined activities of both anterior and terminal determinants are required to establish the acron. Terminal activity is mediated by the torso gene, which encodes a receptor tyrosine kinase (RTK) expressed at the surface of the embryo and activated only at the poles by a locally activated ligand (Perrimon, 1993). The activation of the Torso RTK leads to the activation of the ras signal transduction cascade that results in the zygotic derepression of the tailless (tll) and huckebein (hkb) genes (Liaw et al., 1995; Paroush et al., 1997). tll and hkb are the zygotic mediators of the terminal system posteriorly but a different output of the terminal system has been postulated at the anterior, as anterior terminal structures do form in tll-/hkb- double mutant embryos (Strecker et al., 1986; Weigel et al., 1990). Interestingly, several of the Bcd target genes, including hb and otd, are first expressed during syncytial development as anterior caps that are repressed at the pole upon activation of Torso later on during cellularisation. The persistence of hb at the anterior pole alters the formation of terminal derivatives, such as the labrum and the foregut, and represses the expression of three zygotic genes, cap-n-collar (cnc), wingless (wg) and hedgehog (hh), which are involved in the formation of these structures (Janody et al., 2000a). High levels of Bcd can functionally rescue the lack of terminal activity at the anterior pole (Schaeffer et al., 2000), suggesting that Bcd and Torso can act independently on common target genes. Indeed, cnc, wg and hh are positive targets of Bcd and Torso. These genes are likely to be repressed by the persistence of hb at the anterior pole in torso mutants (Janody et al., 2000a), and over-expression of Bcd might bypass their repression by hb in this mutant background. Thus, Torso-induced repression of hb plays a key role in the interaction between the anterior and terminal systems for the formation of the acron.

The components of the ras signalling cascade, D-sor (MAP kinase kinase; Dsor1 – FlyBase) (Tsuda et al., 1993) and D-raf (phl – FlyBase) are required for the anterior repression of Bcd targets whereas tll and hkb are largely dispensable for this effect (Ronchi et al., 1993; Gao et al., 1996; Janody et al., 2000b). The insertion of three copies of the heterologous activation domain of the yeast GCN4 protein within Bcd renders the resulting fusion protein (Bcd-GCN4) insensitive to Torso inhibition (Ronchi et al., 1993). This indicates that modifications of Bcd could prevent the effect of Torso. Indeed, Bcd transcriptional activity is itself inhibited by the Torso pathway, as shown by Torso-induced downregulation of a Bcd-Gal4 fusion protein, in which the Gal4 DNA-binding domain replaces the homeodomain (Bellaïche et al., 1996). Most phosphorylations of Bcd are dependent on Torso activity (Ronchi et al., 1993) and occur in vivo on MAP kinase (MAPK) sites (Janody et al., 2000b). Surprisingly, however, in vivo mutational analysis indicates that these phosphorylations are not involved in the anterior repression of Bcd target genes but instead contribute to Torso-dependent strengthening of the Bcd morphogenetic gradient in the rest of the embryo (Janody et al., 2000b). This analysis suggests that the molecular mechanism responsible for the downregulation of Bcd by Torso does not rely on direct modifications of the Bcd protein but rather involves modification of a Bcd partner that interacts with Bcd. Besides the HD and the serine/threonine (ST)-rich domain that contains most of the MAPK phosphorylation sites, the Bcd protein contains three putative activation domains located at its C terminus: a glutamine-rich domain (Q), an alanine-rich domain (A) and an acidic domain (C). As a Bcd deletion variant lacking all three activation domains (Q, A and C) rescues the lack of wild-type Bcd to viability (Schaeffer et al., 1999), the N-terminal region of Bcd must contain at least one additional activation domain that mediates the activation of its target genes.

