Functional similarity of Knirps CtBP-dependent and CtBP-independent transcriptional repressor activities

Abstract

Short-range transcriptional repressors are locally acting factors that play important roles in developmental gene expression in Drosophila. To effect repression, Knirps and other short-range repressors bind the CtBP corepressor, but these repressors also function via CtBP-independent pathways. Possible mechanistic differences between CtBP-dependent and -independent repression activities are poorly understood. The distinct activities might provide qualitatively different activities necessary in different promoter contexts, or they might combine to give quantitatively different effects. We analyze separately the CtBP-dependent and CtBP-independent domains of Knirps previously characterized in the embryo to determine possible functional distinctions of the two repression activities. Both domains are active in cell culture and are dependent on the same residues required for activity in the embryo. The domains have similar properties with respect to distance-dependent repression and resistance to inhibition by the deacetylase inhibitor trichostatin A. In tests of repressor-activator specificity, the extent of repression was related not to the chemical nature of the activation domain but to the total activation potential. This result indicates that the balance of competing activation and repression signals is decisive in determining the effectiveness of repressors on genetic switches, suggesting that multiple repression activities are utilized to provide quantitatively, rather than qualitatively, distinct outputs.

Figure 1

Reporters and short-range repressors tested. (A) Assays utilized luciferase reporter genes activated by the actin 5C enhancer or a cluster of Gal4 binding sites. Two Tet operator binding sites are located at –50 bp with respect to the transcriptional initiation site. (B) A schematic diagram of the Knirps repressor, with CtBP binding motif (arrow, PMDLSMK) between residues 331 and 337 as indicated. N-terminal CtBP-independent repression domain (75–330) and C-terminal CtBP-dependent repression domains were fused to the Tet-repressor DNA binding domain (black box). Inactive mutant proteins Nmut (189–330) and Cmut (202–329, 344–358) were also assayed as controls. The entire Giant repression domain (Gt) and the CtBP corepressor protein were also fused to the Tet DNA binding domain. Pluses and minuses (right) indicate the previously determined activity of the repression domains in embryo assays (9,25).

Figure 2

Measurement of Tet-repressor binding activity. Nuclear extracts of S2 Schneider cells transfected with genes for Tet-repressor constructs were assayed by gel electrophoretic mobility shift. Specificity of binding was tested by the addition of 10- or 100-fold molar excess specific or non-specific unlabeled oligonucleotide. Positions of complexes formed by Stop (lanes 1–5, 31–35), Knirps N-terminal CtBP-independent repression domain (N, lanes 11–15), an inactive mutant (Nmut, lanes 16–20), Knirps C-terminal CtBP-dependent repression domain (C, lanes 21–25), an inactive mutant (Cmut, lanes 26–30) and CtBP (dCtBP, lanes 36–40) are marked with arrowheads. Levels of Giant protein were undetectable, even at longer exposures. The inactive Knirps mutant proteins (Nmut and Cmut) were expressed at equal or slightly higher levels than the corresponding active proteins, demonstrating that the inactivity of the mutant proteins is not simply due to poor expression or binding. The Giant and CtBP proteins, which were more active than the Knirps repressors in some assays, were expressed at lower levels than the Knirps N and Knirps C proteins, indicating that their potency is not simply due to higher expression levels.

Figure 3

Both CtBP-dependent and CtBP-independent Knirps repression domains function as repressors in transient assays. An actin 5C activated luciferase reporter construct was cotransfected into Schneider cells with Tet-repressor constructs. Ten-fold or greater repression was noted in the presence of either Knirps N or C repression domains, similar to that exhibited by the CtBP and Giant proteins. Considerably weaker (2-fold) effects were noted with mutant repression domains previously found to be inactive in the embryo (9). Activity of the reporter gene in the presence of doxycycline (Dox) (conditions under which the repressor proteins do not bind DNA) was used to normalize the reporter readout. (The absolute differences between luciferase activities in the presence of doxycycline were <50%.) Transfection efficiency was corrected for by normalizing to the signal from a cotransfected lacZ reporter and total protein (correction for transfection efficiency typically changed the uncorrected values <5%). Experiments in this and subsequent figures were performed in triplicate, with duplicate readings of each extract; standard deviations are shown.

Figure 4

Knirps, Giant and CtBP repressors show strong distance dependence in transfection assays. Actin 5C enhancer reporters containing Tet operator sites at –75 (open bars), –105 (shaded bars) or –160 (black bars) were cotransfected with Tet-repressor constructs. The Tet DNA binding domain and inactive Knirps mutant had little or no activity on all templates, while the Knirps N and C repression domains, as well as Giant and CtBP, showed a sharp distance dependence, with fold repression close to background levels at –160 bp, similar to the distance effects noted with endogenous repressor proteins in transgenic embryos (9,28). Fold repression was calculated by dividing the signal obtained in the presence of doxycycline (no repression) by the signal obtained in the absence of doxycycline (repression).

