InteractiveFly: GeneBrief

hyperplastic discs : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis (Lee, 2002).

Communication is essential for multicellular development. Intercellular signals regulate the timing and pattern of cellular events such as growth, division, movement, and differentiation, allowing large groups of cells to behave in a coordinated manner during events such as organogenesis. Cell-cell signaling can regulate the activity of cells at many different levels, including gene expression, cell division and motility. Within the receiving cell, signal transduction cascades transform the reception of ligand at the cell surface into the proper cellular response. This may involve regulation of protein activity by post-translational modification or by destruction or proteolytic processing. Ubiquitination, the covalent addition of a multimeric chain of the 76-amino-acid Ubiquitin (Ub) protein, is the most common intracellular signal for proteolysis. Ubiquitination is a multi-step process that begins with the activation of a Ub molecule by an E1 or Ub-activating enzyme. The activated Ub is transferred to an E2 enzyme, which is then responsible, either directly or indirectly, for attaching the Ub to a substrate protein. Specificity of the ubiquitination reaction is achieved at the level of the E3 ubiquitin ligase, which is thought to directly bind the substrate. Many such ligases exist and have been classified into families based on the structure of the ubiquitination domain. HECT-domain E3 ligases directly attach Ub to a substrate, while RING domain E3s direct specific substrate ubiquitination by the E2 (Lee, 2002).

A small number of signaling pathways appear to direct most developmental processes. In Drosophila, the BMP family member Decapentaplegic (Dpp) and the founding member of the Hedgehog family (Hh) are used repeatedly throughout development. One function of Hh and Dpp is to direct the progressive differentiation of the eye imaginal disc. In the second instar eye disc, hh is expressed in a complex pattern in both the disc proper and the peripodial membrane, before being refined to a small domain centered on the dorsoventral midline of the disc's posterior margin. Hh signals more anterior cells to express dpp and atonal (ato), which encodes the bHLH transcription factor required for the formation of the R8 'founder' photoreceptor in each cluster. These cells then differentiate as photoreceptors. These cells also express hh, allowing the cycle to propagate toward the anterior of the disc. dpp is expressed in the morphogenetic furrow, an indentation at the front of differentiation, where it is responsible for coordinating the timing of differentiation through synchronization of the cell cycle. Loss of either Hh or Dpp blocks the initiation of differentiation, while loss of both blocks progression. While it is known that Hh activates dpp expression in the furrow, it is not known how hh expression is controlled, nor how dpp is turned off in cells leaving the furrow (Lee, 2002).

To identify novel genes contributing to pattern formation in the Drosophila eye disc, a mosaic genetic screen was carried out in which homozygous mutant clones of cells were generated specifically in the eye disc. In this screen, four alleles were recovered of hyd, which encodes a large protein containing a HECT family E3 ubiquitin ligase domain (Callaghan, 1998; Mansfield, 1994). hyd was initially isolated in a screen for mutations causing imaginal disc overgrowth (Martin, 1977). Adult eyes containing hyd mutant clones show extensive overgrowth of the eye tissue surrounding a hyd clone, although the clones themselves did not persist to adulthood. In the third instar eye disc, premature photoreceptor differentiation is observed, visualized by expression of the markers Elav and Neuroglian, in hyd mutant clones in a region just anterior to the morphogenetic furrow. Very rarely, differentiation is seen in clones lying near the anterior margin. The spatial restriction of this phenotype is consistent with work demonstrating the existence of a preproneural zone anterior to the furrow. Ectopic differentiation spreads beyond the borders of the hyd mutant clones into the surrounding wild-type tissue. The proneural transcription factor Atonal (Ato) is also misexpressed within and surrounding hyd clones anterior to the furrow. The overgrowth of wild-type tissue in adult eyes is visible in third-instar imaginal discs as folding and distortion of the disc epithelium surrounding hyd clones. This effect is more widespread than the ectopic differentiation phenotype, appearing in more anterior regions of the disc (Lee, 2002).

Growth and differentiation of cells in the eye disc both depend on Hh and Dpp secreted by more posterior cells. The non autonomous differentiation and overgrowth caused by hyd clones suggested that the clones might be producing one or both of these molecules; a similar phenotype is produced by ectopic expression of hh or activation of the hh pathway ahead of the furrow. Therefore hh expression was examined in hyd clones using both an enhancer trap line and antibody staining. hyd mutant clones anterior to the furrow indeed expressed hh-lacZ and Hh protein earlier than surrounding wild-type cells. When hyd clones were made in a Minute background, thereby reversing the growth disadvantage of hyd mutant tissue, hh-lacZ expression was observed throughout the eye disc. The widespread hh expression in hyd/Minute clones may explain the accelerated differentiation and small size of these eye discs (Lee, 2002).