In the present study, our aim was to identify the domain of Bcd that mediates its inhibition by the Torso pathway. We first aimed to understand the biological behaviour of the Bcd-GCN4 protein that is insensitive to downregulation by Torso. We show that it is the intrinsic properties of the GCN4 activation domains that render Bcd-GCN4 insensitive to downregulation by Torso. This indicates that the molecular mechanism responsible for the downregulation of Bcd by Torso probably involves an inhibition of the activation process. To better understand the activating potential of Bcd, we performed a structure/function analysis of Bcd in cell culture and in the early embryo using deletion variants (Schaeffer et al., 1999) and fusion proteins containing the Gal4 DNA-binding domain with multiple copies of the Bcd domains. This analysis established: (1) that the Q domain functions as an autonomous activation domain that is not sensitive to downregulation by Torso; (2) that the ST and C domains function as autonomous activation domains that are both downregulated by Torso; and (3) that the A domain functions as a repressor domain in vivo and its activity does not seem to be regulated by Torso at the anterior pole. We propose that the direct downregulation of Bcd transcriptional activity by the Torso pathway results from the competing activity of the Q domain insensitive to Torso with both the C and the ST domains that mediate downregulation by Torso. The contribution of the A domain in this process might be to reduce the general activity of Bcd activation domains, in particular, the activity of the Q domain that would otherwise prevent inhibition by Torso.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA constructs
To obtain the Bcd-GCN4C-coding sequence, we first performed a partial SacII digest of a pKS derivative that contained the bcd genomic sequence (Bellaïche et al., 1996) and inserted at position 3975 (Berleth et al., 1988) of a double-stranded oligonucleotide (5'GCTGCTAGCCGAGGC-3'/5'-CTCGGCTAGCAGCGC-3') that contained a NheI restriction site (underlined). The sequence coding for the three copies of the GCN4 activation domain was amplified by PCR from the Bcd-GCN4 sequence (a gift from G. Struhl (Ronchi et al., 1993)) and it was inserted in this newly created unique NheI restriction site, in frame with the Bcd-coding sequence at its C terminus. Oligonucleotides used for amplification were 5'-CTATGCTAGCTCTAGAGGACCTGGGTCGACGGATCC-3' and 5'-CTAATGCTAGCAGATCTCTCGAGGCCGCCATTGACATTGGTCCC-3' (bcd sequence underlined). To prevent differences due to construct background, the sequence coding for Bcd-GCN4 was first inserted in pPACBcd (Janody et al., 2000b) using XbaI and EcorV partial digestions. The genomic fragments that allow expression of the Bcd-GCN4 and Bcd-GCN4C proteins in the embryo as a maternal anterior gradient were isolated as a BamHI/BamHI cassette from, respectively, the pPACBcd-GCN4 and pKSBcd-GCN4C and cloned into the BglII site of pCasPeRbcdBglII (Bellaïche et al., 1996), which was used for germline transformation. The Bcd deletions used in transfection experiments were obtained by PCR amplification of Bcd-coding fragments cloned at the unique BamHI restriction site of pPAC (Krasnow et al., 1989). Oligonucleotides used for HP/HD and HD/ST amplifications were 5'-AATTGGATCCATGGCGCAACCGCCGCCAG-3', 5'-TTAAGGATCCCTACGATTGGATCTTGTGAC-3'; and 5'-AATTGGATCCATGCCACGTCGCACCCGCACC-3', 5'-TTAAGGATCCCTAGAAGAACTGGCCGCC-3', respectively (bcd sequence underlined). The cloning of {Delta}QAC was described in (Janody et al., 2000b).

The transcription unit pnos-Gal4-Actdomain-Bcd3'UTR in which the sequence coding for the fusion proteins, between the Gal4 DNA-binding domain (1-94) and simple or multiple copies of the putative activation domains of Bcd, are placed under the control of the nanos (nos) promoter and the Bcd 3' untranslated region (UTR), were obtained and processed as pnos-Gal4-GCN4-Bcd3'UTR (Janody et al., 2000a). The oligonucleotides used for amplification of the sequence of each of the Bcd domains were 5'-CTATTCTAGATCGACCAATGTCAATGGCG-3' and 5'-GATAGGATCCGTAGCTAGCGGCTTGCTTTTGCTGG-3' for the Q domain; 5'-CTATTCTAGAGCCAGCGCCTGTCGCG-3' and 5'-GATAGGATCCGTAGCTAGCGTAGACCTCGGAGCCCGG-3' for the A domain; 5'CTATTCTAGAGCTGCCTCGCCGGGC-3'and 5'-GATAGGATCCGTAGCTAGCATTGAAGCAGTAGGC-3' for the C domain; and 5'-TCATCTAGAGTAGATCGCACAAG-3' and 5'-TAGGATCCTTAGCTAGCTAAGCCGCCATTGACATT-3' for the ST domain (bcd sequences underlined). The sequence coding for the B6 and B42 activation domains were amplified by PCR from genomic DNA from the T335 and T337 stocks (Tübingen collection), respectively, using the following oligonucleotides that hybridise in the bcd genomic sequence (underlined): 5'-CTATTCTAGAGGGGGGCCAGGACCTGG-3' and 5'-GATAGGATCCCTAACTTTCTACGCGTAG-3'. The three copies of the GCN4 activation domain were amplified as described above and the unique copy of this domain was amplified using the following oligonucleotides: 5'-CTATGCTAGCTCTAGAGGACCTGGGTCGACGGATCC-3' and 5'-CTATGCTAGCAGATCTCTATCGACCTGCCTTATCAGCG-3'. The amplified fragments were digested by XbaI and either NheI or BamHI (bold) for multimerisation and cloning in frame, downstream of the sequence that codes for the Gal4 DNA-binding domain (XbaI) and upstream of the Bcd 3'UTR (BamHI). The fragment coding for the VP16 activation domain was obtained by XbaI and BamHI restriction digests of the pCG-Gal4 VP16 plasmid (a gift from W. Herr (Das et al., 1995)) and inserted downstream of the Gal4 DNA-binding domain-coding sequence and upstream of the Bcd 3'UTR. The pnos-Gal4-Actdomain-Bcd3'UTR transcription units were inserted as KpnI/NotI fragments in pCasPeR4 for germline transformation and the Gal4-Actdomain-Bcd3'UTR sequences were inserted in pPAC as a NruI/NotI fragment for transfection experiments. In these latter constructs the Gal4 ATG is located approximately a hundred base pairs downstream of the actin promoter transcription start site of pPAC.