Figure 5

Repression by Knirps, Giant and CtBP resistant to HDAC inhibitor TSA. (A) Addition of 0 (open bars), 150 (light-gray bars) or 400 nM TSA (dark-gray bars) had no effect on repression of reporter activity by active Tet repressors. Weak repression activity by Knirps Cmut appeared to slightly increase at the highest levels of added TSA. Activities were normalized to those obtained in the presence of doxycycline (no repression). (B) TSA effect on Groucho repression as a control for TSA treatment effectiveness. Cells grown and transfected under identical conditions as in (A) were cotransfected with a reporter containing binding sites for a Gal4-Groucho corepressor and the VP16 activator, in the presence of 0, 150 or 400 nM TSA (left). In the absence of TSA, reporter activity is repressed 8-fold by Gal4-Groucho (right). Repression effectiveness was decreased to 3-fold by the addition of TSA. A non-specific stimulation of this reporter by TSA was noted previously (31). Results shown are representative of two experiments.

Figure 5

Repression by Knirps, Giant and CtBP resistant to HDAC inhibitor TSA. (A) Addition of 0 (open bars), 150 (light-gray bars) or 400 nM TSA (dark-gray bars) had no effect on repression of reporter activity by active Tet repressors. Weak repression activity by Knirps Cmut appeared to slightly increase at the highest levels of added TSA. Activities were normalized to those obtained in the presence of doxycycline (no repression). (B) TSA effect on Groucho repression as a control for TSA treatment effectiveness. Cells grown and transfected under identical conditions as in (A) were cotransfected with a reporter containing binding sites for a Gal4-Groucho corepressor and the VP16 activator, in the presence of 0, 150 or 400 nM TSA (left). In the absence of TSA, reporter activity is repressed 8-fold by Gal4-Groucho (right). Repression effectiveness was decreased to 3-fold by the addition of TSA. A non-specific stimulation of this reporter by TSA was noted previously (31). Results shown are representative of two experiments.

Figure 6

Inverse correlation between repression and activator strength. A reporter activated by a cluster of five Gal4 binding sites was cotransfected with plasmids expressing Gal4-activator and Tet-repressor proteins. (A) Gal4-Sp1-activated transcription was strongly inhibited (≥5-fold repression) by both Knirps N and C domains, while mutants were inactive. CtBP and Giant exhibited repression activity similar to the Knirps repression domains. (B and C) Repression of Gal4 VP16 and Gal4 AD activators. Knirps C did not exhibit activity greater than the mutant form of the protein, while the Knirps N showed only weak activity. CtBP and Giant mediated 5–20-fold repression. Signals were normalized to those obtained in the presence of doxycyline (no repression).

Figure 6

Inverse correlation between repression and activator strength. A reporter activated by a cluster of five Gal4 binding sites was cotransfected with plasmids expressing Gal4-activator and Tet-repressor proteins. (A) Gal4-Sp1-activated transcription was strongly inhibited (≥5-fold repression) by both Knirps N and C domains, while mutants were inactive. CtBP and Giant exhibited repression activity similar to the Knirps repression domains. (B and C) Repression of Gal4 VP16 and Gal4 AD activators. Knirps C did not exhibit activity greater than the mutant form of the protein, while the Knirps N showed only weak activity. CtBP and Giant mediated 5–20-fold repression. Signals were normalized to those obtained in the presence of doxycyline (no repression).

Figure 6

Inverse correlation between repression and activator strength. A reporter activated by a cluster of five Gal4 binding sites was cotransfected with plasmids expressing Gal4-activator and Tet-repressor proteins. (A) Gal4-Sp1-activated transcription was strongly inhibited (≥5-fold repression) by both Knirps N and C domains, while mutants were inactive. CtBP and Giant exhibited repression activity similar to the Knirps repression domains. (B and C) Repression of Gal4 VP16 and Gal4 AD activators. Knirps C did not exhibit activity greater than the mutant form of the protein, while the Knirps N showed only weak activity. CtBP and Giant mediated 5–20-fold repression. Signals were normalized to those obtained in the presence of doxycyline (no repression).

Figure 7

Activator strength, rather than the nature of the activation domain, dictates repression effectiveness. (A) The weak Gal4-Bicoid activator containing one copy of the glutamine-rich domain was effectively repressed by both Knirps N and C domains (7–15-fold), significantly above that seen with mutant repression domains (∼2-fold). Giant and CtBP both repressed expression, although CtBP was reproducibly weaker in this context than in others tested. (B) A strong Gal4-Bicoid activator containing three copies of the same glutamine-rich domain was not effectively repressed by either Knirps N or C domains, relative to the mutant proteins. CtBP was relatively more effective against this activator, mediating ∼10-fold repression, while Giant mediated ∼4-fold repression.

Figure 7

Activator strength, rather than the nature of the activation domain, dictates repression effectiveness. (A) The weak Gal4-Bicoid activator containing one copy of the glutamine-rich domain was effectively repressed by both Knirps N and C domains (7–15-fold), significantly above that seen with mutant repression domains (∼2-fold). Giant and CtBP both repressed expression, although CtBP was reproducibly weaker in this context than in others tested. (B) A strong Gal4-Bicoid activator containing three copies of the same glutamine-rich domain was not effectively repressed by either Knirps N or C domains, relative to the mutant proteins. CtBP was relatively more effective against this activator, mediating ∼10-fold repression, while Giant mediated ∼4-fold repression.