Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo
1998; Springer Nature; Volume: 17; Issue: 20 Linguagem: Inglês
10.1093/emboj/17.20.5998
ISSN1460-2075
AutoresDavid S. Burz, Rolando Rivera‐Pomar, Herbert Jäckle, Steven D. Hanes,
Tópico(s)Fungal and yeast genetics research
ResumoArticle15 October 1998free access Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo David S. Burz David S. Burz Molecular Genetics Program, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, State University of New York-Albany, NY, 12208 USA Search for more papers by this author Rolando Rivera-Pomar Rolando Rivera-Pomar Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg, D-37077 Göttingen, Germany Search for more papers by this author Herbert Jäckle Herbert Jäckle Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg, D-37077 Göttingen, Germany Search for more papers by this author Steven D. Hanes Corresponding Author Steven D. Hanes Molecular Genetics Program, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, State University of New York-Albany, NY, 12208 USA Search for more papers by this author David S. Burz David S. Burz Molecular Genetics Program, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, State University of New York-Albany, NY, 12208 USA Search for more papers by this author Rolando Rivera-Pomar Rolando Rivera-Pomar Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg, D-37077 Göttingen, Germany Search for more papers by this author Herbert Jäckle Herbert Jäckle Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg, D-37077 Göttingen, Germany Search for more papers by this author Steven D. Hanes Corresponding Author Steven D. Hanes Molecular Genetics Program, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, State University of New York-Albany, NY, 12208 USA Search for more papers by this author Author Information David S. Burz1, Rolando Rivera-Pomar2, Herbert Jäckle2 and Steven D. Hanes 1 1Molecular Genetics Program, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, State University of New York-Albany, NY, 12208 USA 2Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg, D-37077 Göttingen, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5998-6009https://doi.org/10.1093/emboj/17.20.5998 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Bicoid morphogen directs pattern formation along the anterior–posterior (A–P) axis of the Drosophila embryo. Bicoid is distributed in a concentration gradient that decreases exponentially from the anterior pole, however, it transcribes target genes such as hunchback in a step-function-like pattern; the expression domain is uniform and has a sharply defined posterior boundary. A ‘gradient-affinity’ model proposed to explain Bicoid action states that (i) cooperative gene activation by Bicoid generates the sharp on/off switch for target gene transcription and (ii) target genes with different affinities for Bicoid are expressed at different positions along the A–P axis. Using an in vivo yeast assay and in vitro methods, we show that Bicoid binds DNA with pairwise cooperativity; Bicoid bound to a strong site helps Bicoid bind to a weak site. These results support the first aspect of the model, providing a mechanism by which Bicoid generates sharp boundaries of gene expression. However, contrary to the second aspect of the model, we find no significant difference between the affinity of Bicoid for the anterior gene hunchback and the posterior gene knirps. We propose, instead, that the arrangement of Bicoids bound to the target gene presents a unique signature to the transcription machinery that, in combination with overall affinity, regulates the extent of gene transcription along the A–P axis. Introduction Pattern formation during embryogenesis requires the establishment of precise spatial domains of gene expression. These domains, with their characteristic sharp boundaries, arise through a complex set of interactions between transcription regulatory proteins and their cis-acting DNA binding sites. Regulatory proteins use at least three mechanisms to stimulate spatially correct target gene transcription. First, regulatory proteins are localized, thereby restricting their activity to broad regions within the embryo (Driever and Nüsslein-Volhard, 1988a; Roth et al., 1989; Pankratz et al., 1992). Second, regulatory proteins act in combination with other co-activators or repressors of transcription (Riddihough and Ish-Horowicz, 1991; Small et al., 1991; Xue et al., 1993; Sauer and Jäckle, 1995). Third, regulatory proteins interact with themselves in a cooperative manner to activate or repress transcription at subsaturating concentrations and to produce a sharp threshold response (Beachy et al., 1993; TenHarmsel et al., 1993; Wilson et al., 1993). The Drosophila