Hairless-mediated repression of notch target genes requires the combined activity of Groucho and CtBP corepressors

Abstract

Notch signal transduction centers on a conserved DNA-binding protein called Suppressor of Hairless [Su(H)] in Drosophila species. In the absence of Notch activation, target genes are repressed by Su(H) acting in conjunction with a partner, Hairless, which contains binding motifs for two global corepressors, CtBP and Groucho (Gro). Usually these corepressors are thought to act via different mechanisms; complexed with other transcriptional regulators, they function independently and/or redundantly. Here we have investigated the requirement for Gro and CtBP in Hairless-mediated repression. Unexpectedly, we find that mutations inactivating one or the other binding motif can have detrimental effects on Hairless similar to those of mutations that inactivate both motifs. These results argue that recruitment of one or the other corepressor is not sufficient to confer repression in the context of the Hairless-Su(H) complex; Gro and CtBP need to function in combination. In addition, we demonstrate that Hairless has a second mode of repression that antagonizes Notch intracellular domain and is independent of Gro or CtBP binding.

FIG. 1.

Relationship between Hairless and the corepressors CtBP and Gro. (A) Hairless binds to Gro and CtBP in vivo. Proteins immunoprecipitated (IP) from embryonic extracts by anti-Hairless antibodies were probed to detect Gro or CtBP as indicated. The input lane (in) contained 30% of the extract used for IP. C, control precipitate with unrelated antibody. Typically, Hairless is detected as two protein bands, 120 kDa and 150 kDa. (B) Hairless can recruit CtBP and Gro at the same time. Anti-Gro antiserum was used to immunoprecipitate in vitro-translated proteins from a mixture containing Hairless, Gro, and CtBP (input 3) or just Gro and CtBP (input 2). The presence of Gro, Hairless, and CtBP was detected in the immunoprecipitates (arrowheads) in comparison to input (in) (16%). CtBP was only detected when the mix contained H also but was not detected in the double mixture.Control, precipitate with unrelated antiserum, probed with anti-CtBP; M, protein ladder (sizes in kilodaltons). (C and D) Negative regulation of the vg^BE-LacZ by Hairless, Su(H), and Gro. (C) Schematic drawing of a wing disk; the dorsoventral boundary (red), omb expression domain (green), and prospective wing blade (light blue) are indicated. The inset depicts the sector shown in panel D. (D) Effects on vg^BE-LacZ (red) of expressing double-stranded (ds) RNAi constructs, as indicated, in the omb domain (white dotted lines indicate the right boundary of the domain). Note that double-stranded RNAi against Su(H), Hairless, and Gro but not CtBP causes derepression of vg^BE-LacZ (yellow brackets). UAS-redStinger was overexpressed in the control.

FIG. 2.

Both CtBP and Gro are necessary for Hairless-mediated repression in S2 cells. (A) Mapping Gro and CtBP interaction domains. Interactions of Hairless with CtBP or Gro were investigated by yeast two-hybrid assay: positive interactions appear blue on X-Gal indicator plates, negatives and control (pEG, empty vector) remain white. A central domain of about 150 amino acids was necessary (HΔG) and sufficient (GBD) for binding to Gro. This domain contains two eh1-related binding motifs (site 1, EFEKCSLED; site 2, SYSIHSLLG). Mutations within site 1 (F→A; GBD-1) retained Gro binding, whereas mutations within site 2 (Y→A, GBD-2) completely destroyed it. The CtBP interaction domain CBD was confined to a 15-amino-acid interval (HΔC). Conservative substitution within this motif (PLNLSKH to VIQITKR) resulted in a complete loss of CtBP binding (H*C). The Su(H) binding SBD domain is depicted in red. (B) Constructs for assay of residual Hairless activity in cell culture; remaining codons are numbered. (C to F) Effects of Hairless proteins on transcription of Notch reporters (NRE) in the presence of N^ICD (1 μg) (C to E) or Grh (1 μg) (F). S2 cells were transfected with the constructs indicated; control was empty vector (Con). The level of expression from the NRE (blue) (C to E) and NME [mutated Su(H) sites] (red) (C and D) was measured in comparison to cotransfected renilla plasmid; y-axis numbers indicate this relative luciferase activity from the NRE or NME reporters. Values for NRE in the presence of N^ICD or Grh clone were normalized to 100% (D to F). In panel C, differing amounts of each construct are indicated in micrograms. In panel E, the concentrations used were 1.5 μg, 0.5 μg, 0.125 μg, and 0.003 μg. (G) Hairless mutant proteins are expressed at similar levels. Cells transfected with 0.5 μg of the Hairless plasmids indicated (along with the other plasmids described for panel E) were analyzed using Western blots probed with anti-Hairless to detect the expression of the Hairless variants, and anti-tubulin, to detect overall protein levels.

FIG. 3.

Both Gro and CtBP binding motifs are required for Hairless-mediated repression in the wing. (A) In the wild-type (WT) wing, the five longitudinal veins (L1 to L5) are easily discernible. (B) Hairless overexpression (HFL) results in a small wing with thickened veins and clumps of bristles at the tip (see arrow and arrowhead in enlargement at right). (C to F) Effects of deletion constructs (HΔC, HΔG, H*G, HΔGC) are similar and less severe than that of Hairless; wings have some vein thickening (arrow) and disrupted margin (arrowhead). (G) Co-overexpression of HΔGC with Su(H) results in a dramatically enhanced phenotype with ectopic bristles and thickened wing veins and margin defects (arrow and arrowhead, respectively; see enlargement). (H) Su(H) overexpression. Wings lack veins (open arrow) and have balloon-like overgrowth; wings often cannot be inflated well after eclosion, as seen here. (I) Overexpression of Su(H)^WRPW causes more-severe phenotypes than Hairless overexpression, with deep incisions (arrow). In all cases proteins were overexpressed using the omb-Gal4 driver. Wings are shown at same magnification; enlargements are shown in the right panels.

FIG. 4.

Requirement for Gro and CtBP for the repression of the Notch target gene vg. (A) Control disk expressing UAS-redStinger (green). The boxed area depicts a region shown at higher magnification in panels B and C to H. (C to H) Use of full-length Hairless (C) or Su(H)^WRPW (H) results in complete repression of vg^BE-LacZ (red), whereas HΔG (D), HΔC (E), and HΔGC (F) cause only partial repression (yellow arrows). The indicated proteins were overexpressed using the omb-Gal4 driver and detected by anti-Hairless or anti-Su(H) staining (green). The white dotted line indicates the right boundary of expression. (G) Overexpression of Su(H) induces vg^BE-LacZ expression (yellow arrow) and causes increased growth of the wing disk.

FIG. 5.

Two modes of Hairless repression. (A) Active repression requires the combined activity of Gro and CtBP and is independent of Notch. In this example, the activator of transcription is Grh. (B) Competition with N^ICD: specific antagonism of N^ICD can occur independently of corepressors (as shown here) and is dose dependent on Hairless.