1 Institut Jacques-Monod, CNRS UMR7592, Université Paris 6 P. et M. Curie, Université Paris 7-Denis-Diderot, 2, place Jussieu, F-75251, Paris cedex 05, France
2 Institut de Génétique Humaine, CNRS, 141, rue de la Cardonille, F-34396 Montpellier, cedex 5, France
3 Laboratoire de Biologie du Développement, Université Paris 6-P et M. Curie, 9 quai St Bernard, case 24, F-75005 Paris, France
* Present address: Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK
Author for correspondence (e-mail: antoniewski{at}ijm.jussieu.fr)
Accepted April 17, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Drosophila, Ecdysone, Nuclear receptor, GATAb, serpent, AEF-1, Fbp1
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hormone response units (HRU) in nuclear receptor-regulated promoters constitute a particular class of cis-regulatory modules. They are composed of an assembly of binding sites for a variety of transcription factors, including hormone receptor-binding sites, and determine in which tissue(s) and during which developmental period(s) a gene will respond to the hormone (Lucas and Granner, 1992). Drosophila melanogaster is a choice animal for the study of HRUs in the context of a developing organism. At the end of the third larval instar, a pulse of the steroid hormone 20-hydroxyecdysone (hereafter referred to as ecdysone) activates the ecdysone receptor, which is composed of a heterodimer between the nuclear receptors EcR and USP (Koelle et al., 1991; Thomas et al., 1993; Yao et al., 1993). This receptor in turn differentially regulates a number of primary ecdysone response genes in different target tissues (Andres and Thummel, 1992). This hormonally controlled genetic program triggers puparium formation and initiates metamorphosis.
We are interested in understanding the molecular mechanisms whereby ecdysone response units (EcRUs) integrate multiple regulatory inputs to mediate distinct tissue- and time-specific transcriptional responses to circulating ecdysone. Our model gene Fat body protein 1 (Fbp1), which encodes a receptor mediating the uptake of hexamerins from the hemolymph by the larval fat body (Burmester et al., 1999), is transcribed exclusively in this tissue in response to the late third instar ecdysone peak (Andres et al., 1993; Lepesant et al., 1986). Germline transformation analysis has allowed the delimitation in the proximal Fbp1 promoter of an EcRU that is composed of two separable regions (Fig. 1). A 70 bp enhancer (E) is sufficient in itself to specify the spatially and temporally correct ecdysone-controlled pattern of Fbp1 expression (Laval et al., 1993) and an upstream region (A) contains a 32 bp sequence shown to amplify at least fivefold the specific transcriptional response conferred by the enhancer (Lapie et al., 1993).
|
Our previous work had suggested that this factor was perhaps GATAb, a member of the GATA family encoded by the serpent (srp) gene (Reuter, 1994b). GATAb is required for the embryonic development of the endodermal gut and hematopoietic tissues and is involved in the control of the differentiation of the fat body (Lebestky et al., 2000; Moore et al., 1998; Rehorn et al., 1996; Riechmann et al., 1998; Sam et al., 1996). We have shown that GATAb is expressed in a subset of larval tissues, including the fat body, during the third instar, and that it binds in vitro to three sites, GBS1, GBS2 and GBS3, flanking EBS in the Fbp1 enhancer (Brodu et al., 1999). Mutagenesis of these sites, as well as overexpression of GATAb, demonstrate that binding of GATAb to GBS1 is strictly necessary for the activity of the Fbp1 EcRU and contributes to its tissue specificity (Brodu et al., 1999).