morphogen, Bicoid, which directs pattern formation along the anterior–posterior (A–P) axis in the developing embryo, is thought to employ all three mechanisms to establish spatially correct domains of target gene expression (Frohnhöfer and Nüsslein-Volhard, 1986; reviewed in Driever, 1993). The first mechanism forms the basis for the classic morphogen model of Bicoid action. Bicoid is localized along the A–P axis into a protein concentration gradient that spans two to three orders of magnitude decreasing exponentially over the anterior half of the embryo (Driever and Nüsslein-Volhard, 1988a). The importance of Bicoid's localization in a gradient was shown by increasing or decreasing maternal bicoid gene dosage, which resulted in steeper or shallower concentration gradients, thus shifting the cephalic furrow towards the posterior or anterior region of the embryo, respectively (Driever and Nüsslein-Volhard, 1988b). Anterior regions that are exposed to higher levels of Bicoid express a different set of genes (e.g. buttonhead, orthodenticle) than more posterior regions where the Bicoid concentration is lower (e.g. hunchback, Krüppel). It is thought that the extent along the A–P axis to which a Bicoid target gene is expressed will depend on the affinity of Bicoid for its enhancer (i.e. the gradient-affinity model). Despite its vanishingly low concentrations at the posterior end of the embryo, Bicoid also activates posteriorly acting segmentation genes such as the abdominal gap gene knirps (Rivera-Pomar et al., 1995) and the posteriormost stripe of the pair-rule gene hairy (La Rosee et al., 1997). Thus, although Bicoid is localized in a gradient with a peak concentration at the anterior pole, it still functions as a transcriptional activator along the entire A–P axis of the Drosophila embryo. The second mechanism by which Bicoid stimulates spatially restricted gene expression is by acting in combination with other transcription regulatory proteins. For example, the establishment of even-skipped (eve) stripe 2 results from competition between activators Bicoid and Hunchback, and repressors Giant and Krüppel, for overlapping sites within the eve stripe 2 regulatory element, and by a local repression mechanism termed quenching (Small et al., 1991; Stanojevic et al., 1991). In the posterior region of the embryo, Bicoid and the maternal homeodomain protein Caudal, which forms a posterior–anterior concentration gradient, act together in a partially redundant manner to activate the posterior gene knirpsv (reviewed in Rivera-Pomar and Jäckle, 1996). The regulatory region that mediates knirps gene activation contains two cis-acting regulatory elements; one is activated in response to Caudal, the other, termed kni64, is a 64 bp enhancer that is activated in response to Bicoid (Rivera-Pomar et al., 1995). In the third mechanism, Bicoid monomers are thought to interact cooperatively with one another to bind DNA and stimulate target gene expression. Cooperative DNA binding by Bicoid has not been rigorously demonstrated, but has been widely proposed as the explanation for Bicoid's ability to regulate spatial transcription of hunchback (hb) and other genes (e.g. Ptashne, 1986; Driever, 1993). For example, cooperative binding by Bicoid to its sites in the hb upstream regulatory element is thought to result in a sharp posterior boundary of hb expression that specifies the position of the cephalic furrow. Several indirect lines of evidence support the idea that Bicoid binds DNA cooperatively. First, reporter genes require multiple Bicoid binding sites to be activated in yeast (Hanes and Brent, 1989), cultured Drosophila cells (Driever and Nüsslein-Volhard, 1989) and transgenic Drosophila embryos (Driever et al., 1989; Struhl et al., 1989). Secondly, the minimal Bicoid binding site (TAATCCC; Hanes and Brent, 1991) does not define a unique target sequence; this 7 bp site is expected to be present ∼10 000 times in the fly genome. However, an enhancer containing multiple sites would be comparatively rare; such a region would act as a local sink for Bicoid monomers. Thirdly, Bicoid-dependent transcription in yeast and Drosophila is sensitive to the spacing between binding sites (Hanes et al., 1994) and to their relative orientation (G.Devasahayam, Burz & Hanes, unpublished), both hallmarks of proteins that bind DNA cooperatively (Mao et al., 1994). Finally, in vitro evidence consistent with cooperative DNA binding has been reported (Ma et al., 1996). Here we demonstrate that Bicoid binds DNA cooperatively in yeast and in vitro and thus provide the first conclusive evidence for a mechanism proposed to regulate anterior patterning in the embryo (Driever et al., 1989). Our experimental strategy was based on concepts drawn from the study of cI repressor of bacteriophage