1 Division of Developmental Biology, Childrens Hospital Research Foundation, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
2 Department of Molecular Genetics and the Neurobiotechnology Center, The Ohio State University, 125 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210, USA
3 Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA*Present address: Department of Molecular Genetics, University of Cincinnati College of Medicine, 231 Sabin Drive, Cincinnati, OH 45267, USA
Author for correspondence (e-mail: jun.ma{at}chmcc.org)
Accepted 4 January 2002
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SUMMARY |
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Key words: Drosophila, Bicoid, Transcription
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INTRODUCTION |
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Biochemical studies by Sauer and Tjian have demonstrated that TAFs (TATA box-binding protein associated factors) can specifically interact with the putative activation domains located in the C-terminal region of Bcd (Sauer et al., 1995a; Sauer et al., 1995b
). In particular, it was shown that TAF60 interacted with the alanine-rich region, while TAF110 recognized the glutamine-rich region. It was thus proposed that Bcd activated transcription by directly interacting with these TAFs (Sauer et al., 1995a
; Sauer et al., 1995b
; Sauer et al., 1996
). However, the relevance of such interactions in Bcd function during development has been questioned recently (Schaeffer et al., 1999
). Interestingly, it has been shown that a Bcd derivative lacking the entire C-terminal half can rescue the bcd phenotype when expressed at high levels (Schaeffer et al., 1999
). These results further highlight the importance of the N-terminal region of Bcd, suggesting that this region may provide most or all the functions required for Bcd action in vivo. The importance of the N-terminal region of Bcd is also evidenced by the recent finding that this region of Bcd is evolutionarily conserved. The Bcd proteins from Drosophila and a primitive cyclorrhaphan fly, Megaselia abdita, share a highly conserved domain in their N-terminal portions while their C-terminal region diverge dramatically (Stauber et al., 1999
).
In this report, we describe experiments that reveal an unexpected new function provided by the N-terminal region of Bcd. A self-inhibitory domain located immediately N-terminally of the homeodomain can repress Bcd activity in reporter activation assays conducted in Drosophila tissue culture cells. Mutations in this domain, or its removal by deletion, dramatically increase the activity of Bcd. Our experiments demonstrate that this domain operates as an independent module that does not require any other sequences of Bcd and can repress the activity of heterologous activators. We further show that a Bcd derivative with point mutations in the self-inhibitory domain causes severe defects in both embryonic patterning and target gene expression during development. The action of the self-inhibitory domain requires a Drosophila co-factor(s) absent in yeast cells, but our further studies suggest that neither CtBP nor dSAP18 directly target the self-inhibitory domain of Bcd. Our results suggest that proper action of Bcd as a transcriptional activator and molecular morphogen requires a novel co-repressor(s) or complex(es) interacting with its self-inhibitory domain.
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MATERIALS AND METHODS |
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Gel shift assays
The DNA probe used in gel shift assays contains a consensus Bcd binding site A1. Gel shift assays and Scatchard analysis were carried out according to procedures described previously (Dave et al., 2000; Zhao et al., 2000
); all DNA-binding reactions (total volume 30 µl) contained 2 µg poly (dI::dC).
Yeast strain and ß-galactosidase liquid assays
The yeast strain used in this study is CY26::MA630R which contains an integrated hb-lacZ reporter gene in CY26 (Zhao et al., 2000). The effector plasmids were introduced into yeast using the lithium acetate method (Ito et al., 1983
), and three independent colonies were assayed for ß-galactosidase units (Yocum et al., 1984
).
GST pull-down assay
Expression of GST and GST-dSAP18 fusion in bacteria was performed as previously described (Zhang et al., 2000). Equivalent amounts of GST and GST-dSAP18 were used to pull down in vivo translated and 35S-labeled Bcd derivatives. For each Bcd derivative, a similar amount, as estimated by autoradiography, was used in the pull-down assay.
P-element-mediated germline transformation and phenotypic examination
P-element constructs containing either wild-type or mutant bcd genes were injected with transposase helper plasmid into w embryos, and transformant lines were mapped using standard procedures (Rubin and Spradling, 1982; Spradling, 1986
). For cuticle examination, transgenic female flies were crossed with w118 males and allowed to lay on grape agar for 24 hours. The flies were then removed and the vials left at room temperature for 18 to 24 more hours. Cuticles were prepared according to the Hoyers mountant method (Ashburner, 1989
) and photographed by dark-field (whole cuticles) and Nomarski (head regions) microscopy.