We further applied the UAS substitution approach to a detailed investigation of the contribution of GATAb to the highly tissue-specific activity of the Fbp1 EcRU. Unexpectedly, we found that ubiquitously expressed GAL4 could replace GATAb at GBS1 without any change in the tissue specificity of the Fbp1 EcRU. Further combinations of GBS substitutions by UAS enabled us to show that this was due to a redundant tissue-specific role of GATAb through its binding to GBS3 and revealed that GATAb fulfilled two distinct functions at the Fbp1 EcRU: mediating a fat body-specific transcriptional activation and antagonizing (specifically in the fat body) the ubiquitous AEF-1 repressor that maintains the Fbp1 EcRU in an inactive state in other tissues. This points to a mode of repression exerted by AEF-1 that has not been described previously.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Histochemical assays of ß-galactosidase activity
Drosophila stocks were maintained at 25°C on a standard Drosophila medium. Developmental-stage determination of larvae was carried out as described (Andres and Thummel, 1994) and the histochemical staining assay of ß-galactosidase activity was performed essentially as previously described (Ashburner, 1989) using X-Gal.
Gel shift assays
DNA-binding reactions and subsequent gel electrophoresis were performed using 4 µl of late third instar fat body nuclear extract as described previously (Antoniewski et al., 1994). Sequences of the DNA probe and competitors are depicted in Fig. 5. Rabbit polyclonal antibody (1 µl) raised against AEF-1 (kindly provided by T. Maniatis) and 1 µg of protein A (when indicated) were added to the binding reactions for the supershift experiments.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Similarly, when the 5UAS-Fbp1-lacZ construct with five UAS sites fused upstream of the Fbp1 minimal promoter was crossed in the GAL4daG32 line, the lacZ reporter gene was expressed in all tissues except the central nervous system and gut (Fig. 2G). This result excluded the possibility that sequences responsible for a fat body-specific modulation of GAL4 activation were present in the minimal promoter downstream from the enhancer.
Functional redundancy of GBS1 and GBS3
In order to pursue the application of the UAS substitution approach to a further functional dissection of element E, we first ruled out the possibility that the ecdysone receptor EcR/USP played a direct role in the tissue-specificity of GAL4 transactivation by testing the AE[UAS1-UASEBS] construct in which both the GBS1 and EBS sites were replaced by UAS sites. When crossed in the GAL4daG32 line, this construct was still specifically expressed in the fat body of third instar larvae (Fig. 2C), indicating that sequences other than those of GBS1 and EBS in the Fbp1 EcRU were responsible for the tissue-specific modulation of GAL4 activity.
In our previous mutagenesis analysis we found that GBS2 and GBS3 were not essential for the activity of the Fbp1 EcRU when GBS1 was left intact. We showed, however, that they were bound in vitro by GATAb in fat body nuclear extracts (Brodu et al., 1999). This raised the possibility that the binding of GATAb to these sites was responsible for the restricted GAL4 transactivation of AE[UAS1] in the late third instar fat body. To test this hypothesis, GBS2, GBS3 or both GBS2 and GBS3 were disrupted in the AE[UAS1] construct and transgenic lines for the resulting constructs AE[UAS1-GBS2m], AE[UAS1-GBS3m] and AE[UAS1-GBS2m-GBS3m] were established. When crossed in the GAL4daG32 line, AE[UAS1-GBS2m] was still expressed specifically in the late third instar fat body (Fig. 2D). In contrast, no expression of AE[UAS1-GBS3m] and AE[UAS1-GBS2m-GBS3m] was observed at any time during development in the same genetic background (Fig. 2E,F). This led us to conclude that, like GBS1, GBS3 possibly plays a role in the tissue-specificity of the Fbp1 EcRU activity which could only be revealed when the prevalent GBS1 was inactivated by the UAS substitution. In contrast, GBS2 did not seem able to play the same functional role.