λ, in which the cooperative free energy of DNA binding is asymmetrically distributed between strong and weak sites in OR (Ackers et al., 1983, Beckett et al., 1993). We show that Bicoid bound to a strong site promotes occupancy of an adjacent weak site by cooperative DNA binding. Without this cooperative coupling, Bicoid binding to these weak sites, which are similar to Antennapedia-class homeodomain sites, does not occur. Bicoid cooperativity should therefore increase the occupancy of weak, and otherwise unrecognized, sites, leading to a more concerted threshold response. Furthermore, we show that Bicoid cooperativity is pairwise, that is, DNA-bound Bicoid interacts with only one other Bicoid monomer at a time. This binding appears to be sequential: Bicoid binds DNA as a monomer (Kd 0.24 nM) and engages in cooperative interactions only when bound to DNA. Finally, we find that contrary to the explicit prediction of the gradient-affinity model for Bicoid action (Driever et al., 1989), the posteriorly-expressed gene knirps did not have a higher affinity for Bicoid than the more anteriorly expressed gene hunchback. This result suggests that expression of Bicoid target genes at different positions along the A–P axis of the embryo can occur by a mechanism independent of DNA binding affinities. Results In vivo assay to study Bicoid cooperativity To demonstrate that Bicoid activates gene expression cooperatively, we used the assay shown in Figure 1. In this assay, we measure Bicoid concentration-dependent activation of lacZ reporter genes in yeast. The lacZ reporters carry upstream Bicoid binding sites and their activation is quantitated by assaying β-galactosidase (β-gal) activity. To control the amount of Bicoid produced in cells, we used a fusion protein, GAL4–ER–VP16, which consists of the DNA-binding domain of Gal4, the ligand-binding domain of human estrogen receptor and the activation domain of VP16 (Louvion et al., 1993). GAL4–ER–VP16 binds the GAL1 promoter and drives expression of a bicoid cDNA in a hormone-dependent manner. By varying the amount of exogenous hormone (β-estradiol) added to yeast cultures, we can vary the intracellular Bicoid concentration by nearly three orders of magnitude, as assayed by Western analysis of yeast cell extracts (Table I). The ability to generate this broad range of Bicoid concentrations, combined with the use of lacZ reporters that carry particular arrays of Bicoid binding sites, allows us to study Bicoid cooperativity in vivo. Figure 1.An in vivo assay for studying cooperative gene activation in yeast. Yeast cells were co-transformed with plasmids that encode the indicated proteins. Hormone (β-estradiol) was added to mid-log phase cultures, where it entered cells and bound to the fusion protein GAL4–ER–VP16 to drive Bicoid expression in a concentration-dependent manner (Table I). Bicoid binds to different arrays of upstream sites to activate lacZ transcription, which is quantitated by assaying β-galactosidase activity. Download figure Download PowerPoint Table 1. Hormone-induced Bicoid expression Activator [β-estradiol] (μM) [Bicoid] (μM)a GAL4–ER–VP16(mut)b 0 0.5–0.8 0.0005 0.7–1.1 GAL4–ER–VP16 0 1.2–1.9 0.0005 2.4–5 0.0025 11–40 0.01 25–85 1 62–196 a [Bicoid] is the total cellular concentration. b GAL4–ER–VP16(mut) is mutated in the VP16 domain (F442P) to reduce the strength of activation. Increasing the number of Bicoid sites results in cooperative gene activation We used our yeast assay to examine Bicoid-dependent lacZ activation as a function of increasing numbers of strong (S) high-affinity consensus Bicoid sites (TCTAATCCC) derived from analysis of the hunchback promoter (Driever and Nüsslein-Volhard, 1989). At low and intermediate Bicoid concentrations the increase in β-gal activity between a one-site (S) and a two-site (SS) reporter is greater than 100-fold, with three sites (SSS) there is an additional two- to three-fold increase in β-gal activity, and with six sites (6 × S) there is a further 2-fold increase (Figure 2 and Table II). The greater-than-additive increase in lacZ expression with an increasing number of sites indicates cooperative gene activation. As expected, this effect is greater at lower concentrations at which the effects of cooperativity are most pronounced. Figure 2.Cooperative gene activation by Bicoid in yeast. Activation data were obtained using the assay shown in Figure 1. The magnitude and steepness of each activation curve increases with an increasing number of strong consensus sites (TCTAATCCC). S refers to the number of strong sites (1, 2, 3 or 6) present in the lacZ reporter. The curves represent the best fit to a simple concentration-dependent transition. Inset: normalized activation curves show that Bicoid binds more strongly to a two-site