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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01075

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Quantitative contributions of CtBP-dependent and -independent repression activities of Knirps

¹ Department of Biochemistry and Molecular Biology, and Genetics Program, Michigan State University, East Lansing, MI 48824-1319, USA
² Department of Biology, New York University, New York, NY 10003, USA

View larger version (72K): [in a new window]   Fig. 6. Effect of expression of Knirps proteins on hunchback (hb), runt, hairy and fushi tarazu (ftz) demonstrates differential activity of Knirps 1-429 versus Knirps 1-330 on all but the most sensitive target genes. hb, a sensitive target of Knirps (A-C), showed similar repression by both Knirps 1-330 and Knirps 1-429 of the parasegment four zygotic expression pattern (arrow) after 30 minutes of heat shock. On other genes, Knirps 1-330 was much less potent than Knirps 1-429. Knirps 1-330 repressed only stripe 3 of runt (E), while Knirps 1-429 repressed all except runt stripe 5 (F) after 15 minutes of heat shock (similar patterns were observed at 30 minutes). Similarly, 1-330 had a modest effect only on hairy stripes 3/4 (H) and ftz stripe 3 (L), while Knirps 1-429 completely repressed hairy 3,4 7 (I) and extensively disrupted ftz expression (K) after 5 and 10 minutes of heat shock respectively (similar patterns were noted at 15 and 30 minutes). Transcripts of endogenous Knirps target genes were visualized by in situ hybridization. All embryos are oriented with anterior towards the left, dorsal upwards.

View larger version (114K): [in a new window]   Fig. 3. Pattern of endogenous eve expression in embryos expressing full-length Knirps 1-429 (A-D) and CtBP-independent region of Knirps 1-330 (E-G). Phenotypes of increasing severity are illustrated. Class I pattern (A,E), repression of stripe 3; Class II (B,F), repression of stripe 3 and 7; Class III (C,G) repression of stripe 3,4,6 and 7; Class IV, all stripes repressed except stripe 5. Endogenous eve patterns were visualized by in situ hybridization; embryos are oriented with anterior towards the left, dorsal side upwards. More severe phenotypes were produced by expression of full-length Knirps 1-429 than Knirps 1-330, as documented in Table 1.

View larger version (58K): [in a new window]   Fig. 1. CtBP is required for Knirps repression of even-skipped (eve) stripe 4/6 enhancer, but not stripe 3/7 enhancer. Expression patterns of eve stripe 3/7 (A,B) and eve stripe 4/6 lacZ reporter genes (C,D) in wild-type (A,C) and CtBP mutant (B,D) embryos, showing derepression only of the eve stripe 4/6 element in the CtBP mutant. (E) Schematic representation of eve regulatory regions, showing cofactor requirements for Knirps repression. Expression patterns were characterized in transgenic embryos by in situ hybridization. Embryos are oriented anterior towards the left, dorsal side upwards.

View larger version (76K): [in a new window]   Fig. 4. Differential repression of minimal eve stripe enhancers by Knirps1-429 and 1-330, demonstrates differential sensitivities to Knirps activity. Repression of eve stripe 2/3-lacZ reporter gene demonstrates differential sensitivities of eve stripe 2 versus stripe 3 enhancers to Knirps expression. Patterns of lacZ expression in embryos prior to heat shock (A,D) and after heat shock (B,C,E,F). After a 10-minute heat shock, the majority of embryos expressing Knirps 1-429 showed repression of eve stripe 3 (B). After a 30-minute heat shock, the majority of embryos showed repression of both stripe 2 and 3 (C). A significant percentage of embryos showed only partial repression of stripe 3 upon overexpression of Knirps 1-330 (E). The majority of embryos demonstrated a loss of stripe 3, but not stripe 2 after 30 minutes of heat shock (F). (G,J) Unperturbed reporter gene expression. Effects of overexpression of Knirps1-429 and 1-330 in embryos carrying the eve stripe 3/7 lacZ reporter (G-I) or eve stripe 4/6 lacZ reporter (J-L). Full-length Knirps was a more potent repressor, but Knirps 1-330 was capable of repressing the minimal eve stripe 4/6 element (see text for details). The patterns shown in H and K are representative of embryos heat shocked for 15 minutes, whereas the patterns shown in I and L are typical of embryos heat shocked for 30 minutes. Embryos are oriented with anterior towards the left, dorsal side upwards.

View larger version (31K): [in a new window]   Fig. 2. Expression of full-length and CtBP-independent regions of the Knirps transcriptional repressor in transgenic Drosophila. (A) Structure of proteins expressed from hsp70 promoter: 1-429, full-length Knirps protein; 75-429, non-DNA binding control protein; 1-330, CtBP-independent Knirps repression domain; 75-330, non-DNA binding control protein. (B) In situ analysis of expression of knirps mRNA produced from hsp70-knirps transgene before and after heatshock. (C) (Top) Proteins expressed from representative lines of the four constructs measured by western blot. M2 -FLAG antibody was used to detect recombinant proteins. Lines shown in lanes 2 and 4 were used in subsequent experiments. Lane 8, non heat shock control. (Bottom) Coomassie blue stained gel illustrates equal loading.

View larger version (48K): [in a new window]   Fig. 5. Quantitation of proteins expressed from hsp70-knirps transgenes demonstrates that full-length Knirps 1-429 is less abundant than Knirps 1-330. (A) Western blot analysis of embryos subjected to the same heat-shock regimen (0, 5, 10 and 20 minutes) used for analysis shown in Fig. 3 and Table 1. Asterisk marks nonspecific cross-reacting protein that was also present in lysates from non-transgenic Drosophila (presence of nonspecific band appeared to vary with batch of antibody; data not shown). Because this nonspecific band co-migrates with the 1-330 protein, the signal from the nonspecific protein (averaged from lanes 5-8) was subtracted from each of the values in lanes 1-4 to determine levels of 1-330 protein. Below, Coomassie stained gel showing equal loading. (B) Quantitation of western blots demonstrates an approximately twofold higher level of Knirps 1-330 protein at each time point than Knirps 1-429, demonstrating that the higher activity of the 1-429 is not due to higher levels of this protein. Standard deviations are shown in B for four separate gels and quantitations of the heat-shock experiment shown in A.

View larger version (20K): [in a new window]   Fig. 7. Quantitative model for contribution of CtBP activity to repression by Knirps. Protein levels of Knirps (horizontal axis) are plotted against differential levels of repressor activity (vertical axis at top). With CtBP, Knirps repression levels increase more sharply with increasing protein levels, allowing the activity to cross critical thresholds at lower protein levels. The position of the Knirps protein levels in the embryo (lower part of figure indicated by % egg length) then dictates where appropriate stripe boundaries will form (vertical broken lines). This model predicts that, owing to the inherently high threshold of the eve stripe 4/6 enhancer, loss of CtBP activity will move the intercept off of the range of physiological Knirps concentrations, while having little effect on the stripe 3/7 position.