Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed

Abstract

Relatively little is known about the molecular mechanisms involved in transcriptional repression, despite its importance in development and differentiation. Recent evidence suggests that some transcriptional repressors act by way of adaptor molecules known as corepressors. Here, we use in vivo functional assays to test whether different repressor activities are mediated by the Groucho (Gro) corepressor in the Drosophila embryo. Previously, Gro was proposed to mediate repression by the Hairy-related family of basic helix-loop-helix proteins. Our results indicate not only that repression by Hairy requires Gro, but that a repressor domain from the Engrailed (En) homeodomain protein is also Gro dependent. The latter result correlates with an ability of this En domain to bind to Gro in vitro. In contrast, repressor regions from the Even-skipped, Snail, Krüppel, and Knirps transcription factors are effective in the absence of Gro. These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En.

Figure 1

Hairy requires Gro for repression of Sxl. Effect of the hb–h construct in otherwise wild-type (A) or gro^E48 (B) female blastoderm embryos. Efficient repression at the anterior is observed in the presence but not in the absence of Gro. Embryos were stained with an antibody against active Sxl protein. (C) Pattern of lacZ mRNA expression directed by the hb promoter in gro embryos; efficient activity of the promoter is observed in the anterior region of the embryo. In this and subsequent figures anterior is to the left and dorsal is up.

Figure 2

Effects of Hairy repressor chimeras in otherwise wild-type or gro embryos. (A) Diagram of Hairy derivatives expressed under the control of the hb promoter and their effects on female viability and Sxl expression. The degree of female lethality is represented by crosses: (+++) most lines show >80% lethality; (+) most lines show <30% lethality; (−) no lethality detected. (B) Effects on Sxl expression of hb–h^eve, hb–h^en, hb–h^sna, hb–h^Kr, and hb–h^kni in otherwise wild-type or gro^E48 embryos. Repression of Sxl is observed in all cases, except for hb–h^en in gro embryos.

Figure 2

Effects of Hairy repressor chimeras in otherwise wild-type or gro embryos. (A) Diagram of Hairy derivatives expressed under the control of the hb promoter and their effects on female viability and Sxl expression. The degree of female lethality is represented by crosses: (+++) most lines show >80% lethality; (+) most lines show <30% lethality; (−) no lethality detected. (B) Effects on Sxl expression of hb–h^eve, hb–h^en, hb–h^sna, hb–h^Kr, and hb–h^kni in otherwise wild-type or gro^E48 embryos. Repression of Sxl is observed in all cases, except for hb–h^en in gro embryos.

Figure 3

Effects of Hairy, Hairy^En, and Hairy^Eve on ftz transcription in the presence and absence of Gro. The Hairy derivatives were expressed in blastoderm embryos under the control of a heat-shock promoter (see Materials and Methods). The normal patterns of ftz mRNA in control wild-type and gro embryos are also shown. Because of the effects of gro on gap gene expression, ftz is not expressed in stripes in gro embryos, but occupies a broad domain in the trunk region. All three heat-shock constructs repress ftz in wild-type embryos, but only hs–h^eve leads to significant repression in gro embryos. The gro embryos were derived from gro^BX22 (control and hs–h) or gro^E48 (hs–h^en and hs–h^eve) mosaic females.

Figure 4

Ectopic En represses eve expression in wild-type but not in gro embryos. Wild-type pattern of eve mRNA (A) and its repression in a heat-shocked hs–en embryo (C). Pattern of eve in a control gro^E48 mutant embryo (B) and in a heat-shocked gro^E48 embryo carrying the hs–en construct (D); no significant repression is observed. As in the case of ftz, eve transcripts are not expressed in stripes in gro embryos, but accumulate in one or two broad central domains.

Figure 5

Gro binds in vitro to Hairy^En. (A) Radiolabeled Gro protein was incubated with various GST–Hairy derivatives bound to glutathione–Sepharose beads. After washing the beads, the retained Gro protein was visualized by SDS-PAGE and autoradiography. (B) Coomassie staining showing the integrity of the different GST fusions after incubation with Gro.

Figure 6

Mapping of sequences within En^R responsible for binding to Gro. A diagram of different En^R deletion mutants is shown. These mutations were introduced in the original GST–Hairy^En construct and examined for their ability to bind full-length Gro. Deletions of region D (constructs En^168–227 and En^168–258) do not affect the interaction with Gro. In contrast, deletion of region C (construct En^228–298) causes a fourfold decrease in the binding. A similar result is obtained after eliminating 15 or 7 amino acids [En^Δeh1(15) or En^Δeh1(7), respectively] that constitute the conserved eh1 motif (Smith and Jaynes 1996).

Figure 6

Mapping of sequences within En^R responsible for binding to Gro. A diagram of different En^R deletion mutants is shown. These mutations were introduced in the original GST–Hairy^En construct and examined for their ability to bind full-length Gro. Deletions of region D (constructs En^168–227 and En^168–258) do not affect the interaction with Gro. In contrast, deletion of region C (construct En^228–298) causes a fourfold decrease in the binding. A similar result is obtained after eliminating 15 or 7 amino acids [En^Δeh1(15) or En^Δeh1(7), respectively] that constitute the conserved eh1 motif (Smith and Jaynes 1996).

Figure 7

Binding of Hairy and Hairy^En to Gro deletion mutants. A diagram of the different Gro derivatives analyzed is shown. These derivatives were assayed for binding to Hairy, Hairy^1–286 (a truncation similar to Hairy^En but lacking En^R), and Hairy^En. Equivalent aliquots of labeled Gro derivatives are shown as a control for the strength of the interactions (Input lanes). Gro^ΔWD and Gro^WD show weak or no binding to either Hairy or Hairy^En. Gro derivatives with progressive WD repeat deletions also show very weak binding to Hairy, but Gro^Δ6 and Gro^Δ5,6 interact significantly with Hairy^En.