reporter than to a single site. Download figure Download PowerPoint Table 2. Bicoid-dependent reporter gene expression in yeasta Bicoid concentrationb Reporter Genec Low Medium High S S S 57 760 1830 S W S 41 613 1800 S S W 27 442 1350 S S 30 472 834 W S W 13 289 860 S W W 3 249 778 S X S 5 205 518 W S 0 108 427 S W 0 48 269 S 0 4 46 W W W 0 17 44 W W 0 0 3 W 0 0 0 6 × S 336 1060d 2381 kni64 251 1018 2240 Ψkni 417 1085d 2112 2Rkni 277 1169 2287 hb 322 1172 2499 a Data were obtained using the assay shown in Figure 1; results are given in units of β-gal activity. b Low, medium and high Bicoid concentrations correspond to 0, 0.0025 and 1 μM β-estradiol (Table I). c Target sites used: S = TCTAATCCC; W = TCTAATTCC; kni64 = knirps enhancer; Ψkni = knirps enhancer with idealized (S) sites; 2Rkni = idealized knirps enhancer with two sites reversed; 6 × S = enhancer containing six strong sites arranged head-to-tail; hb = hunchback enhancer. d Samples were assayed on different days from others in this group (lower panel, Medium Bicoid Concentration); the values given are extrapolated from several independent experiments. Cooperative gene activation by Bicoid might result from either cooperative DNA binding or from cooperative interactions between DNA-bound Bicoid and the transcription machinery (i.e. transcriptional synergy, Carey et al., 1990; Lin et al., 1990). If cooperative DNA binding occurs, then the apparent midpoint of the normalized activation curve for a two-site reporter will shift to a lower concentration than that of a one-site reporter, reflecting occupancy of these sites at a lower overall Bicoid concentration. This is exactly what is observed (Figure 2, inset): there is an increase of about a half an order of magnitude in the affinity of Bicoid for the two-site reporter relative to that of a single site. By normalizing the data from 0 to 1, which correspond to completely vacant and fully occupied binding sites, we have taken into account the difference in activation potential of a single site versus two sites, allowing us to compare concentration-dependent curves directly, regardless of the number of sites. Finally, we note that the midpoint of the activation curves in vivo is several orders of magnitude greater than the binding affinities measured in vitro (see below). We believe that this is the result of the partitioning of total intracellular Bicoid, which we measured, into cytoplasmic and nuclear compartments. We do not know the actual nuclear concentration of Bicoid, but increasing the total Bicoid concentration ultimately results in saturation of transcription, a condition necessary for interpreting this experiment. We also compared Bicoid binding to one- and two-site templates in vitro using gel-shift assays. The affinity of a homeodomain-containing fragment of Bicoid (Bcd89–154) for a two-site template (Kd = 0.21 nM) was slightly higher than that of a one-site template (Kd = 0.24 nM; Table III). This increase in affinity is in general agreement with our yeast assay; however, the difference observed in vitro is much smaller. The fact that the difference is so small is not unexpected because the magnitude of the cooperative free energy is smallest when the coupled sites are of identical affinity (Ackers et al., 1983; see also below). Thus, in this example, only a small amount of the total cooperative gene activation appears to be due to cooperative DNA binding. In yeast (Figure 2, inset), the apparent difference in affinity might be artificially amplified due to unequal partitioning of Bicoid between the cytoplasm and nucleus, e.g. a 10-fold increase in total cellular Bicoid concentration might correspond to only a 2-fold increase nuclear concentration. Table 3. Bicoid homeodomain (Bcd89–154) binding in vitro Template Kd (nM) nH σ W 1.14 1.0a 0.336 W W W 1.07 1.7 0.034 S 0.24 1.0a 0.125 S S 0.21 2.0a 0.123 S S S 0.17 2.1 0.020 S W S 0.21 3.0a 0.062 S X S 0.88 2.5 0.028 W S W 0.18 1.7 0.095 kni64 0.53 1.4 0.080 Ψkni 0.54 2.1 0.097 2Rkni 0.61 1.1 0.128 hb 0.98 3.0 0.031 nH= Hill Coefficient; σ = standard deviation of fit. a nH was held fixed during analysis, as smaller values increased the standard deviation of the fit; all other values were resolved using the equation given in Materials and methods. Bicoid bound to strong sites promotes Bicoid binding to weak sites The binding of proteins to adjacent DNA sites of different affinities provides a dramatic demonstration of binding cooperativity. This is because the cooperative free energy is distributed asymmetrically, with most of the free energy contributed to binding at the weaker site (Ackers et al., 1983). The result is that weak site saturation occurs at a lower total protein concentration. Therefore, if Bicoid binds DNA cooperatively, then at subsaturating