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)

The Groucho co-repressor is primarily recruited to local target sites in active chromatin to attenuate transcription

Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. This study used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. Gro was found to predominantly bind at discrete peaks (<1 kilobase). It was also demonstrated that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in 'active' chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. It was also found that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. These results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation and polymerase pausing (Kaul, 2014).

Gro was first described as a 'long-range' co-repressor that could inhibit transcriptional initiation of reporter genes while bound to a distant (>1 kb away) enhancer element. However, the model that Gro spreads over multi-kilobase domains to repress transcription was derived from experimental approaches that lacked the resolution to determine if Gro was bound in continuous or clustered peaks around genes. For example, a previous study used ChIP and subsequent qPCR at sites spaced >1 kb apart around their single target gene to test the spreading model. The Gro detected at the promoter and at 1 kb, 2 kb and 4 kb upstream of their target gene may have been derived from distinct, discrete peaks of Gro binding. This study observed that clusters of Gro peaks across the genome are common. One example of this occurs at the E(spl)mβ-HLH locus where distinct Gro peaks lie less than 2 kb apart, either side of the coding region. It seems most likely that these are distinct peaks, as they lie over distinct Su(H) peaks and are separated by peaks of histone H3 and H4 acetylation (Kaul, 2014).

By selecting a superset of high confidence peaks common to all datasets for endogenous Gro and Gro-GFP, this study may have excluded some 'real' peaks from the general analysis. However, the properties of the peaks excluded from the superset did not differ significantly from the peaks in the superset. In general, peaks that were unique to one replicate were narrower than those included in the superset, further supporting the argument that the conditions and analyses were not biased against recovering broad peaks (Kaul, 2014).

A third of the high confidence Gro ChIP-seq peaks overlapped previously published Gro DamID peaks. This overlap is relatively low, however, a comparable level of overlap (34%) is observed between GAF ChIP-seq and GAF DamID peaks. The Dam domain was fused to the C-terminal domain of Gro, which is highly structured and interacts with many classes of transcription factor. Thus, the fusion of the Dam domain to the C-terminal of Gro may have interfered with Gro recruitment to the genome and excluded sites that could not be detected with ChIP-seq (Kaul, 2014).

Consistent with previous studies this study was unable to obtain reproducible ChIP samples for Gro without the use of a two-step crosslinking method. This may reflect that Gro is not directly recruited to chromatin, but rather via intermediate sequence specific DNA binding transcription factors. Use of two cross-linking agents meant that relatively long sonication was required to generate DNA fragments of a suitable size for sequencing. Extended sonication may disrupt indirect chromatin interactions and select only for high affinity binding sites. However this study recovered peaks with widths up to 2.9 kb from Kc167 cells indicating that the sonication regime was not inhibiting the recovery of broad peaks per se. Furthermore, previously published Gro-Dam peaks that overlapped the current ChIP-seq peaks tended to be broader than those that did not, indicating that the current analysis was not biased against detecting any broad low affinity Gro peaks (Kaul, 2014).

While some peaks of Gro binding were observed in intergenic regions that may be associated with enhancer elements that are more than 1 kb from the nearest annotated TSS, the data support a model in which Gro is recruited locally by transcription factors and does not spread along the chromatin by oligomerization when it acts on a distant target promoter. Thus, it is most likely that Gro recruited to distant regulatory elements is brought into the proximity of target promoters by 'looping' of the DNA. It is well established that chromatin looping can facilitate gene activation by bringing factors bound at intergenic enhancers into contact with the transcription machinery and also facilitate repression by distant regulatory elements. Future studies using chromatin capture techniques in wild-type and Gro depleted cells will determine if Gro contributes to the formation and stability of chromatin loops from distant cis-regulatory elements to target promoters (Kaul, 2014).

The RNA-seq experiments did not reveal a general upregulation of genes closely associated with Gro ChIP-seq peaks in cells treated with gro RNAi in either Kc167 or S2 cells. Indeed treatment with gro RNAi led to very few significant changes in gene expression. Similarly, widespread Gro-related changes to histone acetylation status or RNAP II recruitment or pausing were not detected. Only highly significant changes to gene expression and RNAP II recruitment were observed at a single known Gro target, E(spl)mβ-HLH. It is possible that loss of Gro may have led to increased variability in target gene expression, and the average expression values from many cells in the two biological replicates is unlikely to be sufficient to show any change in variability. However, genome-wide loss of Gro from its targets may not facilitate recruitment of activating factors in the absence of other changes in the nuclear environment (e.g de novo expression of transcription factors in response to cell-cell signaling). In addition, the residual Gro in these cells may be sufficient to maintain repression (Kaul, 2014).