Embryo staining and Drosophila germline clones
Drosophila embryos were collected and stained for hb or otd mRNA using digoxigenin-labeled antisense RNA probes as previously described (Jiang et al., 1991). CtBP germline clones were generated according to Nibu et al. (Nibu et al., 1998a
).
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RESULTS |
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Our deletion analysis shown in Fig. 1B further suggests that residues 52-61 play a most crucial role in the self-inhibitory function. Five residues in this region (52-56) share significant homology with a consensus motif interacting with CtBP, a major co-repressor in Drosophila (see below). To determine the importance of residues 52-56 in regulating Bcd activity, we generated a full-length Bcd derivative, Bcd(A52-56), with these five amino acids changed to alanines (Fig. 2A). A systematic titration assay of this Bcd derivative and the wild-type protein was performed, using increasing amounts of effector DNA for transfection. Our results show that, under all protein concentrations analyzed, Bcd(A52-56) was 18-24 times more active than the wild-type protein (Fig. 2B,C). More importantly, this mutant protein was more active than the wild-type protein even when it was expressed at lower levels than the wild-type protein (Fig. 2C,D). As Bcd mutants with critical residues individually mutated also exhibited higher activity (not shown), it is unlikely that the five alanine residues in Bcd(A52-56) may have created fortuitously an alanine-rich activation domain responsible for the observed strong Bcd activity. Together, our experiments demonstrate an essential role for residues 52-56 of Bcd in repressing its own activity.
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The self-inhibitory domain is an independent module that does not require any specific Bcd sequences and can repress the activity of heterologous activators
To further determine whether the self-inhibitory domain of Bcd specifically targets any other regions of the protein, we measured the activities of various deletion derivatives in transient transfection assays (Fig. 4A). For each deletion derivative, a pair was tested, one with residues 52-56 mutated to alanines [Bcd(A52-56)] and the other wild type. Our experiments demonstrate that the mutant proteins in each pair were always more active than their wild type counterparts (Fig. 4A). Specifically, these experiments show that neither residues 246-489 (lines 2-4) nor residues 152-246 (line 5) are required for the action of the self-inhibitory domain. As shown already in Fig. 1C, the first 51 N terminal amino acids are not required for the action of the self-inhibitory domain.
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We conducted experiments to determine further whether the self-inhibitory domain of Bcd is transferable, i.e. whether it can work on entirely heterologous activators. For this analysis, the N-terminal domain of Bcd (residues 1-91), either wild type or Bcd(A52-56), was attached to two different activators, GAL4-B6 and GAL4-B42. These two activators contain the DNA-binding domain of GAL4 (residues 1-147) fused to bacterially derived activation domains that have different activation potentials (Ma and Ptashne, 1987b). When assayed on the CAT reporter gene containing GAL4 binding sites, both activators with the Bcd(A52-56) N terminus exhibited higher activity than their wild-type counterparts (Fig. 4B), demonstrating that the self-inhibitory domain can function on entirely heterologous activators.
The function of the self-inhibitory domain requires a Drosophila factor(s) absent in yeast cells
Our studies described thus far demonstrate that the self-inhibitory domain of Bcd is an independent module that does not target any specific sequences from Bcd and can work on heterologous activators. One attractive model consistent with these findings is that this module provides a docking site for a Drosophila co-repressor(s) or complex(es) that can inhibit transcription. To test this hypothesis, we analyzed the activities of full-length Bcd proteins, either wild type or Bcd(A52-56), in yeast cells that contain an integrated hb-lacZ reporter gene. Our experiments show that, in striking contrast to its behavior in Drosophila S2 cells, Bcd(A52-56) did not have an increased activity (Table 2); it actually exhibited a moderately reduced activity when compared with the wild-type protein. These results suggest that the action of the self-inhibitory domain requires a co-factor(s) that is present in Drosophila S2 cells but missing in yeast cells.