concentrations, Bicoid bound to a strong site should promote occupancy of a nearby weak site. We tested whether this was true using lacZ reporter genes that carry different combinations of strong and weak Bicoid sites. The strong site (S) used in these experiments was the consensus Bicoid site (TCTAATCCC) described above. The weak site (W) contained a C:G to T:A change at position 7 in the Bicoid site (TCTAATTCC); the C:G base pair is required for specific recognition by Bicoid in yeast and in flies (Hanes and Brent, 1991; Hanes et al., 1994). The W site resembles an Antennapedia (Antp) class homeodomain binding site that is recognized poorly if at all by Bicoid in yeast (Hanes and Brent, 1991). Gel-shift analyses using Bcd89–154 confirm that the affinity of Bicoid for the W site is indeed lower than for the S site (Table III). Control experiments show that reporter genes that carry three weak sites (WWW) are not activated, suggesting that Bicoid does not occupy these sites in vivo (Figure 3A). In contrast, reporters that carry three strong sites (SSS) are activated strongly, suggesting that Bicoid does occupy these sites well. If Bicoid binding is cooperative, then reporters that carry a mixture of sites (SWS) should be activated to levels comparable to that of a three strong-site reporter (SSS), rather than to reporters that carry only two strong sites. Indeed, this is what we observed at all Bicoid concentrations examined (Figure 3A). The level of transcription elicited by SWS was always greater than that of either SS (Table II) or SXS, where the X site lacks all base pairs required for recognition by homeodomains, but preserves the spacing between S sites. Activation of SWS was virtually identical to that of SSS, particularly at lower Bicoid concentrations. The simplest explanation for these results is that Bicoid bound to strong sites promotes Bicoid binding to a weak site by cooperative coupling. The effect of transcriptional synergy is ruled out because we compare reporter genes with identical numbers of sites; therefore, the maximum level of activity attainable in this experiment corresponds to three saturated sites, while lower activity reflects partial vacancy of these sites. Figure 3.Bicoid binds DNA in a cooperative, pairwise manner in yeast, and distinguishes between sites of different affinity from the hunchback enhancer. (A) Bicoid bound to strong (S) sites promotes binding to a weak (W) site. (B) Bicoid binds with pairwise cooperativity. (C) Cooperative interactions amplify differences between the intrinsic affinities of strong (A1) and weak (X1) sites. The data were generated as shown in Figure 1, and the curves represent the best fit of the data to a simple concentration-dependent transition. Reporter constructs contained the indicated sites inserted upstream of lacZ. Download figure Download PowerPoint The level of β-gal activity obtained for SS is greater than that of SXS, confirming the previous observation that, in yeast, Bicoid activates gene expression better from closely spaced sites than from more widely spaced sites (Hanes et al., 1994). Site-spacing preferences are often indicative of cooperative DNA binding; changes in site-spacing may disrupt or enhance protein–protein interactions that stabilize DNA-bound monomers, thereby affecting levels of gene activation (Mao et al., 1994). To confirm the idea of cooperative coupling between strong and weak sites, we compared in vitro binding of Bicoid89–154 to SWS versus SSS and SXS using gel-shift assays. The results (Table III) indicate that binding to SWS (Kd = 0.21 nM) is much more similar to that of SSS (Kd = 0.17 nM) than to that of SXS (Kd = 0.88 nM), corroborating the results observed in vivo. Site loading, as detected by the formation of three distinct complexes, occurs at the same Bicoid concentration for SWS as for SSS, and these complexes are formed in the same relative proportions at a given Bicoid concentration (Figure 4). However, loading of SXS requires a Bicoid concentration about 10-fold higher than for SWS, and the complexes form at different relative proportions. For example, at the highest concentrations, the ratios of triply bound to doubly bound complexes (C3:C2) for SSS, SWS and SXS are 2.6:1, 2.5:1 and 1:1, respectively. These results indicate that the W site is readily occupied in SWS, despite the intrinsically low affinity of Bicoid for an isolated W site, or a W site flanked by other weak sites (WWW; Table III). Similar experiments using full-length Bicoid show that it also exhibits cooperative coupling, forming a triply bound complex with SWS and SSS, but not with SXS (data not shown). Thus, in vitro experiments demonstrate that Bicoid bound to strong sites helps Bicoid bind to a weak site, confirming the in vivo