The ectopic hh expression observed in hyd mutant clones could be a consequence, rather than a cause, of their premature differentiation. To rule out this possibility, hyd;ato double mutant clones were generated. Clones mutant for ato cannot form the R8 photoreceptor, which is itself required for the recruitment of photoreceptors R1-R7. Thus ato mutant clones do not differentiate, unless a cell at the margin of a clone is recruited by a neighboring wild-type R8. It was confirmed that ato single mutant clones do not express hh. While hyd;ato mutant clones do not differentiate, they nevertheless expressed hh-lacZ and are capable of directing ectopic differentiation in surrounding wild-type tissue. This demonstrates that loss of hyd function has a direct effect on hh expression that is not simply due to differentiation (Lee, 2002).

As an E3 ubiquitin ligase, Hyd is likely to promote the degradation of one or more proteins. Based on the ectopic expression of hh in hyd mutant clones, it was hypothesized that Hyd was acting in the anterior of the third instar eye disc to prevent premature expression of hh. hyd is expressed in proliferating tissues in the embryo and larva, but its expression in the eye disc had not been described in detail (Mansfield, 1994). Using in situ hybridization, it was found that hyd RNA is highly expressed in the anterior of the eye imaginal disc, especially around the dorsoventral midline. hyd is expressed at lower levels towards the dorsal and ventral margins but was still restricted to the anterior. This expression pattern is consistent with a role for hyd in preventing the premature expression of hh (Lee, 2002).

If the ectopic differentiation and overgrowth associated with hyd clones is due to ectopic hh expression, it should be possible to rescue this phenotype by removing hh function from the clones. The phenotype of hyd;hh double mutant clones was determined. Similar results were obtained with three hh alleles, hh^rJ413, hh^ts2 grown at 29°C, and the null allele hh^AC. While hh mutant clones appear wild type unless located on the margin of the eye disc, hyd;hh double mutant clones show a partial suppression of the hyd phenotype. Ectopic photoreceptors are no longer present in or around hyd;hh clones, and hyd;hh double mutant clones generated in a Minute background have only a few photoreceptors associated with the remaining wild-type tissue. Thus hyd mutant tissue requires Hh in order to differentiate. However, some hyd;hh mutant clones are still able to stimulate proliferation of surrounding tissue, leading to overgrowth of the adult eye (Lee, 2002).

dpp has also been shown to stimulate proliferation in the eye disc. The remaining non autonomous overgrowth induced by hyd;hh double mutant clones might therefore be due to ectopic expression of dpp. Using a dpp-lacZ reporter construct, ectopic dpp expression was observed in and around hyd mutant clones anterior to the furrow. Ectopic dpp expression appears more widespread than ectopic hh expression and occurs in more anterior regions of the disc, raising the possibility that dpp misexpression is not merely induced by ectopic Hh in the clones but is an independent consequence of the loss of hyd. Indeed, dpp is still expressed in some hyd;hh mutant clones, although its expression is limited to clones close to the morphogenetic furrow. Thus in the absence of hyd function, dpp expression is no longer strictly regulated by Hh signaling (Lee, 2002).

The persistence of dpp expression in hyd;hh double mutant clones has suggested that dpp regulation by Hyd is at least partially independent of hh. One molecule that is known to transcriptionally regulate both hh and dpp is Ci. In the wing imaginal disc it has been shown that Hh controls dpp expression both by suppressing the production of Ci₇₅, which inhibits dpp expression, and by activating Ci₁₅₅, which activates dpp expression. In addition, Ci₇₅ inhibits hh expression in the anterior compartment of the wing disc; ci transcription is repressed by en in the posterior compartment, allowing hh to be expressed there. To test whether hyd acts through Ci to affect hh and dpp expression in the eye disc, the expression of a truncated constitutive repressor form of Ci (Ci₇₆) was driven specifically in hyd mutant clones using the MARCM system. dpp-lacZ is no longer expressed in hyd clones expressing UAS-ci₇₆ in or anterior to the furrow. However, Ci₇₆ does not prevent the ectopic expression of hh in hyd mutant clones. Continued hh expression in these clones sometimes led to ectopic differentiation in tissue surrounding the clone. Thus Ci₇₆ is sufficient to block dpp but not hh expression in hyd clones, suggesting that Hyd regulates hh and dpp expression through at least partially independent mechanisms (Lee, 2002).

