dCtBP-Dependent and -Independent Repression Activities of the Drosophila Knirps Protein

Abstract

Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila melanogaster embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, we have undertaken a detailed structure-function analysis of the Knirps protein. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions were identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities.Transcriptional repression is a critical component of genetic regulation during development, and the Drosophila melanogaster embryo has served as an important model for elucidation of basic repression mechanisms (7,19). Differential gene expression in the early embryo is controlled in large part by the activity of repressor proteins encoded by gap, pair-rule, and other genes (42, 45). Repression of transcription can involve reactions occuring off the DNA, such as the formation of inactive heteromeric complexes. Another mechanism involves competition between activators and repressors for binding sites on DNA. DNA-binding repressors that function by mechanisms other than competitive binding have been termed active repressors (24).An active repressor can repress basal promoters or enhancer elements over a short range (Ͻ100 bp) or, alternatively, over long ranges (Ͼ1,000 bp) (7,17). One model of repression in the embryo suggests that the short-range-long-range distinction results from the recruitment of distinct classes of cofactors (36, 55). Short-range repressors may interact with dCtBP, while long-range repressors interact with Groucho.Long-range repressors are typified by the Hairy protein, a transcription factor that binds the Groucho cofactor (5, 27, 40). Long-range repression complexes regulating the dpp, tld, and zen genes also recruit Groucho (7, 27), as do Engrailed, Runt, and dTCF, Drosophila repressors whose range of action has not yet been determined (3, 9, 50).Short-range repressors present in the early Drosophila em-

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

“…In the case of Knirps, a form of the protein that lacks the CtBP-binding motif exhibits weak activity when overexpressed (Nibu et al, 1998b). The CtBP independent activity has been mapped to an N-terminal repression domain that lacks a CtBP-binding motif and is able to repress in the absence of CtBP (Keller et al, 2000). Although many transcriptional repressors have been found to possess multiple activities, the functional relevance of such activities is not well understood.…”

“…The even-skipped stripe 2/3 lacZ reporter used in Fig. 4 contains ~500 bp minimal elements separated by a 340 bp spacer sequence and 2 UAS sites (not used in this experiment) fused to the eve basal promoter (Keller et al, 2000). The eve stripe 3/7 reporter used in Fig.…”

“…1 were described elsewhere (Small et al, 1996;Fujioka et al, 1999). Germline mutants of CtBP were generated as previously described (Keller et al, 2000) using CtBP 03463 /TM3, Sb (Bloomington stock no. P1590).…”

“…CtBP-mediated repression is crucial for full activity of shortrange repressors; however, Drosophila short-range repressors also possess CtBP-independent repression activities (La Rosee-Borggreve et al, 1999;Keller et al, 2000;Strunk et al, 2001;Nibu et al, 2003). In the case of Knirps, a form of the protein that lacks the CtBP-binding motif exhibits weak activity when overexpressed (Nibu et al, 1998b).…”

“…The Knirps protein is able to regulate at least one known target, the stripe 3 enhancer of the even-skipped (eve) gene in a CtBP-independent fashion, yet this CtBP-independent activity is not sufficient to supply the full biological function of Knirps (Keller et al, 2000;Nibu et al, 1998b). For example, transheterozygous knirps and CtBP embryos have disruptions in eve expression, suggesting that Knirps function is partially impaired, and a frameshift mutation in knirps encoding a protein lacking the CtBP binding motif is a strong hypomorph (Gerwin et al, 1994;Nibu et al, 1998a).…”