The demonstration of the requirement for GBS3 in the modulation of GAL4 activity when this factor was targeted to a UAS site replacing GBS1, prompted us to analyze the symmetrical situation in which GAL4 was targeted to a UAS site replacing GBS2/GBS3, while GBS1 was either left intact or disrupted. As expected from our previous finding that neither GBS2 nor GBS3 were essential for the activity of the Fbp1 EcRU (Brodu et al., 1999), the AE[UAS2-3] transgenic construct, which carried a UAS site in place of both GBS2 and GBS3, was expressed in late third instar fat body, even in the absence of GAL4 (Fig. 3A). When crossed in the GAL4daG32 line, the expression of AE[UAS2-3] was strongly reinforced but remained restricted to the late third instar fat body (Fig. 3B), indicating that this further transactivation of the construct brought about by GAL4 was modulated in a tissue-specific manner. In contrast, the AE[GBS1m-UAS2-3] construct in which both GBS2 and GBS3 were replaced by a UAS site and GBS1 was disrupted, remained completely silent, even when crossed in the GAL4daG32 line (Fig. 3C).
|
|
The possibility that target sites for this putative repressor were located between 68 and +80 in the minimal Fbp1 promoter was ruled out, because of the full responsiveness of the 5UAS-Fbp1-lacZ construct to GAL4 in all but a few tissues throughout development (see Fig. 2G). Because it had been reported that the ecdysone receptor has a repressing activity in the absence of its ligand (Cherbas et al., 1991; Dobens et al., 1991; Tsai et al., 1999) we tested whether the ecdysone receptor itself was the putative repressor by mutating the EcR/USP binding site (EBS) in the AE-UAS1-UAS2-3 construct. In a GAL4daG32 context, the resulting AE[UAS1-EBSm-UAS2-3] construct remained silent (Fig. 4B), indicating that the hormonal receptor was not involved in the silencing of GAL4 transactivation.
Finally, we examined the possibility that element A was the target for the putative repressor by deleting it in the AE[UAS1-UAS2-3] construct. When placed in a GAL4daG32 genetic context, the resulting E[UAS1-UAS2-3] construct was expressed in all tissues but the central nervous system and gut, from embryogenesis to puparium formation (Fig. 4C; data not shown), indicating that element A probably contains a target sequence for a ubiquitous repressor strongly antagonizing transactivation by GAL4.
The AEF-1 repressor binds to sequence A
We noted that element A contains a putative binding site 5'CAACAA3' for the AEF-1 protein (Fig. 5), which had been identified as a factor that negatively regulates the Alcohol deshydrogenase (Adh) and Yolk protein 1 and Yolk protein 2 (Yp1 and Yp2) genes of Drosophila melanogaster (An and Wensink, 1995; Falb and Maniatis, 1992a). AEF-1 is expressed throughout the Drosophila life cycle in all tissues examined, including the fat body (Falb and Maniatis, 1992b). This prompted us to examine the possibility that AEF-1 was the factor that mediated the repressing activity of element A.
The double-stranded oligonucleotide A1 that encompassed the putative AEF-1 site gave rise to the formation of a strong retarded complex C1 when used as a radioactive probe in a gel shift assay with a fat body nuclear extract (Fig. 5, lane 1). The formation of this complex was inhibited in the presence of a 200-fold molar excess of unlabeled oligonucleotide A1 indicating that it was sequence specific (Fig. 5, lane 2). In contrast, the formation of complex C1 was not inhibited by using, as a competitor, the oligonucleotide A1m, in which the putative AEF-1 binding site was mutated (Fig. 5, lane 3). When the binding reaction was performed in the presence of a rabbit polyclonal antibody raised against AEF-1, complex C1 was not formed and two slower migrating complexes S1 and S2 were revealed (Fig. 5, lane 4). Neither of these complexes was formed in the presence of the AEF-1 antibody alone (Fig. 5, lane 7). However, the migration of complex S1 but not that of complex S2 was further retarded in the presence of protein A, which has a strong affinity for IgGs (Fig. 5, lane 5) but does not bind to DNA by itself (Fig. 5, lane 6). This confirmed that S1 resulted from a specific supershift of complex C1 by the AEF-1 antibody and S2 from a nonspecific DNA binding activity that occurred when rabbit serum was incubated in the presence of fat body nuclear extract. Taken together, these results demonstrate that the AEF-1 repressor is present in fat body nuclear extracts and binds specifically to element A upstream of the Fbp1 enhancer.