Drosophila stocks and transgenics
Mutant stocks were bcdE1 (Frohnhofer and Nusslein-Volhard, 1986), torPM and tor4021 (Klingler et al., 1988). P-element transformed lines were Bcd3-lacZ (Ronchi et al., 1993), UAS-placZ (Rorth, 1998), Bcd-Gal4 (Bellaïche et al., 1996), {Delta}Q, {Delta}A, {Delta}C, {Delta}QA, {Delta}AC, {Delta}QC and {Delta}QAC (Schaeffer et al., 1999). Injections for transgenics were performed as described in (Bellaïche et al., 1996). For each Gal4 fusion construct at least two independent insertions were analysed and the line giving the strongest expression of the UAS-placZ was chosen for further analysis. The nature of the transgene was verified by genomic PCR amplification using primers hybridising within the sequence of the Gal4 DNA-binding domain (5'-GCATTGTTAACACTGCCAGGC-3') and the Bcd 3' UTR (5'-ACTAGACCTAACTTTCTACGCG-3'). The size of the amplified fragments was shown on agarose gel to be approximately 1007 bp for Gal4-3ST, 1421 bp for Gal4-3C, 404 bp for Gal4-2Q, 530 bp for Gal4-3A, 1889 bp for Gal4-3A3C, 1043 bp for Gal4-3Q3A, 1934 bp for Gal4-3Q3C, 347 bp for Gal4-GCN4, 911 bp for Gal4-3GCN4, 290 bp for Gal4-VP16, 287 bp for Gal4-B6 and 296 bp for Gal4-B42. The amplified fragments were sequenced on each side on 300 bases with the primers used for PCR, in order to confirm the nature of the transgenes (data not shown).

For the Bcd-GCN4 and Bcd-GCN4C lines, several P-element insertions were obtained. Some of the lines exhibited a dominant female sterile phenotype whereas others exhibited a recessive female sterile phenotype. These differences were probably due to the expression level of the transgene in each case. Indeed, decreasing the number of copy of wild-type bcd in the lines exhibiting a recessive phenotype reveal a dominant phenotype, and increasing the number of copies of bcd (using additional transgenes allowing expression of wild-type Bcd) allowed rescue of the dominant female sterile phenotype. The female sterile phenotype was, thus, only observed for a given ratio of wild-type to mutant (Bcd-GCN4 or Bcd-GCN4C) protein concentration.

Whole-mount in situ hybridisation
Diogoxigenin-labelled RNA probes and in situ hybridisation were as described (Bellaïche et al., 1996) from pKS derivatives containing the coding sequences of hb, otd and lacZ. The anti-digoxigenin antibody, coupled to alkaline phosphatase (Boehringer) was pre-adsorbed and used at 1/2000 dilution. Embryos were mounted in 80% glycerol and photographed with Nomarski optics.

Tissue culture and transactivation assay
Drosophila S2 cells were grown at 25°C in M3 medium supplemented with 10% fetal calf serum. Transfections were performed at 50-70% confluence with the calcium phosphate procedure (Wigler et al., 1979). For the transactivation assay, each plate was transfected with 1 µg of the hsp83-lacZ plasmid (internal control), 1 µg of either the Bcd responder plasmid (Bcd3-CAT) (Ronchi et al., 1993) or the Gal4 responder plasmid (UAS-CAT) (Bellaïche et al., 1996) and variable amounts of the producers plasmids (pPAC and derivatives). CAT assays were performed as described (Bellaïche et al., 1996) on 20 µl or normalised quantities of cytoplasmic extracts (relative to ß-galactosidase activity). Radiolabelled and acetylated forms of chloramphenicol were detected using a Fuji Bioimaging Analyser, and quantification was made using Mac Bas V2.2 computer software. Fold activation is the ratio of CAT activity obtained with a given fusion protein to background CAT activity obtained with the pPAC plasmid producing no protein. Indicated values are the means of three independent experiments.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Bcd-GCN4 protein is not inhibited by the Torso pathway
When three copies of the yeast GCN4 activation domain (Hope and Struhl, 1986) were fused downstream the ST domain of Bcd (Bcd-GCN4, Fig. 1A) and expressed under the control of bcd regulatory sequences (Ronchi et al., 1993), a head phenotype was induced in the embryo (compare Fig. 1B with 1C). In addition, Torso-dependent repression of the Bcd target genes hb (compare Fig. 1E with 1G) and otd (compare Fig. 1F with 1H) was no longer observed in these embryos. This indicates that the Bcd-GCN4 protein is insensitive to downregulation by Torso at the anterior pole, and that the absence of repression of hb and otd might lead to head phenotype. To understand whether this lack of repression was due to the site of insertion of the GCN4 activation domains or to their presence in Bcd-GCN4, these domains were fused to the C-terminal end of Bcd and the activity of the new fusion protein (Bcd-GCN4C, Fig. 1A) was analysed in vivo. As shown in Fig. 1, the expression of Bcd-GCN4C as a maternal anterior gradient also induced a head phenotype (Fig. 1D), which was correlated with the lack of anterior repression of the Bcd target genes (Fig. 1I,J). This indicates that both Bcd-GCN4 and Bcd-GCN4C are insensitive to downregulation by Torso at the anterior pole, and suggests that it is unlikely that the insertion of the GCN4 activation domains in Bcd-GCN4 disrupts the domain of Bcd, mediating its downregulation by Torso. More probably, the intrinsic properties of the GCN4 activation domains render Bcd-GCN4 and Bcd-GCN4C insensitive to Torso. These observations suggest two hypotheses:



