BIOLOGICAL OVERVIEW

groucho is a neurogenic gene and member of the Enhancer of split complex (E[spl]-C). While it is ancestrally unrelated to other genes of E(spl)-C, it is a distinct neurogenic gene that happens to occur in the E(spl)-C neighborhood (Schrons, 1992). groucho is named for a mutant that developed an increased number of supraorbital bristles (bristles around the eye), reminiscent of the bushy browed Marx brother (Schrons, 1992). It encodes a nuclear protein expressed ubiquitously in both embryos and imaginal discs, and acts as a transcriptional repressor of several important genes in Drosophila development.

groucho interacts with helix-loop-helix protein Hairy, one of the Enhancer of split complex genes and Deadpan, and thus regulates transcription as a transcriptional corepressor, in partnership with other proteins. Groucho-E(SPL) protein complexes promote epidermal cell fate by repressing transcription of proneural AS-C genes (Paroush, 1994). In wing discs, hedgehog and engrailed are repressed in anterior cells by the activity of Groucho (de Celis, 1995). Thus Groucho, lacking a DNA binding domain, acts as a transcription factor by combining with other transcription factors to form an active complex repressing the transcription of target genes.

A transgenic embryo assay was employed to discover the mode of repression mediated by Hairy. Hairy can act as a dominant repressor capable of functioning over long distances to block multiple enhancers. Hairy is shown to repress a heterologous enhancer, the rhomboid enhancer sequence, when bound 1 kb from the nearest upstream activator. The binding of Hairy to a modified NEE leads to the repression of both the rhomboid and a distintly linked mesoderm-specific enhancer with a synthetic modular promoter. Two models are proposed for Hairy's long distance repressive function. (1) Hairy could recruit a cofactor that mediates repression at a distance. This factor would inhibit specific upstream activators bound within the proximal promoter. (2) Hairy could interact directly with one or more components of the basal transcriptional complex (Barolo, 1997).

How then does Hairy function? Hairy has been shown to interact with the co-repressor protein Groucho through the C-terminal WRPW motif. Gro is not known to bind DNA, but fusions of GRO with heterologous DNA binding domains have revealed that GRO can act as a transcriptional repressor. The Gro protein contains several repeats of a 40-residue motif, termed the WD40 repeat, that is thought to mediate protein-protein interactions. Tup1, a yeast corepressor protein that also contains WD40 repeats, is recruited to DNA by the alpha2 repressor in alpha-type cells for the silencing of alpha-specific genes. Similarly, Hairy may recruit Gro for silencing specific genes in the Drosophila embryo. The yeast mating-type repressors alpha2 and Tup1 have been reported to interact with histones. This observation raises the possibility that Gro mediates transcriptional silencing by influencing chromatin structure (Barolo, 1997)

GENE STRUCTURE

Bases in 5' UTR - 265

Exons - five

Bases in 3' UTR - 1310

PROTEIN STRUCTURE

Amino Acids 719

Structural Domains

Unlike other proteins of the Enhancer of split complex, Groucho has no bHLH domain. It does contain a WD40 domain, generally used in G protein mediated signal transduction as a protein interaction domain (Tata, 1993). Extra sex combs, a member of the Polycomb group, is another Drosophila transcription factor with a WD motif (Sathe, 1995).

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 that should therefore have specific repression domains. The WRPW motif is a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. The WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter. Thus Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW (Fisher, 1996).

Molecular recognition of transcriptional repressor motifs by the WD Domain of the Groucho/TLE corepressor

The Groucho (Gro)/TLE/Grg family of corepressors operates in many signaling pathways (including Notch and Wnt). Gro/TLE proteins recognize a wide range of transcriptional repressors by binding to divergent short peptide sequences, including a C-terminal WRPW/Y motif (Hairy/Hes/Runx) and internal eh1 motifs (FxIxxIL; Engrailed/Goosecoid/Pax/Nkx). This study identifies several missense mutations in Drosophila Gro, which demonstrate peptide binding to the central pore of the WD (WD40) β propeller domain in vitro and in vivo. These interactions were defined at the molecular level with crystal structures of the WD domain of human TLE1 bound to either WRPW or eh1 peptides. The two distinct peptide motifs adopt markedly different bound conformations but occupy overlapping sites across the central pore of the β propeller. Thise structural and functional analysis explains the rigid conservation of the WRPW motif, the sequence flexibility of eh1 motifs, and other aspects of repressor recognition by Gro in vivo (Jennings, 2006).

This paper presents genetic, biochemical, and structural evidence that the WRPW and eh1 motifs interact specifically with the C-terminal WD β propeller domain of Gro. All the missense mutations isolated in the genetic screen lie within this domain, and most are expected to disrupt the structure of the WD domain (Jennings, 2006).

All the mutations are defective in binding to a WRPW motif in vitro and disrupt the processes mediated by WRPW repressors in vivo: segmentation, neurogenesis, and sex determination. These effects should be due to direct binding between the WD domain and the WRPW motif because such binding is sufficient for repression in vivo: a C-terminal WRPW converts unrelated nuclear proteins into transcriptional repressors (Jennings, 2006).