AEF-1 silences the activation of the Fbp1 EcRU by GAL4
In order to test whether AEF-1 was the repressor antagonizing in vivo the activation of the Fbp1 EcRU by GAL4, we established transgenic lines for the AE[AEF-1m-UAS1-UAS2-3] construct bearing the mutation that abolished AEF-1 binding in gel shift experiments. In striking contrast to the AE[UAS1-UAS2-3] construct (Fig. 4A), the AE[AEF-1m-UAS1-UAS2-3] construct was expressed in the same way as the E[UAS1-UAS2-3] construct in all tissues except the central nervous system and gut, and throughout development, when placed in a GAL4daG32 genetic context (Fig. 4D; data not shown).
In summary, our experiments demonstrate that activation of the Fbp1 EcRU by GAL4 is blocked by the binding of AEF-1 to element A. This silencing is relieved by the binding of GATAb to either GBS1 or GBS3.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
What are the mechanisms involved in the competence function of GATAb, as revealed by the UAS substitution approach? Numerous studies have shown that the yeast transcription factor GAL4 is able to activate reporter transgenic constructs under the control of UAS sites in all tissues, including those in which GATAb is not expressed (Brand and Perrimon, 1993; Phelps and Brand, 1998; Rorth, 1998). These data make it very unlikely that one of GATAb functions is to specifically potentiate the transactivating activity of GAL4 in the fat body tissue. The restriction of the expression of the AE[UAS1], AE[UAS2-3] and AE[UAS1-GBS2m] constructs to the fat body in a GAL4daG32 genetic context provides a strong argument in favor of the idea that specific sequences in these constructs target a potent ubiquitous repressor of GAL4 activity, which is antagonized solely in this tissue by means of a GATAb-dependent mechanism. The observation that the 5UAS-Fbp1-lacZ control construct is expressed throughout development in most tissues when crossed in the GAL4daG32 animals indicates that the Fbp1 minimal promoter is fully responsive to GAL4 and does not contain any such repressor binding sequences. Similarly, the strong and fat-body specific expression of the AE[UAS1-UASEBS] construct excludes the notion that the binding site for the EcR-USP receptor plays this role. In contrast our results clearly demonstrate that element A mediates the binding of the strong repressor AEF-1.
It is interesting to note that other instances of modulation of heterologous activators by Ultrabithorax-flanking regulatory sequences have been described (McCall and Bender, 1996; Szuts and Bienz, 2000). Together, these results provide strong support for the use of heterologous transactivators in transgenic systems as a tool to reveal and analyze the activity of cis-regulatory modules in promoters.
A novel function of the AEF-1 repressor
The UAS substitution approach reveals that element A initially identified as a positively acting element (Lapie et al., 1993; Fig. 1) also possesses the properties of a negatively acting element. Deletion of element A from the completely inactive AE[UAS1-UAS2-3] construct leads to the ubiquitous GAL4-driven expression of the resulting E[UAS1-UAS2-3] construct. The similar ubiquitous expression of the AE[AEF-1m-UAS1-UAS2-3] construct in which the AEF-1-binding site is disrupted, provides direct evidence that AEF-1 is the factor responsible for the complete blocking of GAL4 transactivation of the Fbp1 EcRU. This is consistent with the initial characterization of AEF-1 as a repressor (Falb and Maniatis, 1992a) expressed throughout development in all tissues examined (Falb and Maniatis, 1992b). Our results, however, provide novel insight into the mechanisms whereby AEF-1 represses the transcription of its target genes.