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Fig. 1. Bcd-GCN4 is insensitive to inhibition by the Torso pathway. (A) The Bcd protein and its chimeric derivatives were wild-type Bcd protein (1) composed of the histidine/proline-rich repeat (H/P, amino acids 11-42), the homeodomain (HD, amino acids 91-151), the serine/threonine-rich domain (ST, amino acids 152-252), the glutamine-rich domain (Q, amino acids 252-300), the alanine-rich domain (A, amino acids 300-349) and the acidic C-terminal domain (C, amino acids 340-489); the Bcd-GCN4 and Bcd-GCN4C proteins in which three copies of the GCN4 activation domain (amino acids 54-141) have been fused to Bcd, respectively, downstream of the ST domain (2) or at its C terminus (3). (B-D) Cuticle preparation of embryos. Anterior is at the top. (E-J) In situ hybridisations on whole-mount embryos at the beginning of cellularisation with hb (E,G,I) and otd (F,H,J) probes. Embryos were from wild-type females (B,E,F), from females carrying one copy of wild-type bcd and one copy of the bcd-GCN4 transgene (C,G,H) and from females carrying two wild type copies of bcd and the bcd-GCN4C transgene (D, I and J).

 
(1) There are certain types of activation domains, such as those of Bcd, that are downregulated by Torso, while others, such as those of GCN4, remain insensitive to Torso. The presence in Bcd-GCN4 and Bcd-GCN4C of the GCN4 activation domains, which are insensitive to Torso, might overcome or mask the inhibition of the endogenous activation domains of Bcd by Torso.

(2) The Bcd protein might contain a repression domain induced by Torso. This putative repression domain might reduce the activity of the endogenous activation domains of Bcd upon Torso activation, whereas it might be unable to act on the heterologous GCN4 activation domains.

Sensitivity of heterologous activation domains to inhibition by Torso
To determine the effect of Torso activity on heterologous activation domains, we analysed the activity of fusion proteins between the Gal4 DNA-binding domain and either one or three copies of the yeast GCN4 activation domain, one copy of the VP16 activation domain (Das et al., 1995) or one copy of the B42 artificial (Ma and Ptashne, 1987) activation domain (Fig. 2A). The transcriptional activity of these proteins was first analysed in co-transfection experiments in S2 cells. These proteins were expressed under the control of the actin promoter and their activating potential, indicated by their capacities to induce the expression of a reporter for a CAT reporter gene containing five Gal4-binding sites (UAS-CAT), was determined by CAT assays performed on cytoplasmic extracts of transfected cells. As shown Fig. 2B, most of these proteins activated the expression of the UAS-CAT reporter gene in cell culture. These proteins were then expressed as maternal anterior gradients in the early embryo. As shown Fig. 2C-F, these proteins allowed expression of a lacZ reporter gene (UASp-lacZ) (Rorth, 1998) to very variable extends. Surprisingly, although the VP16 activation domain is considered as one of the most potent eucaryotic activation domain, Gal4-VP16 only very weakly directed expression of lacZ (Fig. 2E). Gal4-GCN4 and Gal4-B42 allowed expression of lacZ at the tip of the embryo (Fig. 2C,F) and Gal4-3GCN4 allowed expression of lacZ as a strong anterior cap (Fig. 2D). Despite the variable levels of activity of these proteins, none of them appeared to be inhibited by Torso at the anterior pole, indicating that the GCN4, VP16 and B42 activation domains are not downregulated by Torso in vivo.



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Fig. 2. Heterologous activation domains are not affected by the Torso pathway. (A) Structure of the fusion proteins between the Gal4 DNA-binding domain and one (1) or three (2) copies the GCN4 activation domain (amino acids 54-141), the VP16 activation domain (amino acids 413-490) (3) and the B42 activation domain (amino acids 1-79) (4). (B) transfections were performed with 1 µg of the UAS-CAT reporter and with 1 µg of pPAC (-) or its producer derivatives for the Gal4-GCN4 (1), Gal4-3GCN4 (2), Gal4-VP16 (3) and Gal4-B42 (4) proteins. (C-F) In situ hybridisations were performed on blastoderm embryos with a lacZ probe. Embryos were from female carrying the Gal4-GCN4 (C), Gal4-3GCN4 (D), Gal4-VP16 (E) and Gal4-B42 (F) transgenes. Females were crossed with males carrying the UASp-lacZ transgene. Anterior is to the left in this and subsequent figures showing in situ hybridized whole mounts.

 
The additional activation domain of Bcd is the ST domain
As deletion of the Q, A and C domains still results in a Bcd protein that can rescue Bcd function to viability, we wanted to identify the additional activation domain located in the N-terminal half of the Bcd protein. For this purpose, we analysed in cell culture the trans-acting potential of truncated versions of Bcd (Fig. 3A) using a CAT reporter gene driven by a minimal promoter containing three Bcd-binding sites (Bcd3-CAT). As previously described (Janody et al., 2000b), {Delta}QAC (Fig. 3A), which removes the entire C-terminal part of Bcd (including Q, A and C), retained significant trans-acting properties (Fig. 3B, compare lane 2 with lane 3). A protein containing the histidine/proline-rich repeat and the DNA-binding domain (HP/HD, Fig. 3A) did not activate CAT expression (Fig. 3B, lane 4). In contrast, a protein containing only the DNA-binding and the ST domains (HD/ST, Fig. 3A) was as potent as {Delta}QAC (Fig. 3B, compare lanes 3 with 5). This indicates that the HP repeat does not have a transcriptional potential in cell culture and that the additional activation domain of Bcd located in the N-terminal region is likely to be the ST domain.