Previous overexpression studies in S2 cells and in the fly indicate that oligomerization affects how Gro acts in cells. For example, ectopic expression of wild-type Gro leads to ectopic repression of the vgQ-lacZ reporter gene whereas overexpression of the non-oligomerizing GroL38D,L87D variant has no detectable effect on vgQ-lacZ expression. No dramatic differences were seen in the breadth or location of Gro peaks with a variant that does not oligomerize (L38D,L87D-GFP), lending support to the alternative models that it is the efficiency of Gro recruitment or overall structure of the co-repressor complex that is compromised in the presence of non-oligomerizing variants. An apparent reduction was observed in the amount of L38D,L87D-GFP binding with respect to Gro-GFP at the Rh5 locus although this effect is not observed at E(spl)mβ-HLH. This indicates that the level of Gro binding may be dependent on oligomerization at a subset of targets. Genetic evidence indicates that gro is not expressed in vast surplus to requirement as many genetic interactions can be detected with gro heterozygotes. For example, multiple gro mutations were isolated in screens for dominant suppressors of ro^DomThese results are generally consistent with those from previous studies that identified an association of Gro with hypoacetylated histones H3 and H4. However, this study did not detect significant changes in the histone acetylation status of histones H3 and H4 at Gro target sites when Gro levels were reduced in Kc167 cells. It cannot be formally ruled out that the residual Gro left in cells treated with RNAi against gro is sufficient to maintain histones in a hypoacetylated state or that there are subtle changes to acetylation levels that cannot be accurately detected by ChIP-seq methods. Furthermore, loss of repression and gene activation are separable processes and depletion of Gro did not facilitate the recruitment and activity of histone acetylases at levels that could be detected (Kaul, 2014).

Recent studies have revealed that regulation of promoter proximal pausing by RNAP II is a major point of control of the expression of many genes that respond to developmental and environmental cues. Paused polymerase is highly enriched at genes in stimulus-responsive pathways and in genes involved with patterning the axes in the early Drosophila embryo. Strikingly, Gro has critical functions regulating gene expression in stimulus-responsive pathways (e.g. Notch and Wnt signaling) and both AP and DV patterning. It has been proposed that pausing contributes to the plasticity of gene expression by keeping genes that must be repressed transiently in a state permissive for rapid reactivation. Gro-mediated repression is frequently dynamic and rapidly reversible during animal development. For example, the serial production of Drosophila embryonic neuroblasts relies on five short pulses of Notch signaling that occur within 4 hours. Activation of primary Notch target genes repressed by the Su(H)/Gro complex occurs within 5 minutes of triggering the Notch pathway in Drosophila DmD8 cells, and this activation is correlated with reduced RNAP II pausing. This study has demonstrated that Gro peaks frequently overlap with peaks of a known regulator of RNAP II pausing (GAF) and that Gro is required to maintain RNAP II pausing at E(spl)mβ-HLH, a gene known to be a target of Gro repression via recruitment by Su(H) in Kc167 cells. Although much is known about the molecular mechanisms that control the P-TEFb checkpoint and RNAP II pausing, very little is known about which contextual factors determine the extent of RNAP II pausing. Future studies will address whether Gro interacts with known regulators of the P-TEFb checkpoint to promote RNAP II pausing in a gene-specific manner (Kaul, 2014).

Finally, the finding that Gro target genes are transcribed is consistent with several other genome-wide studies that show association of repressors with actively transcribed loci. It is thought that this class of repressor allows cells to make rapid responses to developmental and environmental cues and to fine-tune levels of active gene expression. The data indicates that Gro belongs to this class and behaves like a modulator rather than an off switch at its target genes. This work adds to the growing body of evidence that fine-tuning of gene expression is a general mechanism of co-repressor function (Kaul, 2014).

Wnt-Dependent inactivation of the Groucho/TLE co-repressor by the HECT E3 ubiquitin ligase Hyd/UBR5

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF; see Pangolin) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin (see Armadillo) binds to TCF to overcome this repression, but how it achieves this is unknown. This study discovered that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. Groucho/TLE was identified as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription (Flack, 2017).

