Defining the Hairless binding domain in Su(H). (A) Left, ribbon diagram of mouse CSL-DNA (Protein Data Bank [PDB] ID: 3IAG) structure, showing overall fold (Friedmann and Kovall, 2010). All CSL proteins, including Drosophila Su(H), have three distinct domains: the N-terminal domain (NTD; cyan), the β-trefoil domain (BTD; green), and the C-terminal domain (CTD; orange). A β-strand that makes hydrogen bonds with all three domains is colored magenta. Both BTD and NTD contact the DNA (gray). Right, ribbon diagram of worm CSL-ICN-Mam-DNA (PDB ID: 2FO1) activation complex (Wilson and Kovall, 2006). CSL is colored green. Notch ICN contacts the BTD with its RAM domain (yellow) and the CTD with its ANK domain (blue). Mam is colored red and binds a groove that is formed by the CTD–ANK interface and the NTD of CSL. (B) Yeast two-hybrid assays demonstrate the interactions between Su(H) and full-length Hairless (HFL) or Notch ICN I, respectively. A set of Su(H) deletion constructs and replacement mutants as indicated were tested to define the domains of Su(H) that interact with Hairless. (C) The CTD domain of CSL proteins derived from Drosophila [D.m., Su(H)], mouse (M.m., CBF1), and C. elegans (C.e., Lag-1) were assayed for interaction with HFL or Notch ICN I. Empty vectors were included as negative controls.

Mapping Su(H)-binding sites in Hairless. (A) Scheme of the HFL protein containing the Su(H)-binding domain (SBD; pink), the Groucho binding domain (GBD; blue) and a binding site for the C-terminal binding protein (CBD; yellow). Bottom, the primary sequence of the SBD is shown; the NT and CT boxes, defined by their conservation in insects, are framed in red. Amino acids that are identical in all known/predicted Hairless proteins are shown in blue and red, with the red residues (L235 and F243) indicating the mutation sites tested for Su(H) binding. (B) Yeast two-hybrid assay on the binding activity of wild-type and mutant NTCT constructs (amino acids 171–357) with full-length Su(H); empty vector served as a negative control. Note that the lack of binding for the ΔNT deletion construct is comparable to the single-site mutant L235D, whereas the F243A mutant binds Su(H) similar to wild-type NTCT.

Characterization of ICN and Hairless competition for binding Su(H). For all EMSAs, unless otherwise noted, the concentrations of DNA, Su(H), Hairless (NT), ICN (RAMANK), and Mam are 1, 0.1, 4, 4, and 5 μM, respectively. (A) ICN efficiently displaces Hairless from Su(H); control lanes 1–4: Su(H), Su(H)–ICN, Su(H)–ICN–MAM, and Su(H)–Hairless complexes, respectively; lanes 5–9: preformed Su(H)–Hairless complexes titrated with increasing concentrations of ICN (0.5, 1, 2, 4, and 6 μM). (B) Hairless is less efficient in displacing ICN from Su(H); control lanes 1–5: Su(H), Su(H)–Hairless, Su(H)–ICN–MAM, Su(H)–ICN, and Su(H)–ICN (1 μM ICN) complexes, respectively; lanes 6–10: preformed Su(H)–ICN (1 μM ICN) complexes titrated with increasing concentrations of Hairless (0.5, 1, 2, 5, and 20 μM). (C) Hairless NT competes with ICN for binding to endogenous CSL in mammalian cells. Cultured MEFs were transiently transfected with an activated form of murine Notch1 (ICN1), the 4xCBS luciferase reporter, and increasing concentrations of GFP (light gray bars) or GFP–Hairless (NTCT; dark gray bars) constructs (lanes 2–5). Potent activation of the reporter is observed from the 4xCBS reporter with ICN (lane 1). Increasing concentrations of GFP–Hairless, but not the GFP control, result in strong inhibition from the reporter (lanes 2–5). Lanes 2–5 represent 25, 50, 100, and 250 ng of transfected GFP or GFP–Hairless DNA, respectively. The y-axis represents relative activity derived from normalizing the data to experiments performed in the absence of GFP or GFP–Hairless (lane 1). Data are derived from three independent experiments (n = 3), and the error bars represent the SE of the mean.

In vivo activity of Hairless mutant constructs. Wild-type and mutant constructs as indicated were expressed in the thorax of the developing fly using the MS1096-Gal4 driver line. Effects of Su(H) and Hairless on bristle development were studied singly and in combination to assay for the formation of a repressor complex. Open arrows (missing bristles due to transformation of external to internal fate) and closed arrows (ectopic bristles or double shafts) point to Notch loss-of-function phenotypes, and arrowheads (loss of bristles, double sockets) point to Notch gain-of-function phenotypes. Compared to the control (A), overexpression of wild-type Su(H) causes typical Notch gain-of-function phenotypes affecting the majority of macrochaetae and microchaetae (B). (C) Overexpression of full-length Hairless HFL results in Notch loss-of-function phenotypes affecting most macrochaetae and many microchaetae. (D) Combined overexpression of HFL and Su(H) enhances this effect strongly, and bushes of bristles appear as a result of reduced lateral inhibition. Flies resulting from overexpression of either Hairless mutant H^LD (E) or H^ΔNT (G) have nearly wild-type phenotype. Moreover, in combination with Su(H) (F, H) the phenotypes are similar to the sole Su(H) overexpression (see B).