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Bcd(A52-56) causes severe alterations in target gene expression during embryonic development
To determine how Bcd(A52-56) affects the expression of Bcd target genes, we conducted in situ hybridization assays for hb and orthodenticle (otd) in embryos from females that carry one copy of bcd(A52-56)18A. These embryos were chosen because 100% of them exhibited developmental defects (Table 3). Our data shown in Fig. 6 reveal the following results. First, the Bcd-dependent expression domains of both hb and otd in the anterior are dramatically expanded towards the posterior at different developmental stages (e.g. compare C with D and I with J), demonstrating that Bcd(A52-56) can activate these target genes much more effectively in embryos. The posterior shift of the expression domains of these target genes is consistent with both a posterior shift of segmentation gene expression stripes (not shown) and our observed cuticle phenotypes resulting from a failed or incomplete head involution (Fig. 5). In embryos containing Bcd(A52-56), the parasegment 4 (PS4) domain of hb at a later stage is dramatically shifted toward the posterior (Fig. 5F), further illustrating a posterior shift of the fate map of these embryos.
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Third, the expression domain of hb at the posterior is greatly reduced (Fig. 5D,F) or missing. Both this expression domain and the PS4 domain of hb are thought to be regulated in a Bcd-independent manner (Margolis et al., 1995; Schroder et al., 1988
; Tautz, 1988
). In particular, previous studies have shown that the posterior expression domain of hb is repressed by the Hunchback protein (Hb) itself (Margolis et al., 1995
; Struhl et al., 1989
). It is possible that the dramatic posterior expansion of the Bcd-dependent anterior hb expression domain may contribute to the reduction or elimination of the posterior domain. Taken together, our phenotypic and staining analysis of embryos containing Bcd(A52-56) demonstrate that the self-inhibitory domain of Bcd is required for proper embryonic patterning and target gene activation during development.
The self-inhibitory domain of Bcd is targeted by a novel co-factor(s), rather than CtBP or dSAP18
As discussed above, residues 52-56 of Bcd, PFDLL, share similarity with the consensus motif for CtBP interaction, PLDLS, where the underlined residues are invariable (Postigo and Dean, 1999). CtBP is a major co-repressor that mediates the activity of a variety of transcriptional repressors in Drosophila and other organisms (Criqui-Filipe et al., 1999
; Deconinck et al., 2000
; Meloni et al., 1999
; Nibu et al., 1998a
; Nibu et al., 1998b
; Poortinga et al., 1998
; Postigo and Dean, 1999
; Schaeper et al., 1995
; Sollerbrant et al., 1996
; Turner and Crossley, 1998
). To test whether Drosophila CtBP is involved in modulating Bcd function, we analyzed the expression of hb and otd in embryos containing disruptions in CtBP activity. CtBP is expressed maternally and zygotically, and northern blots show a complex expression pattern in early Drosophila embryos (Poortinga et al., 1998
). To disrupt maternal CtBP activity, we generated germline clones (GLCs) that are homozygous for the P-element insertion (P1590) using the FRT-ovoD technique (Chou et al., 1993
). Previous experiments have shown that this technique disrupts the early functions of those repressors known to interact with CtBP, including Hairy, Kruppel (Kr), Knirps (Kni) and Snail (Morel et al., 2001
; Nibu et al., 1998a
; Nibu et al., 1998b
; Poortinga et al., 1998
).
Fig. 7 shows the in situ staining results for hb and otd in wild type or embryos from P1590 GLCs. There is no detectable change in the early expression patterns of these two genes, as judged by both their posterior borders and relative expression levels (Fig. 6B and 6F, compare with 6A and 6E, respectively). In addition, both genes appear to be downregulated at the anterior tip, indicating that CtBP does not mediate the repression of Bcd activity in this region. These results are consistent with the previous finding that the disruption of maternal CtBP does not grossly affect gap gene expression in Drosophila embryos (Nibu et al., 1998b; Poortinga et al., 1998
). Furthermore, most embryos from P1590 GLCs exhibited normal hb expression in both the PS4 domain and the posterior domain at a later stage (not shown). However, in a small percentage (
10%) of embryos from P1590 GLCs, the PS4 stripe (marked *) expands posteriorly, and the posterior domain is expanded toward the anterior (Fig. 7D). These alterations may be due to disruptions of the functions of the gap proteins Kr and Kni, which are expressed in the region between these two hb domains. Consistent with this hypothesis, previous experiments have shown that Kni may act as a repressor of the PS4 stripe (Kosman and Small, 1997
; Wimmer et al., 2000
).
