The WRPW Motif of the Hairy-Related Basic Helix-Loop-Helix Repressor Proteins Acts as a 4-Amino-Acid Transcription Repression and Protein-Protein Interaction Domain

Abstract

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Gal4. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.Basic helix-loop-helix (bHLH) transcription factors control cell fate decisions, such as myogenesis or neurogenesis, in many animal species (9-11, 21, 33, 34, 38, 60, 80, 81). These proteins can be classified into two groups, the activator bHLH proteins and the repressor bHLH proteins, on the basis of biological function (56). Remarkably, the DNA binding specificities of the activator and repressor bHLH proteins directly correlate with their biological functions (56). The activator proteins, such as MyoD or the proteins of the Achaete-Scute complex, promote differentiation by binding to class A binding sites and activating transcription (21,50,52,53,56,77,81). The repressor bHLH proteins are Hairy-related proteins such as Hairy, the proteins of the Enhancer of Split [E(spl)] complex, and the homologous mammalian Hes proteins (2,19,23,32,41,43,56,61,62,70,71). These proteins antagonize the activator proteins and prevent differentiation by binding to specific class B or C sites and repressing transcription (2,32,54,56,70,73,75). Thus, Hairy-related proteins are distinct from the emc and Id HLH repressors, which lack basic regions and repress by forming non-DNA-binding heterodimers with the activator bHLH proteins (7,17,22,25,76,77). Drosophila neurogenesis is regulated by both activator and repressor bHLH genes (9-11, 33, 34, 38). The activators are proneural genes (33) and include daughterless (13), the four genes of the ach...

Hes1: a key role in stemness, metastasis and multidrug resistance

“…Hes1 binds to N box by forming complexes with co-repressor transducin-like enhancer of split (TLE), homologs of Drosophila Groucho via WRPW motif. 17,18 The transcriptional inhibition is mediated by this interaction, and occurs by recruitment of histone deacetylases (HADCs). 3,19 In addition, Hes1 may recruit HADCs using the bHLH domain to repress transcription.…”

Bowline, a novel protein localized to the presomitic mesoderm, interacts with Groucho/TLE in Xenopus

“…Together, these findings (1-4) indicate that Bowline downregulates X-Delta-2 expression in cooperation with XGrg-4, which is localized in the PSM at tailbud stage (Molenaar et al, 2000). In this downregulation, it is considered that Bowline regulates the activity of XGrg-4 by (i) modulating interaction of Groucho/TLE with a DNA binding transcription factor which interacts with Groucho/TLE via the WRPW or WRPY motif (Hairy/HES for example), or by (ii) mediating interaction of Groucho/TLE with a DNA binding protein which alone does not interact with Groucho/TLE, since no DNA binding motifs are identified in the Bowline sequence (Chen and Courey, 2000, Fisher et al, 1996, Grbavec and Stifani, 1996. Note that Groucho/TLE proteins are transcriptional corepressors that lack DNA binding motif, but interact with DNA-bound transcription factors (Chen and Courey, 2000).…”

Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates

“…These family members have been shown to be widely expressed both during development and in the adult, in contrast to the more limited expression pattern of their DNA-binding partners (Hartley et al 1988;Stifani et al 1992;Miyasaka et al 1993;Schmidt and Sladek 1993;Choudhury et al 1997;Pflugrad et al 1997;Sharief et al 1997). In addition, both Groucho and the human family member TLE1 have been shown to actively repress transcription when fused to a heterologous DNA-binding domain and directly bound to DNA (Fisher et al 1996). Hence, the Groucho proteins are recruited to target gene promoters by direct binding to specific DNA-binding repressors, and once recruited, the Groucho proteins repress transcription via a conserved intrinsic repression domain.…”

“…The Groucho proteins serve as non-DNA binding corepressors for specific subsets of DNA-binding transcription factors, including the Hairy-related proteins, Runt domain proteins, Engrailed, and Dorsal, and they are essential for certain aspects of repression by each of these repressors (Paroush et al 1994;Fisher et al 1996;Aronson et al 1997;Dubnicoff et al 1997;Jimenez et al 1997). These family members have been shown to be widely expressed both during development and in the adult, in contrast to the more limited expression pattern of their DNA-binding partners (Hartley et al 1988;Stifani et al 1992;Miyasaka et al 1993;Schmidt and Sladek 1993;Choudhury et al 1997;Pflugrad et al 1997;Sharief et al 1997).…”