An essential step enabling Wnt-dependent transcription is the conversion of the Wnt enhanceosome from silent to active. This involves the binding of the Wnt effector β-catenin to TCF, which releases the transcriptional silence imposed on the linked genes by TCF-bound Groucho/TLE. This study has discovered a crucial role of Hyd/UBR5 in this process, and the evidence suggests that β-catenin directs the activity of this HECT ubiquitin ligase toward Groucho/TLE, to block its repressive activity. The evidence also implicates VCP/p97 in this UBR5-dependent inactivation of Groucho/TLE during Wnt signaling (Flack, 2017).

By generating UBR5 null mutant cell lines, it was possible to resolve previous inconsistencies regarding the effects of UBR5 depletion on Wnt/β-catenin responses in human cell lines. UBR5 KO cell lines consistently showed reduced Wnt responses, but no changes in β-catenin levels. This parallels the results from hyd mutant clones in flies, providing unequivocal evidence for Hyd/UBR5 as a positive regulator of Wnt signaling in fly and human cells (Flack, 2017).

Three strands of evidence implicate Groucho/TLE as a physiologically relevant substrate of Hyd/UBR5 during Wnt signaling. First, epistasis analysis revealed that Hyd/UBR5 acts below Armadillo/β-catenin, and thus likely targets a substrate in the nucleus, consistent with its nuclear localization. Second, the activity of UBR5 in ubiquitylating Groucho/TLE is triggered by Wnt/β-catenin signaling. Third, in Drosophila wing discs, hyd is largely dispensable in the absence of Groucho (as revealed by hyd gro double mutant clones), which provides powerful evidence that Hyd acts by antagonizing Groucho (Flack, 2017).

Two possible mechanisms by which β-catenin might activate UBR5 toward TLE3 during Wnt signaling are considered. Either, β-catenin might disinhibit UBR5 if this enzyme were normally autoinhibited, like the NEDD4 family HECT ligases. Indeed, one of these ligases (WWP2) is disinhibited by Dishevelled, which, upon polymerization, engages in multivalent interactions with WWP2 to release its cognate binding sites from autoinhibitory contacts. However, the strong activity of UBR5 toward PAIP2 in the absence of Wnt signaling argues against this mechanism. An alternative mechanism is favored, namely that β-catenin apposes enzyme and substrate, e.g., via triggering a conformational change of the Wnt enhanceosome that results in proximity between UBR5 and Groucho/TLE. Support for this mechanism comes from previous proximity labeling experiments that revealed a β-catenin-dependent rearrangement of some of the components within the Wnt enhanceosome (van Tienen, 2017), and from coIP assays showing that β-catenin promotes the association between UBR5 and TLE3 (Flack, 2017).

How does UBR5-dependent ubiquitylation of Groucho/TLE inactivate its co-repressor function? The most obvious mechanism involves proteasomal turnover of Ub-TLE, given the specificity of UBR5 in generating K48-linked Ub chains, which are efficient proteasomal targeting signals. In support of this, the levels of UBR5-dependent Ub-TLE3 are elevated after proteasome inhibition. However, negative results from the cycloheximide chase experiments argue against rapid proteosomal degradation being the primary mechanism underlying the UBR5-dependent inactivation of Groucho/TLE (Flack, 2017).

It was also considered that the ubiquitylation of the WD40 domain might interfere with its binding to cognate ligands, and thus weaken the association of Groucho/TLE with the Wnt enhanceosome. However, this does not seem to be the case since Ub-TLE3 appears to bind to its ligands as efficiently as unmodified TLE, including a K-only mutant which can only be ubiquitylated at K720, a WD40 pore residue that is crucial for ligand binding and co-repression. Evidently, the extended C terminus through which ubiquitin is attached to K720 is flexible enough to allow simultaneous ligand binding. However, for technical reasons, it was not possible to test the binding of Ub-TLE to the key ligand through which Groucho/TLE exerts its repressive function -- namely the nucleosomes to which Groucho/TLE binds via both its structured domains, to promote chromatin compaction. Nevertheless, it is plausible that the attachment of multiple ubiquitin chains to the WD40 domain would loosen up the binding of Groucho/TLE to nucleosomes, and thus attenuate its ability to compact chromatin (Flack, 2017).