Two mechanisms of gene repression by transcriptional interference have been characterized so far for AEF-1. It has been shown that the binding of AEF-1 to the Adult Adh enhancer negatively regulates the Adh gene by interfering with the binding of an activator of the C/EBP family to an overlapping site (Falb and Maniatis, 1992a; Falb and Maniatis, 1992b). A similar binding interference between AEF-1, C/EBP and the female-specific Doublesex protein was proposed for the downregulation of the yolk protein genes Yp1 and Yp2 (An and Wensink, 1995). More recently, Ren and Maniatis (Ren and Maniatis, 1998) showed that AEF-1 also binds to the initiator region (Inr) of the Adh proximal promoter and represses transcription by a distinct mechanism thought to involve steric interference with the binding of general transcription factors. In contrast, our finding that AEF-1 is able to block the activation of the Fbp1 EcRU by GAL4 targeted to a site more than 50 bp downstream from the AEF-1 site, provides evidence that AEF-1 has yet another function, which is to repress enhancers at a distance.
Transcriptional repressors have been characterized by their range of action on promoters and enhancers (Gray and Levine, 1996b; Mannervik et al., 1999). Short-range repressors, including Snail, Knirp and Krüppel, interact over distances of 50-150 bp to inhibit, or quench, either transcriptional activators or the basal transcription complex (Gray and Levine, 1996a; Nibu et al., 1998a; Nibu et al., 1998b). These repressors share a conserved PXDLSXK sequence motif, responsible for interaction with the corepressor dCtBP. In contrast, long-range repressors, including Dorsal and Hairy, act over distances of several kilobases to silence basal promoters (Barolo and Levine, 1997; Cai et al., 1996) and interact with the corepressor Groucho through a conserved WRPW motif. Our data suggest that AEF-1 belongs to the short-range repressor family. Whether AEF-1 is also able to act as a long-range repressor requires additional experiments.
The lack of PXDLSXK or WRPW motives in AEF-1 suggests that it mediates repression through an interaction with corepressors other than dCtBP and Groucho. Evidence that histone deacetylation plays a role in gene silencing has accumulated in recent years (Ng and Bird, 2000). It has been shown in particular that the histone deacetylase Rpd3, and the Sin3A and SMRT/NcoR proteins are part of a corepressor complex of mammalian transcriptional repressors (reviewed in Ahringer, 2000). Similarly, Rpd3 and the Drosophila SMRT homolog SMRTER were shown to interact with Groucho (Flores-Saaib and Courey, 2000) and the unliganded EcR/USP ecdysone receptor (Tsai et al., 1999). In this context, a possible link between AEF-1 and complexes displaying a histone-deacetylase activity deserves investigation.
We were unable to demonstrate a direct protein-protein interaction between GATAb and AEF-1 in either an in vitro GST pull-down assay or two-hybrid screens in yeast using GATAb as a bait (data not shown). In addition, it should be noted that GATAb exerts its antagonizing effect on AEF-1 even when bound to GBS3, as in the AE[UAS1] construct where this site is located more than 100 bp downstream of the AEF-1-binding site with a GAL4 binding at an intervening site. It appears thus very unlikely that the interaction between GATAb and AEF-1 is direct.
GATAb performs two distinct functions in the tissue-specific regulation of the Fbp1 EcRU
A functional hierarchy between the GBS1 and GBS3 sites emerges from our results. GBS3 is dispensable but can redundantly supply the AEF-1 antagonizing GATAb effect when GBS1 is non-functional or absent. In contrast, GBS1 is essential for the activity of the natural Fbp1 EcRU. This suggests that GBS1 not only supplies the AEF-1 antagonizing GATAb function, but is also involved in mediating another GATAb function essential to the specific activity of the Fbp1 EcRU.