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Amino acids 52-56 of Bcd, PFDLL, are not similar to the previously defined motifs for interaction with Groucho, another major co-repressor present in the early embryo (Chen and Courey, 2000; Tolkunova et al., 1998
; Zhang and Levine, 1999
; Zhang et al., 2001
). However, a Drosophila protein called Bin1 was recently isolated as a Bcd-interacting protein in a custom-design yeast two-hybrid system (Zhu and Hanes, 2000
). This protein shares homology with the SAP18 component of a mammalian histone deacetylase complex (Zhang et al., 1997
). Histone deacetylase complexes represent another major mechanism of transcription repression (for a review, see Ahringer, 2000
). Interestingly, dSAP18 (Bin1) has also been shown to interact with another Drosophila transcription factor GAGA both biochemically and genetically (Espinas et al., 2000
). To determine whether dSAP18 directly targets our delineated self-inhibitory domain of Bcd, we conducted a GST pull-down analysis. In this assay, bacterially expressed GST-dSAP18, or GST alone, was used to pull down in vitro translated and radioactively labeled Bcd derivatives. Our results (Fig. 8) demonstrate that, as expected, wild-type Bcd can interact with dSAP18 (lanes 1, 2). However, Bcd(A52-56), which has a defective self-inhibitory function, interacted with dSAP18 similarly, suggesting that dSAP18 does not target Bcd through the delineated self-inhibitory domain. Consistent with this suggestion and the findings described in a recent report (Zhu et al., 2001
), our experiments further show that dSAP18 can interact with Bcd(92-489), which lacks the entire N-terminal domain (lanes 7, 8). Taken together, these studies suggest that self-inhibitory domain of Bcd delineated in this report represses its own activity by interacting with a novel Drosophila factor(s) or complex(es), other than CtBP and dSAP18.
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DISCUSSION |
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We currently favor a co-repressor model based on the following results and considerations; in this model, a co-repressor(s) or complex(es) specifically interacts with the self-inhibitory domain of Bcd, thus inhibiting its transcriptional activation function. First, our results argue against a role of the self-inhibitory domain in subcellular localization and DNA binding (Fig. 3). Second, our data show that this domain works as an independent module that does not specifically target any other sequences of Bcd and can repress the activity of heterologous activators (Fig. 4). We note that the magnitude of repression by the self-inhibitory domain in some of our hybrid activators is decreased (Fig. 4), suggesting that Bcd sequences (particularly the neighboring homeodomain), though not necessary, may play a contributory role. Third, our experiments further suggest that the action of the self-inhibitory domain requires a Drosophila protein(s) that is missing in yeast cells (Table 2). Our results are consistent with the idea that CtBP and dSAP18 do not directly target Bcd through the self-inhibitory domain (Figs 7 and 8). These findings suggest the existence of a novel co-repressor(s) or complex(es) that regulates Bcd activity in Drosophila. The isolation and characterization of such co-repressor molecule(s) will enhance our future understanding of the molecular mechanisms of transcriptional activation and pattern formation by Bcd during embryonic development.
Our analysis of embryos from bcd(A52-56) transgenic females reveals a dominant, gain-of-function effect causing developmental defects in both head and abdominal regions. These phenotypes share resemblance to those caused by a Bcd-VP16 fusion protein which contains the strong activation domain VP16 (Driever et al., 1989). Interestingly, an excessive amount of wild-type Bcd produced from six copies of bcd can also cause head and abdominal defects in a fraction of the embryos (Namba et al., 1997
). It is relevant to note that a bcd cDNA transgene in our P-element vector pCaSpeRBcdBglII was estimated to produce, on average, approx. half the amount of Bcd protein as an endogenous bcd gene (Driever et al., 1990
). Compared with wild-type Bcd expressed from six copies of bcd, Bcd(A52-56) can cause embryonic defects at a much higher penetrance (100% in line 18A) and at a much lower concentration (
1/8). We note that two copies of wild-type bcd cDNA transgene only caused moderate abdominal defects at a low frequency in most of the lines examined (Table 3). This observation is consistent with the estimate that two copies of our transgene are equivalent to only one copy of genomic bcd.