Evidence based on dominant-negative VCP/p97 and two distinct VCP/p97 inhibitors implicates this ATPase in the Wnt-dependent inactivation of Ub-TLE. Intriguingly, a recent proteomic screen for NMS-873-induced VCP/p97-associated proteins identified TLE1 and TLE3 as the only Wnt signaling components, along with VCP/p97 adaptors and other putative substrates, consistent with the notion of Groucho/TLE is a substrate of this ATPase. VCP/p97 regulates the folding of ubiquitylated proteins, to promote their segregation from large structures, such as endomembranes, and also from large protein complexes, including DNA repair and chromatin complexes. It is therefore conceivable that VCP/p97 unfolds Groucho/TLE upon its ubiquitylation, especially if this modification loosened the interaction of Groucho/TLE with nucleosomes. Whatever the case, unfolding of the Groucho/TLE tetramer by VCP/p97 is likely to destabilize it, which would disable its repressive function. This is consistent with a recent proposal that the relief of Groucho-dependent repression is based on kinetic destabilization of the Groucho complex (Chambers, 2017), which may be facilitated by its ubiquitylation and unfolding by VCP/p97 (Flack, 2017).

One other E3 ligase has been shown to ubiquitylate TLE3, namely the RING ligase XIAP, which constitutively monoubiquitylates the Q domain of TLE3, apparently stimulating Wnt-dependent transcription by blocking its binding to TCF4. This contrasts with the Wnt-induced activity of UBR5 toward TLE3 revealed by this study. Evidently, the two ligases act distinctly, and also independently, given that the UBR5-dependent polyubiquitylation of TLE3 is normal in XIAP KO cells. However, it is also noted that the reduction of Wnt-dependent transcription in the XIAP KO cells was modest at best, compared to the substantial reduction in UBR5 KO cells. Either XIAP plays a lesser role in promoting transcriptional Wnt responses or a compensating E3 ligase was upregulated during the process of establishing XIAP KO cells. It is noted that the XIAP KO mice are viable, and without any overt mutant phenotypes, and that the Drosophila XIAP mutants do not show wg-like phenotypes, in contrast to the hyd mutant clones that phenocopy strong wg-like mutant phenotypes. All in all, it appears that UBR5 has a more profound role than XIAP in enabling transcriptional Wnt responses (Flack, 2017).

Inactivation of Groucho/TLE by UBR5 and VCP/p97 could also underlie other signaling-dependent gene switches that involve Groucho/TLE-dependent repression, e.g., Notch signaling, which depends on binding of Groucho/TLE to HES repressors. Indeed, recent genetic screens in C. elegans have identified the UBR5 ortholog sog-1 as a negative regulator of Notch signaling during nematode development. Although it is conceivable that hyd also affects Notch responses in flies, this study found that the derepression of the Notch target gene wg in hyd mutant wing disc clones is not sensitive to blockade by dominant-negative Mastermind, which argues against a role of Hyd in Notch-dependent transcription in this tissue. It is also noted that Ubr5 has been linked to defective Hedgehog signaling in mice, following an earlier lead of Groucho as a putative Hyd target in the context of Hedgehog signaling, although these links between Hyd/Ubr5 and Hedgehog signaling appear to be indirect (Flack, 2017).

However, UBR5 clearly also modifies substrates other than Groucho/TLE, including proteins with PAM2 motifs that are recognized by its MLLE domain, e.g., PAIP2 involved in translational control. Furthermore, via its UBR domain, UBR5 may recognize substrates of the N-end rule pathway, though few of these have been identified to date. Given the nuclear location of UBR5, it seems highly likely that most of its physiologically relevant substrates are nuclear proteins, e.g., the RING E3 ligase RNF168, which is ubiquitylated and destabilized by UBR5 during the DNA damage response (Flack, 2017).

UBR5 has been heavily implicated in cancer, although it is somewhat unclear whether it promotes or antagonizes tumor progression, which may depend on context. However, UBR5 amplification is the predominant genetic alteration in many types of cancers (far more prevalent than loss-of-function UBR5 mutations), and amplified UBR5 correlates with poor outcomes in breast cancer. This implies a tumor-promoting role of UBR5, consistent with its role in relieving Groucho/TLE-dependent repression of Wnt responses. It will be interesting to test whether UBR5 loss-of-function inhibits β-catenin-dependent tumorigenesis, e.g., in the intestine. This might be expected, given the results from the colorectal cancer cell line HCT116 whose β-catenin-dependent transcription is attenuated by UBR5 KO and whose proliferation is slowed down by VCP/p97 inhibition. If this were to apply generally to other colorectal cancer lines, this would indicate the potential of UBR5 and VCP/p97 as new enzymatic targets for therapeutic intervention in colorectal and other β-catenin-dependent cancers. It could widen the application of CB-5083, an orally bioavailable VCP/p97 inhibitor currently in phase 1 clinical trials (Flack, 2017).

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:  25  October 2023

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