The E construct remains expressed in a tissue-restricted manner in the late third instar fat body (see Fig. 1), indicating that the Fbp1 enhancer can act independently of element A as an autonomous time- and tissue-specific positive element. Disruption of GBS1 in the E construct leads to its inactivation (V. B., unpublished). Consistently, UAS substitution of the GATAb-binding sites in the E[UAS1-UAS2-3] construct also results in its total inactivation in the absence of GAL4. Together, these results point out to a GBS1-mediated specific activating function of GATAb on the Fbp1 enhancer, in addition to its AEF-1 antagonizing function (Fig. 6). It is worth noting that both these functions are effective in the third instar fat-body only, leading to a highly tissue-specific regulatory output. The expression and developmental functions of GATAb are not, however, restricted merely to the larval fat body tissue. It also plays roles in the embryonic development of the gut (Reuter, 1994a), hematopoietic lineage (Lebestky et al., 2000; Rehorn et al., 1996) and gonads (Moore et al., 1998; Riechmann et al., 1998). Consistently, we have shown that GATAb is expressed in a number of third larval instar tissues, gonads, gut, lymph glands and pericardial cells in addition to the fat body. Hence, it is clear that the fat body-specific regulation of Fbp1 by GATAb does not rely solely on the mere presence of this factor in the fat body. That additional factors are probably involved in fat body specification besides GATAb is further supported by the observation that ubiquitous overexpression of GATAb leads to an ectopic expression of the AE Fbp1 construct limited to two additional tissues only, gut and salivary glands (Brodu et al., 1999). Together, these data suggest that in the fat body GATAb co-factors strictly target its dual Fbp1 regulatory functions (Fig. 6). Our previous observation that GATAb interacts in complexes with as yet unidentified factors in the fat body (Brodu et al., 1999) strongly supports this hypothesis. Whether these factors have any effect on the fat body-specific functions of GATAb remains, however, to be determined.
|
Nevertheless, our results point out to another level of temporal regulation for the Fbp1 EcRU. On the one hand, the observation that GAL4-driven expression of the AE[UASEBS] and AE[UAS1-UASEBS] constructs takes place throughout the third larval instar but remains restricted to this stage indicates that a stage-specific competence for transactivation of the Fbp1 EcRU is acquired at the third larval instar, independently from the hormonal control. On the other hand, the complete inactivity of the AE[UAS1-GBS3m], AE[UAS1-GBS2m-GBS3m] and AE[UAS1-UAS2-3] constructs strongly suggests that this stage-specific competence is dependent upon GATAb. In addition, the AE[AEF-1m-UAS1-UAS2-3] construct, which is not submitted to AEF-1 repression and therefore GATAb-independent, is ubiquitously expressed in embryos and throughout larval development in the GAL4daG32 context. Together, these data reveal the role of the GATAb/AEF-1 interplay in the acquisition of the capacity of the Fbp1 EcRU to respond to transactivation during the third larval instar. However both GATAb and AEF-1 are expressed well before the third instar during embryogenesis. GATAb itself performs essential regulatory functions during this period of early development. Hence, it is clear that the stage-specific activation of the Fbp1 EcRU does not rely solely on the combined functions of AEF-1 and GATAb. It is conceivable that GATAb co-factors such as those mentioned above are themselves temporally restricted to the third larval instar and required to potentiate these functions at the appropriate stage (Fig. 6).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahringer, J. (2000). NuRD and SIN3 histone deacetylase complexes in development. Trends Genet. 16, 351-356.[Medline]
An, W. and Wensink, P. C. (1995). Integrating sex- and tissue-specific regulation within a single Drosophila enhancer. Genes Dev. 9, 256-266.[Abstract]
Andres, A. J., Fletcher, J. C., Karim, F. D. and Thummel, C. S. (1993). Molecular analysis of the initiation of insect metamorphosis: a comparative study of Drosophila ecdysteroid-regulated transcription. Dev. Biol. 160, 388-404.[Medline]
Andres, A. J. and Thummel, C. S. (1992). Hormones, Puffs and flies: the molecular control of metamorphosis by ecdysone. Trends Genet. 8, 132-138.[Medline]
Andres, A. J. and Thummel, C. S. (1994). Methods for quantitative analysis of transcription in larvae and prepupae. Methods Cell Biol. 44, 565-573.[Medline]
Antoniewski, C., Laval, M., Dahan, A. and Lepesant, J. A. (1994). The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor. Mol. Cell. Biol. 14, 4465-4474.[Abstract]
Antoniewski, C., Mugat, B., Delbac, F. and Lepesant, J. A. (1996). Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster. Mol. Cell. Biol. 16, 2977-2986.[Abstract]
Arnone, M. I. and Davidson, E. H. (1997). The hardwiring of development: organization and function of genomic regulatory systems. Development 124, 1851-1864.