The head defects caused by Bcd(A52-56), like those by Bcd-VP16 and excessive amounts of wild-type Bcd (Driever et al., 1989; Namba et al., 1997
), are presumably due to a failed or incomplete head involution resulting from the posterior shift of the fate map. It is possible that both Bcd-VP16 and Bcd(A52-56) may have additional molecular consequences associated with their strong activation functions. It remains to be determined whether, for example, Bcd(A52-56) causes the developmental defects, in part, by activating zygotic genes that are normally not targets of Bcd in embryos.
The expression domains of hb and otd in embryos containing Bcd(A52-56) are expanded dramatically towards the posterior (Fig. 6). Interestingly, we did not observe any obvious increase in the intensity of their expression in these embryos. It is possible that hb and otd are expressed, in response to the Bcd gradient, at levels that are already maximal in wild-type embryos. According to this idea, the consequence of the stronger activator Bcd(A52-56) is not an elevated level of hb and otd expression, but rather, a posterior shift of their expression domains. It has been shown that the activating strength of an activator can actually influence its in vivo DNA-binding ability (Tanaka, 1996). In particular, activators with stronger activation domains can bind DNA at lower concentrations in vivo, presumably because a stronger interaction with the basal transcription machinery can facilitate their DNA binding function at low concentrations. Although our experiments demonstrate that both wild type Bcd and Bcd(A52-56) have a similar affinity to a single Bcd binding site in vitro (Fig. 3), a dramatic posterior shift of the hb and otd expression domains in embryos containing Bcd(A52-56) suggests that Bcd(A52-56) may have a significantly higher in vivo affinity to both enhancers. Furthermore, as Bcd(A52-56) is a much stronger activator, it is possible that, as proposed previously (Arnosti et al., 1996
; Lehman et al., 1998
; Ma et al., 1999
), hb and otd can be activated by fewer Bcd(A52-56) molecules (than wild-type molecules) in the more posterior part of the embryo.
Another domain of Bcd (residues 300-340, alanine-rich) was shown recently to also exhibit an inhibitory function (Janody et al., 2001). Besides their different physical locations and amino acid compositions, there are several other important differences between the self-inhibitory domain delineated in this report and that newly described domain. First, the self-inhibitory domain described here represses transcription over 20-fold in deletion assays (Fig. 1), whereas a single alanine-rich domain only represses transcription threefold (its effect is significantly enhanced when multimerized). We note that a Bcd derivative lacking the alanine-rich region also causes a posterior shift of the hb expression domain in embryo (Schaeffer et al., 1999
), though less dramatically than Bcd(A52-56). Second, the self-inhibitory domain can work on heterologous activation domains (Fig. 4B), in addition to those from Bcd, suggesting an active repression mechanism. Third, this domain has been systematically dissected by deletion and point mutations (Figs 1 and 2; C. Z. and J. M., unpublished). Finally and most importantly, while point mutations in the self-inhibitory domain cause severe developmental defects (Fig. 5), the entire C-terminal half of Bcd, including the alanine-rich domain, can be deleted (Schaeffer et al., 1999
).
Although our transgenic studies demonstrate that the self-inhibitory function of Bcd is important for proper embryonic pattern formation in Drosophila, it is not completely clear how this function is regulated by other developmental cues. Our results show that the action of the self-inhibitory domain is not responsible for Tor-dependent repression upon cellularization (Fig. 6), although we cannot rule out the possibility that the self-inhibitory domain may play a contributory role in this process. In addition, it has been shown that self-inhibitory domains of other proteins are involved in synergistic activation with co-factors (Amendt et al., 1999; Durocher et al., 1997
). The self-inhibitory domain of Bcd may similarly participate in synergistically activating transcription with other Drosophila factors, such as Hb (Simpson-Brose et al., 1994
). Furthermore, as the N-terminal region of Bcd is also engaged in self-association and cooperative DNA binding (Yuan et al., 1996
; Zhao et al., 2000
), enhancer architecture [i.e. the arrangement of DNA sites for Bcd (Yuan et al., 1999
) and other factors] may influence how Bcd molecules are positioned on different enhancers and, thus the availability of the self-inhibitory domain for interacting with the proposed co-repressor(s). Given its intricate morphogenetic role in instructing embryonic patterning, an intriguing possibility exits that Bcd itself may function as an active repressor in a context-dependent manner during embryonic development.
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ACKNOWLEDGMENTS |
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