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Fig. 3. The serine-threonine-rich domain of Bcd is an activation domain. (A) Structure of the Bcd deletion variants. (B) Transfections were performed with 1 µg of the Bcd3-CAT reporter and with 5 µg of pPAC (1) or its producer derivatives for the Bcd (2), {Delta}QAC (3), HP/HD (4) and HD/ST (5) proteins.

 
Q and C are activation domains in cell culture, whereas A is a repression domain
To further characterise the trans-acting potential of Bcd, fusion proteins containing the Gal4 DNA-binding domain and simple or multiple copies of the Bcd domains (Fig. 4A) were expressed in S2 cells and analysed for their capacity to induce expression of UAS-CAT. As indicated in Fig. 4B, the Gal4-Q and Gal4-3Q were potent activators of CAT expression (Fig. 4B, lanes 1-2). The Gal4-C protein does not activate CAT expression (Fig. 4B, lane 7) but the Gal4-3C significantly activates transcription (Fig. 4B, lane 8). This indicates that the C domain of Bcd, although probably weaker than the Q domain, is also an activation domain in S2 cells. Similar results were obtained with the Gal4-ST (not shown) and the Gal4-3ST (Fig. 4B, lane 11) proteins. Surprisingly, although it has been reported that the A domain mediates transactivation in a cell free system (Sauer et al., 1995), the Gal4-3A protein did not activate CAT expression in this assay (Fig. 4B, lane 6). On the contrary, when the A domain was fused to the Q or C domains, it dramatically reduced the activating potential of the fusion protein (Fig. 4B, compare lanes 1 and 3, 1 and 4, 2 and 5, and 8 and 9). This indicates that the A domain is not an activation domain in cell culture but rather inhibits the activity of two activation domains of Bcd. The position of the A domain does not appear crucial, as it inhibited the Q domain activity, when positioned at its C terminus (Fig. 4B, lanes 3-5), and the C domain activity, when placed at its N terminus (Fig. 4B, lane 9). These observations strongly argue that the A domain of Bcd behaves as a repression domain in a cellular context.



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Fig. 4. trans-acting potential of Bcd domains in S2 cells. (A) Structure of the fusion proteins between the Gal4 DNA-binding domain and simple or multiple copies of the Bcd domains. (B) Transfections were performed with 1 µg of the UAS-CAT reporter and with 1 µg of pPAC (-) or its producer derivatives for the Gal4-Q (1), Gal4-3Q (2), Gal4-QA (3), Gal4-Q3A (4), Gal4-3Q3A (5), Gal4-3A (6), Gal4-C (7), Gal4-3C (8), Gal4-3A3C (9), Gal4-3Q3C (10) and Gal4-3ST (11) proteins.

 
C and ST are sensitive to inhibition by Torso whereas Q is not
We then aimed to determine whether Bcd functional domains have similar behaviour in cell culture and in the embryo. Several Gal4 fusion proteins containing the Gal4 DNA-binding domain and the Bcd domains were expressed in transgenic animals as maternal anterior gradients in the early embryo. Their transcriptional activity was determined through the expression of the UASp-lacZ reporter transgene. As the Bcd-Gal4 protein in which the homeodomain was replaced by the Gal4 DNA-binding domain (Bellaïche et al., 1996) allowed only very weak expression of the UASp-lacZ (Fig. 5A), we were worried that the detection level in this system might require strong activators. Thus, most of the fusion proteins tested in this assay contained multiple copies of the Bcd domains. As shown in Fig. 5, Gal4-3ST (B), Gal4-3C (C) and Gal4-2Q (D) significantly activated the expression of lacZ. Interestingly, whereas expression driven by Gal4-2Q (Fig. 5D) remained present at the pole during cellularisation, the same transgene was repressed from the pole when driven by Gal4-3ST (Fig. 5B) or by Gal4-3C (Fig. 5C). Pole repression of lacZ driven by Gal4-3ST and Gal4-3C was dependent on torso activity (Fig. 5E,F). Finally, although both Gal4-3ST and Gal4-3C proteins were downregulated by Torso at the anterior pole, this process occurs in a slightly different way for each of them. Whereas anterior inhibition of Gal4-3C was clearly observed (Fig. 5C) during the whole process of cellularisation, anterior inhibition of Gal4-3ST was detected without any ambiguity at the beginning of the cellularisation process (Fig. 5B); but most stained embryos regained a strong anterior cap of lacZ expression by the end of cellularisation (not shown). Altogether, these experiments establish that the C and ST domains of Bcd function as autonomous activation domains downregulated by Torso in the early embryo, whereas the Q domain is an activation domain that is insensitive to Torso.



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Fig. 5. trans-acting potential of the Bcd domains in the embryo. In situ hybridisations were performed on blastoderm embryos with a lacZ probe. Embryos were from females carrying the Bcd-Gal4 (Bellaïche et al., 1996) (A), Gal4-3ST (B,E), Gal4-3C (C,F), Gal4-2Q (D), Gal4-3A (G), Gal4-3A3C (H), Gal4-3Q3A (I) and Gal4-3Q3C (J) transgenes. Females were crossed with males carrying the UASp-lacZ transgene. Females were torPM (E,F).