Ashburner, M. (1989). Drosophila: A Laboratory Handbook. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Barolo, S. and Levine, M. (1997). Hairy mediates dominant repression in the Drosophila embryo. EMBO J. 16, 2883-2891.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
Brodu, V., Mugat, B., Roignant, J. Y., Lepesant, J. A. and Antoniewski, C. (1999). Dual requirement for the EcR/USP nuclear receptor and the dGATAb factor in an ecdysone response in Drosophila melanogaster. Mol. Cell. Biol. 19, 5732-5742.
Burmester, T., Antoniewski, C. and Lepesant, J. A. (1999). Ecdysone-regulation of synthesis and processing of Fat Body Protein 1, the larval serum protein receptor of Drosophila melanogaster. Eur. J. Biochem. 262, 49-55.
Cai, H. N., Arnosti, D. N. and Levine, M. (1996). Long-range repression in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 93, 9309-9314.
Cherbas, L., Lee, K. and Cherbas, P. (1991). Identification of ecdysone response elements by analysis of the Drosophila Eip28/29 gene. Genes Dev. 5, 120-131.[Abstract]
Dobens, L., Rudolph, K. and Berger, E. (1991). Ecdysterone regulatory elements function as both transcriptional activators and repressors. Mol. Cell. Biol. 11, 1846-1853.[Medline]
Falb, D. and Maniatis, T. (1992a). A conserved regulatory unit implicated in tissue-specific gene expression in Drosophila and man. Genes Dev. 6, 454-465.[Abstract]
Falb, D. and Maniatis, T. (1992b). Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers. Mol. Cell. Biol. 12, 4093-4103.[Abstract]
Flores-Saaib, R. D. and Courey, A. J. (2000). Analysis of groucho-histone interactions suggests mechanistic similarities between groucho- and Tup1-mediated repression. Nucleic Acids Res. 28, 4189-4196.
Gray, S. and Levine, M. (1996a). Short-range transcriptional repressors mediate both quenching and direct repression within complex loci in Drosophila. Genes Dev. 10, 700-710.[Abstract]
Gray, S. and Levine, M. (1996b). Transcriptional repression in development. Curr. Opin. Cell Biol. 8, 358-364.[Medline]
Kirchhamer, C. V., Yuh, C. H. and Davidson, E. H. (1996). Modular cis-regulatory organization of developmentally expressed genes: two genes transcribed territorially in the sea urchin embryo, and additional examples. Proc. Natl. Acad. Sci. USA 93, 9322-9328.
Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P. and Hogness, D. S. (1991). The drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67, 59-77.[Medline]
Lapie, P., Nasr, F., Lepesant, J. A. and Deutsch, J. (1993). Deletion scanning of the regulatory sequences of the Fbp1 gene of Drosophila melanogaster using P transposase-induced deficiencies. Genetics 135, 801-816.
Laval, M., Pourrain, F., Deutsch, J. and Lepesant, J. A. (1993). In vivo functional characterization of an ecdysone-response enhancer in the proximal upstream region of the Fbp1 gene of D. melanogaster. Mech. Dev. 44, 123-138.[Medline]
Lebestky, T., Chang, T., Hartenstein, V. and Banerjee, U. (2000). Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science 288, 146-149.
Lepesant, J. A., Maschat, F., Kejzlarovà-Lepesant, J., Benes, H. and Yanicostas, C. (1986). Developmental and ecdysteroid regulation of gene expression in the larval fat body of Drosophila melanogaster. Arch. Insect Biochem. Physiol. S1, 133-141.