If the overgrowth phenotype induced by hyd;hh double mutant clones is indeed due to their misexpression of dpp, it should be blocked by introducing Ci₇₆ into the mutant cells. Indeed, when hyd;hh mutant clones expressing UAS-ci₇₆ were generated, the adult eyes no longer exhibited any overgrowth. Thus all the ectopic growth and differentiation caused by loss of hyd in the eye can be attributed to independent effects on hh expression and activation of the Hh pathway (Lee, 2002).

If hyd regulates dpp expression by altering Ci activity, loss of hyd should lead to upregulation of full-length, active Ci. Increased levels of full-length Ci are indeed observed in hyd mutant clones in the anterior of the eye disc. However, this could be due to misexpression of hh in the same clones. To determine whether hyd has a direct effect on Ci, hyd;hh double mutant clones anterior to the morphogenetic furrow were examined. High levels of full-length Ci accumulated in these clones, confirming that Hyd normally reduces Ci levels independently of Hh activity (Lee, 2002).

Hyd might act directly on Ci to promote its proteolytic cleavage or degradation. An alternative possible target for Hyd activity is Smoothened (Smo). Smo is a transmembrane protein that acts positively in Hh signaling. Smo levels are kept low by the receptor protein Patched (Ptc) in the absence of Hh, but Smo is stabilized and localized to the membrane when Hh binds to Ptc. To test whether Hyd normally contributes to Smo degradation, eye discs containing hyd;hh clones were stained with Smo antibody. No Smo accumulation is apparent in the clones. Thus loss of hyd leads to accumulation of full-length Ci without altering the level of Smo (Lee, 2002).

Since hyd is expressed in the wing disc and is required for its normal growth (Mansfield, 1994), whether its effects on wing development might also be mediated by alterations in hh expression and Ci levels was examined. In wild-type wing discs, hh is expressed uniformly throughout the posterior compartment of the wing pouch, while dpp is expressed in the anterior compartment in a stripe along the AP border. Ci₁₅₅ is present at high levels in a similar stripe at the AP border and at lower levels elsewhere in the anterior compartment. Expression of hh, dpp and Ci₁₅₅ in hyd clones remains restricted to the correct compartment. However, some hyd mutant clones in the posterior compartment express elevated levels of hh-lacZ. This misexpression of hh is correlated with a rounded shape and apparent overgrowth of the clones. The only known regulator of hh expression in the wing disc is Ci, which is restricted to the anterior compartment by En-mediated repression; Ci₇₆ represses hh there. These results suggest that a Ci-independent activator of hh expression must be present in the posterior compartment and kept in check by Hyd activity (Lee, 2002).

In addition, Ci₁₅₅ is upregulated in anterior hyd mutant clones. In contrast to the eye disc, no hh misexpression was observed in anterior hyd clones; thus Ci upregulation in hyd clones must be independent of hh. This is consistent with findings that hyd regulates Ci and hh independently in the eye disc. Smo levels were not significantly increased in hyd mutant clones in the anterior compartment of the wing disc, suggesting that as in the eye disc, hyd affects Ci independently of Smo (Lee, 2002).

Thus hyd acts as a negative regulator of both hh and dpp expression in the anterior of the Drosophila eye disc. Loss of hyd function leads to the ectopic expression of both genes, resulting in non autonomous overgrowth of the disc and premature photoreceptor differentiation that propagates into the surrounding tissue. The ability to suppress this overgrowth by preventing both expression of hh and activation of the Hh pathway indicates that the hyperplastic effects of hyd in the eye are entirely mediated by Hh signaling. This is probably not the case in the wing disc; in this tissue hh and dpp expression are also blocked by hyd, but hyd clones in the posterior compartment can autonomously overgrow without expressing Ci, the transcription factor required for the response to Hh. hyd may thus have an independent effect on the cell cycle in wing disc cells. The human homolog of hyd, EDD, is located in a chromosomal region that is disrupted in a variety of cancers (Callaghan, 1998). It will be of interest to determine whether loss of hyd is responsible for any of these syndromes, and if so, whether the tumorous growth can be attributed to misregulation of hh or dpp homologs (Lee, 2002).

Little is known about the control of hh expression in the eye disc. In the embryo and other imaginal discs hh expression is controlled by en, which defines the posterior compartment in a lineage-dependent manner. The eye disc has no anterior-posterior compartment boundary, and loss of en function in the eye has no effect. Since hh expression is repressed by Ci₇₆ in the anterior wing disc, it seemed possible that this was also the case in the eye disc; Hh could then activate its own expression in more anterior cells by blocking Ci cleavage. However, it was not possible to prevent hh expression by providing Ci₇₆ to hyd mutant cells in the eye disc, although this does suffice to repress hh target genes such as dpp. In agreement with this result, ci mutant clones anterior to the furrow do not induce ectopic differentiation, indicating that loss of the repressor form of Ci is not sufficient to allow hh transcription in the eye disc. hyd must therefore be a component of the Ci-independent mechanism that restricts hh expression (Lee, 2002).