 
A is a repression domain in the early embryo
As shown Fig. 5G, the Gal4-3A protein was not able to activate expression of the UAS-placZ transgene. This result was obtained in three independent lines homozygous for the P-element insertion, allowing expression of Gal4-3A. Similarly, Gal4-3A3C (Fig. 5H) and Gal4-3Q3A (Fig. 5I) were also not able to activate expression of lacZ. Thus, as observed in cell culture (Fig. 4B), the presence of three copies of the A domain abolishes the transcriptional activity of three copies of either the Q or the C domain. This indicates that the A domain represses the activity of at least two distinct activation domains (Q and C) when (respectively) placed either at their C or N terminus. Finally, as the activity of Gal4-3Q3A and Gal4-3A3C was repressed by A not only at the pole but in the whole anterior domain (Fig. 5H,I), the repressive activity of the A domain is not spatially regulated by activation of the Torso pathway.

Sensitivity of the Bcd deletion variants to the Torso pathway
We then analysed Torso-induced anterior repression of Bcd target genes in transgenic flies bearing deletion variants of bcd rescue constructs (Schaeffer et al., 1999). These deletion variants removed either only one single domain of Bcd ({Delta}Q, {Delta}A or {Delta}C) or pairs of domains ({Delta}QA, {Delta}AC or {Delta}QC). A C-terminal truncated version ({Delta}QAC) removed simultaneously the Q, A and C domains (Schaeffer et al., 1999). The transgenic lines bearing each of these Bcd deletion variants in rescue constructs were analysed in a bcd mutant background by in situ hybridisation for the expression of three Bcd target genes: hb, otd and the Bcd reporter transgene Bcd3-lacZ, which places lacZ under the control of a naive promoter containing three Bcd-binding sites (Ronchi et al., 1993).

As mentioned by Schaeffer et al. (Schaeffer et al., 1999), the position of the posterior border of expression of hb, otd and Bcd3-lacZ was variable in different Bcd deletion variants. Despite these quantitative differences, which probably reflected the different level of expression of the various proteins, anterior repression of hb and otd at the anterior pole was observed in each of the lines tested (data not shown). Interestingly, although the expression of Bcd3-lacZ was repressed in the {Delta}QAC (Fig. 6B), the {Delta}A (Fig. 6C), the {Delta}C (Fig. 6D) and the other deletion variants tested (not shown), it persisted at the pole during cellularisation in the {Delta}AC deletion variant line (Fig. 6A). To confirm that the {Delta}AC protein was insensitive to downregulation by Torso, the expression of Bcd3-lacZ was also analysed in embryos from tor4021 females where the Torso cascade is activated throughout the embryo. As mentioned previously (Janody et al., 2000b; Ronchi et al., 1993), Bcd3-lacZ expression is totally repressed in a tor4021 mutant background (Fig. 6G). As Bcd3-lacZ contains only Bcd-binding sites upstream of a naive promoter, its expression probably reflects the intrinsic transcriptional activity of the Bcd protein (Bellaïche et al., 1996) and its extinction in tor4021 mutants indicates a complete inhibition of Bcd transcriptional activity by the constitutively activated Torso pathway (Janody et al., 2000b; Ronchi et al., 1993). In contrast, in this same tor4021 background, Bcd3-lacZ remained expressed when activated by {Delta}AC (compare Fig. 6F with 6H). Torso-induced anterior repression of Bcd target genes is generally observed during the first half of cellularisation, when the activation of the Torso pathway is optimal (Gabay et al., 1997). In Fig. 6E-H, slightly younger embryos were stained. At this stage, anterior repression of Bcd3-lacZ in wild type background is observed (Fig. 6E) and the effect of the constitutive allele of torso (torso4021) on Bcd3-lacZ expression is fully visible (Fig. 6G). This observation is consistent with the results of Gabay et al. (Gabay et al., 1997) who detected Torso induced ERK activation in tor4021 background at cycle 12. Altogether, these observations indicate that the {Delta}AC protein is not downregulated by Torso and that the deletion of both the A and C domains is sufficient to prevent the direct downregulation of Bcd transcriptional activity by the Torso pathway.



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Fig. 6. {Delta}AC is insensitive to inhibition by Torso. In situ hybridisations were performed on blastoderm embryos with a lacZ probe. Embryos were from bcdE1 females carrying two copies of the {Delta}AC (A), {Delta}QAC (B), {Delta}A (C) and {Delta}C (D) transgenes (Schaeffer et al., 1999), from wild-type females (E), from bcdE1 females carrying one copy of {Delta}AC (F), from tor4021 females (G), and from tor4021 bcdE1 females carrying one copy of {Delta}AC (H). Females were crossed with males carrying the UASp-lacZ transgene.