Lucas, P. C. and Granner, D. K. (1992). Hormone response domains in gene transcription. Annu. Rev. Biochem. 61, 1131-1173.[Medline]
Mannervik, M., Nibu, Y., Zhang, H. and Levine, M. (1999). Transcriptional coregulators in development. Science 284, 606-609.
McCall, K. and Bender, W. (1996). Probes of chromatin accessibility in the Drosophila bithorax complex respond differently to Polycomb-mediated repression. EMBO J. 15, 569-580.[Abstract]
Merika, M. and Orkin, S. H. (1993). DNA-binding specificity of GATA family transcription factors. Mol. Cell. Biol. 13, 3999-4010.[Abstract]
Moore, L. A., Broihier, H. T., Van Doren, M. and Lehmann, R. (1998). Gonadal mesoderm and fat body initially follow a common developmental path in Drosophila. Development 125, 837-844.
Mugat, B., Brodu, V., Kejzlarova-Lepesant, J., Antoniewski, C., Bayer, C. A., Fristrom, J. W. and Lepesant, J. A. (2000). Dynamic expression of broad-complex isoforms mediates temporal control of an ecdysteroid target gene at the onset of drosophila metamorphosis. Dev. Biol. 227, 104-117.[Medline]
Ng, H. H. and Bird, A. (2000). Histone deacetylases: silencers for hire. Trends Biochem. Sci. 25, 121-126.[Medline]
Nibu, Y., Zhang, H., Bajor, E., Barolo, S., Small, S. and Levine, M. (1998a). dCtBP mediates transcriptional repression by Knirps, Kruppel and Snail in the Drosophila embryo. EMBO J. 17, 7009-7020.
Nibu, Y., Zhang, H. and Levine, M. (1998b). Interaction of short-range repressors with Drosophila CtBP in the embryo. Science 280, 101-104.
Phelps, C. B. and Brand, A. H. (1998). Ectopic gene expression in Drosophila using GAL4 system. Methods 14, 367-379.[Medline]
Rehorn, K. P., Thelen, H., Michelson, A. M. and Reuter, R. (1996). A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila. Development 122, 4023-4031.
Ren, B. and Maniatis, T. (1998). Regulation of Drosophila Adh promoter switching by an initiator- targeted repression mechanism. EMBO J. 17, 1076-1086.
Reuter, R. (1994a). The gene serpent has homeotic properties and specifies endoderm versus ectoderm within the Drosophila gut. Development 120, 1123-1135.
Reuter, R. (1994b). The gene serpent has homeotic properties and specifies endoderm versus ectoderm within the Drosophila gut. Development 120, 1123-1135.
Riechmann, V., Rehorn, K. P., Reuter, R. and Leptin, M. (1998). The genetic control of the distinction between fat body and gonadal mesoderm in Drosophila. Development 125, 713-723.
Rorth, P. (1998). Gal4 in the Drosophila female germline. Mech. Dev. 78, 113-118.[Medline]
Sam, S., Leise, W. and Hoshizaki, D. K. (1996). The serpent gene is necessary for progression through the early stages of fat-body development. Mech. Dev. 60, 197-205.[Medline]
Szuts, D. and Bienz, M. (2000). LexA chimeras reveal the function of Drosophila Fos as a context- dependent transcriptional activator. Proc. Natl. Acad. Sci. USA 97, 5351-5356.
Thomas, H. E., Stunnenberg, H. G. and Stewart, A. F. (1993). Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature 362, 471-475.[Medline]
Tsai, C. C., Kao, H. Y., Yao, T. P., McKeown, M. and Evans, R. M. (1999). SMRTER, a Drosophila nuclear receptor coregulator, reveals that EcR-mediated repression is critical for development. Mol. Cell 4, 175-186.[Medline]
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82, 67-76.[Medline]
Yao, T. P., Forman, B. M., Jiang, Z., Cherbas, L., Chen, J.-D., McKeown, M., Cherbas, P. and Evans, R. M. (1993). Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 336, 476-479.