Since Gro^MB31 (L692F) can bind to the eh1 repressor motif in vitro and in vivo, its WD domain must retain its overall β propeller structure. Thus, the residue mutated in Gro^MB31 (equivalent to Leu-743 in TLE1) highlights a candidate region in the WD domain for interacting directly with WRPW. Similarly, the ability of Gro^MB41 to mediate trunk repression via Gro-dependent repressor Capicua (Cic) indicates that the mutation (R483H) also does not disrupt the β propeller structure and that the mutated Arg (R534 in TLE1) lies in a region that associates with both WRPW and eh1. As expected if both repressor peptides bind to the same region of the WD domain, it is found that soluble WPRW peptide can compete with GST-eh1 for pulling down Gro in vitro (Jennings, 2006).

The residues mutated in Gro^MB31 and Gro^MB41 both lie in a depression at the mouth of the central pore of the β propeller, suggesting that the repressor motifs bind near this region of the WD domain. Crystallographic analysis of WRPW and eh1 peptide complexes with TLE-WD confirm that this is indeed the case and that both of these residues are directly involved in interaction with the bound repressors (Jennings, 2006).

The crystal structure also reveals how Gro recognizes both C-terminal and internal repressor motifs. One α-carboxyl oxygen of the C-terminal tryptophan in the WRPW motif makes an ion-pair/hydrogen bonding interaction with the side chain of Lys 718 at the edge of the binding pocket. However, the other carboxyl oxygen points out to solvent, so that the continuing peptide chain is accommodated in those examples of Hairy and Hes transcription factors where the WRPW motif is not at the extreme C terminus (Jennings, 2006).

According to the genetic and structural data, the outer edges of the TLE-WD propeller appear not to play direct roles in recognizing repressor peptides. Thus, a previous suggestion based on missense mutations of the C. elegans Gro homolog UNC-37, that eh1 motifs might bind to the edge of blade 6 of the WD domain, is not supported by the data presented in this study. Most likely, the unc-37 mutations cause general conformational disruption, paralleling some of the Gro mutations described here (Jennings, 2006).

WD domains are widespread in eukaryote biology and have been implicated in mediation of protein-protein interactions in a very diverse range of cellular processes. Comparison of the WD-peptide complexes described in this study with structures of other WD-domain complexes shows the recurring use of the 'top' face of the β propeller as a site for specific interaction with other proteins. Indeed, use of the compact and invaginated interaction pocket provided by the WD β propeller pore to bind small structural motifs may be common. For example, recognition of phosphorylated β-catenin or IκB by β-TrCP1, and recruitment of Gα, phosducin, or G coupled receptor kinase to Gβγ, both involve the interaction of a small peptide motif from the interacting protein with the mouth of the central pore of the β propeller. Recognition of short peptide motifs may be a general feature of the WD domain (Jennings, 2006).

All residues involved in direct interaction with the WRPW motif are identical in human, Drosophila, and C. elegans Gro/TLE sequences and are very strongly conserved in the distantly related yeast Tup1 corepressor. There are no obvious WRPW-motif proteins in yeast, suggesting that interaction by yeast transcription factors with Tup1 is mediated by embedded amphipathic helical motifs similar to eh1. This mode of corepressor recruitment must be evolutionarily ancient. The more recent WRPW motif is readily appended to the C termini of already functional proteins and confers new regulatory potential with minimal need for further structural adaptation (Jennings, 2006).

This analysis also clarifies how Gro recognizes other repressor motifs. Gro binds to WRPY, the variant C-terminal motif found in Runx proteins, with substantially lower affinity than to WRPW. The genetic data argue that the two motifs bind to the same site on the WD domain, because like repression by WRPW, Runt activity is sensitive to the gro^MB31 mutation. However, the phenolic hydroxyl of a C-terminal tyrosine in WRPY would be buried in the base of the binding pocket without the opportunity to make an obvious compensating hydrogen bond, consistent with the lower affinity of the WRPY motif. Nevertheless, a C-terminal Tyr is absolutely conserved in the Runx family of transcription factors, suggesting that this particular amino acid is functional, perhaps as a point of recognition for accessory proteins (Jennings, 2006).

Other aspects of the structure explain the activity of the internal FRPW motif found, for example, in Hkb. Genetic analysis indicates that Hkb activity is lost in gro^MB41 and gro^MB31 embryos, arguing that the mode of internal FRPW binding resembles that of WRPW. The crystal structure of bound WRPW peptide can readily accommodate both the replacement of the N-terminal tryptophan with phenylalanine and the continuation of the polypeptide beyond the C-terminal tryptophan (Jennings, 2006).

In summary, the WRPW motif forms a remarkably compact structure when bound to Gro/TLE, which contrasts with the helical conformation adopted by the bound eh1 motif. Nevertheless, both bind to the same region of the WD domain, which seems able to recognize a broad range of peptide motifs. This versatility is reflected by use of Gro/TLE as a corepressor by diverse repressors in a wide variety of developmental contexts. The protein family is also active in signaling pathways that are associated with human disease. The compact interface between repressor peptide motifs and Gro/TLE indicates that it might provide a suitable target for therapeutic intervention (Jennings, 2006).

date revised:  3 July 97

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