 
ST and C mediate downregulation of Bcd by Torso
The experiments described above provide interesting support for the identification of the domain(s) of Bcd that mediate its downregulation by Torso. They indicate that (1) as Bcd3-lacZ was repressed at the anterior pole in {Delta}QAC (Fig. 6B), whereas it remained expressed in {Delta}AC (Fig. 6A), the Q domain must be insensitive to Torso and this is consistent with the previous observation indicating that the Gal4-2Q protein was insensitive to downregulation by Torso (Fig. 5D); (2) as Bcd3-lacZ was repressed at the anterior pole in the {Delta}QAC line (Fig. 6B), this indicates that the N-terminal region of Bcd contains all the sequences sufficient for Torso induced downregulation, which is consistent with the downregulation of the Gal4-3ST protein by Torso (Fig. 5B); and (3) as Bcd3-lacZ was repressed at the anterior pole in both the {Delta}A and the {Delta}C lines (Fig. 6C,D), whereas it remained expressed in the {Delta}AC line (Fig. 6A), this indicates that the A and C domains both independently facilitates the effect of Torso. With respect to the C domain, this is consistent with the previous observation that the Gal4-3C protein was downregulated by Torso (Fig. 5C). Concerning the A domain, intriguingly, the Gal4 fusion analysis indicated that it functions as a repression domain whose activity was not modulated by Torso (Fig. 5G-I). Our interpretation of this discrepancy is that the {Delta}AC protein, that only contains the ST and the Q activation domains, is not downregulated by Torso, because the activity of Q, insensitive to Torso, is strong enough to mask the downregulation of ST by Torso. A similar situation was observed with Gal4-3Q3C, which is insensitive to Torso and contains both sensitive (3C) and insensitive (3Q) domains (Fig. 5J). In contrast, in {Delta}C the additional presence of A reduces Q activity and reveals the activity of ST, as well as its downregulation by Torso. The A domain might thus contribute only indirectly to the downregulation of Bcd by reducing the activity of the Q domain that is insensitive to Torso. Finally, with the {Delta}A protein, the activity of the Q domain insensitive to Torso is not strong enough to mask Torso downregulation of both the ST and C domains that appear to act very strongly together within this deleted mutant of Bcd.


    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Downregulation of Bcd activity by the Torso pathway
In the present study, we have aimed to gain insight into the molecular mechanism that leads to the transcriptional downregulation of the Bcd protein by the Torso pathway, and to identify the domain of Bcd that mediates this effect. Functional analysis of the Bcd-GCN4 fusion protein (Ronchi et al., 1993) that is insensitive to inhibition by Torso suggests that it involves either the inhibition of an activation domain or the activation of a repression domain. Analysis of the transcriptional activity of the Gal4 fusion proteins in the early embryo indicates that Bcd possesses three autonomous activation domains. Two of these domains, C and ST, are downregulated by Torso, whereas the third, Q, is not. This analysis also indicates that the A domain of Bcd functions as a repression domain whose activity is not regulated by Torso. We propose that the downregulation of Bcd by Torso is mediated by the ST and C domains and results from a competition between these domains and the Q activation domain insensitive to Torso. The A domain might contribute to this process only indirectly by reducing the general activity of the Bcd activation domains, particularly the activity of the Q domain which might otherwise prevent inhibition by Torso.

The molecular mechanism that leads to the downregulation of the ST and C domains by the Torso pathway remains to be uncovered. As ST and C are large domains, composed of 100 and 149 amino acids, respectively, they might be bipartite and might contain, in addition to an activation domain, a repression domain stimulated by Torso. Alternatively, the ST and C domains might function with positive co-factors whose activities are directly downregulated by activation of the Torso pathway. A simple scenario might be that both the ST and C domains of Bcd function through the same co-factor. Alternatively, there might be different co-factors for each of these domains. The sensitivity to Torso of the Gal4-3C and the Gal4-3ST proteins was slightly different: although the Gal4-3ST protein was clearly repressed by Torso at the beginning of cellularisation, it became insensitive to Torso during the second half of this process. The reason for this unique dynamic sensitivity of Gal4-3ST to Torso is not clear. One trivial explanation might be that Gal4-3ST is an unusually stable protein compared with the other Gal4 fusion proteins that we have analysed. If the Gal4-3ST protein persists in the embryo during the second half of cellularisation, it might escape the Torso pathway activity that decays notably during this period (Gabay et al., 1997). In contrast, other Torso-sensitive proteins, such as Gal4-3C or Bcd, might be degraded during the second part of cellularisation and the detection of lacZ, in these cases, might only reflect the persistence and the stability of the lacZ message.

Is the alanine-rich domain of Bcd a repression domain?
We have accumulated evidence that points to the A domain of Bcd as having a repressive function. These results are surprising as several studies have indicated that the A domain of Bcd had features of an activation domain, interacting in vitro with TAF60 and mediating transcriptional activation and synergism in a cell free system (Sauer et al., 1995). We do not fully understand the reasons for these discrepancies but several observations enlighten them. First, a role for the TAFIIs in transcriptional activation by Bcd in vivo has been recently questioned (Schaeffer et al., 1999) as the {Delta}QAC deletion variant line, lacking TAF110 and TAF60 interaction domains, can rescue the lack of bcd to viability. Second, although the alanine-rich domain (A) of Bcd binds TAF60, this type of domain is also frequently implicated in transcriptional repression in vivo (Hanna-Rose and Hansen, 1996). Finally, expression of Bcd3-lacZ in the {Delta}A deletion variant line was not only expanded towards the posterior but the intensity of the lacZ message was also much stronger than in the other lines tested (Fig. 6C). The shift of the posterior border of Bcd3-lacZ expression observed in this line is consistent with a two to three time increase in Bcd concentration when compared with wild type (Schaeffer et al., 2000). The increase in the intensity of the lacZ message indicates that the {Delta}A protein is probably a stronger activator than the wild-type Bcd and confirms that the A domain might function as a repression domain.