results. Figure 4.Bicoid cooperative DNA binding shows strong-site/weak-site coupling in vitro. The upper three panels are gel-shifts of Bicoid89–154 binding to oligonucleotide templates SSS, SWS and SXS. The lower panel shows isotherms resolved from data in the upper panels and data from other experiments. Note that the isotherm for SWS is nearly coincident with that of SSS, implying occupancy of the W site. Bicoid concentration ranges are the same for all three gels (10−12 M–10−9 M, with five steps per log unit). Free DNA, and singly (C1), doubly (C2) and triply bound (C3) complexes are indicated. The templates are identical to oligonucleotides used to make the reporter genes tested in Figure 3. Download figure Download PowerPoint Surprisingly, at the highest Bicoid concentrations the X site can be loaded to form a triply bound complex. We suspect that cooperative interactions between Bicoid bound to the adjacent S sites promotes binding to the non-specific X site. Loading of the X site might also occur in vivo; we observe that the level of activation of an SXS reporter at very high Bicoid concentrations (higher than shown in Table III) exceeds that of SS (data not shown). Bicoid cooperative interactions are pairwise We tested whether Bicoid cooperativity is pairwise, that is, does Bicoid bound to a strong site interact cooperatively with only one additional Bicoid monomer at a time. To do this, we measured the level of β-gal activity resulting from a three-site reporter gene containing a strong site in the middle position (WSW). If Bicoid bound to the S site facilitated Bicoid binding to both W sites, then we would expect the level of gene expression to be comparable to that of SSS. Instead, we find that the level of activation by WSW is similar to that of SS, particularly at high Bicoid concentrations (Figure 3B, Table II), suggesting that only one or the other weak site is occupied, consistent with the idea of pairwise cooperativity. Our assay cannot determine whether a single weak site is being occupied, or whether both weak sites are occupied part of the time with equal probability; however, these two conditions are statistically equivalent. Likewise, the activity of SWW is similar to that of SS, again suggesting that only one of the weak sites is occupied. Taken together, these data support the idea that a Bicoid monomer bound to a strong site mediates predominantly pairwise cooperative DNA binding. Pairwise cooperativity predicts that under certain conditions, discrete pairs of adjacently bound Bicoid monomers will dominate the binding process. Indeed, we found that the levels of activity elicited by SSW at low and intermediate Bicoid concentrations are similar to that of SS, implying that the W site is vacant at these concentrations. However, at the highest Bicoid concentrations examined, SSW stimulates more gene expression than SS, but still less than SSS (or SWS), which suggests that the W site in SSW is only partially occupied. Thus, most of the cooperative free energy is shared between monomers bound to the S sites, with little available to facilitate binding to the W site, which loads only at very high Bicoid concentrations. Bicoid pairwise cooperativity is directional If Bicoid cooperativity is strictly pairwise, then the maximum activity resulting from Bicoid binding to a strong and weak site (SW) should yield transcription activity comparable to that of SS. However, the maximum level of activity elicited by SW is 3 to 4-fold weaker than that of SS, although it is still 5-fold greater than that of a single S site (Table II). The reason for this is unclear. One explanation is that Bicoid cooperativity is directional. To test this idea, we reversed the orientation of the strong and weak sites (i.e. WS) and found that the activity of the WS reporter is more similar to that of SS (about twice that of SW) at all Bicoid concentrations (Table II). Gel-shift experiments gave similar results: WS sites are bound with slightly higher affinity than are SW sites (data not shown). Thus, a Bicoid monomer bound to a strong site prefers to interact with a Bicoid monomer positioned in the 5′ direction. Yeast assay distinguishes between A1 and X1 sites in hunchback Driever et al. (1989) identified two classes of Bicoid sites in the hb promoter region: strong sites (termed A1, A2 and A3) and weak sites (termed X1, X2 and X3), and a third uncharacterized class (termed B1, B2 and B3). However, in vitro measurements by others of Bicoid binding to the so-called strong and weak sites failed to resolve a difference in affinity between them (Ma et al., 1996). In contrast, our assay easily resolves differences between Bicoid recognition of A1 and X1 sites in vivo. For example, Bicoid activation of lacZ reporters containing th
Referência(s)