Fine mapping of the Hairless contact site on Su(H) CTD. (A) Structure of the human CSL-ICN-Mam activator complex (PDB ID: 2F8X) (Nam et al., 2006). CSL is represented as a gray surface, and ICN and Mam are colored blue and red, respectively. Mutations in the CTD that lie at the interface with ICN and Mam are colored magenta. (B) Mutant CTD constructs were tested in a yeast two-hybrid assay for binding to full-length Hairless (HFL) and to intracellular Notch (ICN I). Moreover, the mutant CTD constructs were tested in a yeast three-hybrid assay for their potential to assemble the trimeric activator complex with Notch ANK and Mam. Empty vectors served as negative controls. Position of mutations within CTD is indicated. Note that mutations Ank2 and mN are competent for binding HFL but are considerably reduced for ICN binding and also fail to assemble the trimeric activator complex. Hence, Notch and Hairless contact different sites on Su(H). (C) Primary sequence of the Su(H)–CTD construct; the CTD is shown in bold. Amino acids interacting with ANK/Mam that were mutated are colored blue. (D) Schematic for yeast three-hybrid assay; the N-terminal domain of Mam (mamN) was fused to the LexA DNA-binding domain, and the CTD of Su(H) was fused with the trans-activation domain (TAD). Notch ANK was provided from the plasmid pESC. Su(H) CTD constructs carried mutations as indicated in B. Assembly of the trimeric complex results in activation of the lacZ reporter.

Biophysical analysis of Hairless interactions with Su(H). (A) Far-UV circular dichroism spectroscopy for Hairless (NT), Notch ICN (ANK), and Su(H) NH-CTD. The normalized root-mean-square deviation (NMRSD) parameter values for analysis of the Hairless NT, ANK, and NH-CTD CD data are 0.03, 0.02, and 0.08, respectively. The CD spectrum of Hairless NT has a minimum at ∼200 nm, which is characteristic of random coil. Consistent with its largely helical structure and previously published results (Zweifel et al., 2003), the CD spectrum of ANK shows characteristic minima for α-helix at 207 and 222 nm. The CD spectrum of NH-CTD displays a minimum at 215 nm, consistent with this domain of Su(H) being largely composed of β-sheet. (B) Representative thermograms, raw heat signal, and nonlinear least squares fit to the integrated data for full-length Su(H) and NH-CTD constructs interacting with Hairless (NTCT). Forty titrations were performed per experiment, consisting of 7-μl injections that were spaced 120 s apart. Thermodynamic parameters are shown for each type of experiment; the average N (ligand/macromolecule) values for both experiments were 0.9. Values are the mean of at least three independent experiments and errors represent the SDs of multiple experiments.

Mutant Hairless fails to assemble functional repressor complexes. (A) Effects of wild-type or mutant Hairless protein, in the absence or presence of Su(H), on Notch ICN-mediated expression from the Notch reporter (NRE) were studied in Drosophila S2 cell culture. S2 cells were transiently transfected with constructs indicated; empty vector was used as a negative control. Luciferase activity is represented on the y-axis, and transfection efficiency was normalized by cotransfection of the Renilla plasmid. Values for NRE expression in the presence of Notch ICN were normalized to 100% (lane 1). (B) Effects of the overexpression of wild-type and mutant Hairless constructs, singly or together with Su(H), on in vivo expression of the Notch target gene vestigial. Wild-type and mutant UAS constructs as indicated were induced in the central region of wing imaginal disks using the omb-Gal4 driver line and visualized by antibody staining. UAS-GFP served as control (red). Expression of vg^BE-lacZ reporter is expected along the dorsoventral boundary and shown in green (bottom). In the merged picture (top), overlap appears yellow. Overexpressed Su(H) is shown in red, and Hairless protein is shown in blue; overlap appears magenta, and with vg^BE-lacZ (green) it appears white. Overexpression of Su(H) causes an overgrowth of the disk and an activation of vg^BE-lacZ (asterisk). Conversely, overexpression of HFL results in a repression of vg^BE-lacZ (blunt arrow). However, overexpression of the Hairless mutant constructs H^ΔNT or H^LD has little effect on vg^BE-lacZ expression, reflecting the loss of Su(H)-binding activity (arrow). A combination of HFL with Su(H) strongly affects growth of the wing disk and vg^BE-lacZ expression. In contrast, a combination of Su(H) with either H^ΔNT or H^LD results in overgrowth and vg^BE-lacZ induction that resembles the sole Su(H) overexpression, indicating that these Hairless mutants fail to form repressor complexes in vivo.