There are several possible molecular mechanisms to explain the repressive activity of the A domain. It is possible that the presence of the A domain modifies targeting of the protein to a cellular compartment or to degradation and indirectly prevents it from activating transcription. However, we favour the possibility that the repressive effect of the A domain directly involves the transcriptional process and that a specific co-repressor interacts with it. Indeed, it has recently been shown that the Bcd protein interacts in a two-hybrid screen with dSAP18, a member of the Sin3/Rpd3 deacetylation complex involved in repression mechanisms (Zhu and Hanes, 2000); it is tempting to speculate that dSAP18 might be this co-repressor.

Torso-induced repression of hb and otd involves two parallel processes
Our analysis indicates that hb and otd are repressed at the anterior in the {Delta}AC deletion variant line, whereas Bcd3-lacZ is not. Similarly, whereas expression of Bcd3-lacZ is totally repressed by the constitutive activation of Torso in tor4021 background (Ronchi et al., 1993; Janody et al., 2000b), expression of hb and otd is only slightly affected in this mutant (Gao et al., 1996; Janody et al., 2000b). This indicates that the repression of hb and otd by Torso at the anterior pole occurs through a slightly different mechanism from that mediating the regulation of Bcd3-lacZ. The Bcd3-lacZ reporter transgene contains only three Bcd-binding sites upstream of a naive promoter. In contrast, hb and otd promoters are more complex and probably involve, in addition to Bcd binding sites, response elements for other regulators. The observation that hb and otd are still repressed in the {Delta}AC deletion variant line suggests two hypotheses. As {Delta}AC still carries the ST domain sensitive to Torso, ST might contribute to hb and otd repression but not to Bcd3-lacZ repression. Alternatively, anterior repression of hb and otd might also occur indirectly by Torso-induced zygotic expression of a repressor that will bind to the regulatory sequences of these genes. Such a mechanism has been proposed for otd by Gao et al. (Gao et al., 1996) who have observed that otd anterior repression is reduced in hkb mutants although not eliminated. This process would be parallel and partially redundant with Torso-induced direct inhibition of Bcd transcriptional activity, mediated by the ST and C domains.

Finally, the {Delta}AC deletion variant line does not rescue the lack of bcd to viability leading to small head defects (Schaeffer et al., 1999). These defects might be due to a low expression level of the {Delta}AC protein in this line. We believe that this is unlikely as the morphogenetic activity of this line (as indicated by the position of the posterior border of Bcd3-lacZ expression) is comparable with that of the {Delta}QAC deletion variant line (compare Fig. 6A with 6B) that rescues the lack of bcd to viability (Schaeffer et al., 1999). The head phenotype observed with {Delta}AC is more probably due to the absence of the A and C domains of Bcd, and to the lack of Torso-induced anterior repression of Bcd transcriptional activity. As hb and otd are normally repressed by Torso in {Delta}AC, this indicates that another Bcd target gene might exist that is not properly regulated by Torso in this mutant background.

Transcriptional activation by Bcd in the early embryo
We have dissected the activating potential of the Bcd protein in vivo and have shown that the morphogen possesses three autonomous activation domains with different sensitivity to Torso, as well as a repression domain. These functional domains probably regulate transcription through interaction with different and specific co-factors. The reason for this complexity is not evident. It has been proposed that the Bcd morphogenetic activity (as measured by the position of the posterior border of a given Bcd target gene) is dependent on Bcd concentration and on the affinity of Bcd-binding sites found in the control region of each target gene (Driever and Nusslein-Volhard, 1988a; Struhl et al., 1989). In contrast, the strength of expression of Bcd target genes might be modulated by their promoter context. It is probable that the transcription factors binding in the proximity of the Bcd protein in these control regions might have synergistic or antagonistic interactions with some of the Bcd activation and repression domains or co-factors bound to these domains. These interactions might selectively affect the levels of expression of Bcd target genes. Our results therefore suggest a new level of complexity in the mechanism of action of the Bcd morphogen that resides in the multiple and diverse trans-acting potential of the Bcd protein.


    ACKNOWLEDGMENTS
 
We are indebted to Claude Desplan for his constant support during the process of this work and for allowing us to start this work in his laboratory. We gratefully acknowledge Pernille Rorth for the UASp-lacZ transgene. We thank Ernst Wimmer for stimulating discussions and sharing unpublished results and materials; Winshipp Herr and Gary Struhl for constructs; Faye Williams (Erasmus) and Christelle Guiraud (DU) for their participation to the molecular aspect of this work as undergraduate students from the Aix-Marseille II University; and Françoise Catala, Claude Desplan and Yacine Graba for helpful comments on the manuscript. F. J. was supported by MRT, ARC and FRM fellowships; R. S. by the CNRS; V. S. by a MRT fellowship; Y. A. by the French Ministry of Education; and N. D. by INSERM. This work was supported by grants from the CNRS (ATIPE), the FRM and ARC to N. D.


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