Regulation of hh by hyd is also clearly independent of Ci in the wing disc, since loss of hyd leads to hh upregulation in the posterior compartment, where Ci is not present, and not in the anterior compartment. The Groucho (Gro) corepressor has been proposed to contribute to a Ci-independent mechanism of hh repression in cells close to the compartment boundary. However, the effects of loss of hyd differ from those of loss of gro, which affects only the anterior compartment of the wing disc and promotes excessive photoreceptor differentiation only posterior to the furrow in the eye disc, suggesting that a third mechanism of hh regulation may exist. hh expression may not be merely a default state resulting from the absence of the Ci repressor and Gro, but may require another activator, the levels of which are normally kept in check by Hyd (Lee, 2002).

Control of dpp expression by hyd, in contrast, appears to be mediated by Ci. Ci₁₅₅ is upregulated in hyd mutant cells in the eye disc in a hh-independent manner, and ectopic dpp expression in these cells can be blocked by Ci₇₆. In the wing disc, dpp misexpression is limited to the ci-expressing anterior compartment, and is again associated with upregulation of Ci₁₅₅. Thus hyd acts on Hh signaling as well as hh expression, preventing full activation of the Hh pathway in anterior cells (Lee, 2002).

Hyd independently regulates hh and dpp expression, suggesting either that Hyd has multiple substrates or that its substrate has multiple functions. dpp expression in hyd clones is blocked by Ci₇₆, placing the effect of Hyd on dpp upstream or at the level of Ci activity. Ci₁₅₅ but not Smo accumulates to high levels in hyd;hh mutant cells in the eye disc and in anterior cells in the wing disc; thus Hyd may act on Ci itself, on a component of the Hh pathway between Smo and Ci, or on a nuclear cofactor that stabilizes Ci. Consistent with an effect on Ci or a cofactor, Hyd protein has been detected in both the cytoplasmic and nuclear compartments (Mansfield, 1994). The human Hyd homolog EDD appears to be predominantly localized in the nucleus, where it interacts with the progesterone receptor and DNA topoisomerase II-binding protein (Henderson, 2002; Honda, 2002). Ubiquitination can function to enhance the potency of transcriptional activation domains; however, the ectopic gene expression observed in hyd mutant clones would be difficult to explain by this mechanism (Lee, 2002).

The SCF ubiquitin ligase complex containing the F-box protein Slmb and the RING finger protein Roc1 has been implicated in the ubiquitination of Ci that mediates its processing to Ci₇₅. There are several possible explanations for the apparent overlap between Slmb and Hyd functions. Slmb may directly ubiquitinate Ci, while Hyd acts on another substrate; the more dramatic effect of slmb than hyd clones on Ci accumulation argues for this possibility. However, it is also possible that Hyd, rather than Slmb, is the direct ubiquitin ligase for Ci. The consensus sequence for Slmb is not present in Ci, although it has been suggested that several weakly matching sequences might suffice for its recognition. In addition, two groups have obtained inconsistent genetic evidence as to whether slmb acts upstream or downstream of smo and protein kinase A (PKA). It is unlikely that Hyd and Slmb carry out the same process in different cells, since hyd is expressed throughout the wing pouch (Mansfield, 1994), and slmb is clearly active in the same region, although its expression has not been examined. Finally, Hyd and Slmb could both act on Ci, either additively or with different outcomes. For example, ubiquitination by Slmb promotes processing of Ci to Ci₇₅, while ubiquitination by Hyd might promote complete degradation of the Ci protein. Unfortunately, it was not possible to obtain large enough quantities of hyd;hh mutant tissue to test this possibility directly by Western blotting. The lack of hh misexpression in hyd clones in the anterior compartment of the wing disc suggests that these cells still contain Ci₇₅ as well as Ci₁₅₅; loss of hyd may therefore stabilize both forms of the Ci protein rather than altering their ratio. However, this is not a definitive test of hyd function, as loss of PKA does not lead to ectopic hh-lacZ expression despite its effect on Ci processing. Hyd-mediated degradation of both forms of Ci, or its redundancy with Slmb for Ci cleavage, would explain the limited effect on dpp expression in hyd;hh double mutant clones in the eye disc and in hyd mutant clones in the wing disc; dpp misexpression in these cases is restricted to a region in which endogenous Hh may contribute to altering the ratio of the two forms of Ci (Lee, 2002 and references therein).

Ubiquitination is a mechanism commonly used to regulate protein activity by targeting proteins for degradation or processing, during the cell cycle and in a number of signaling pathways (reviewed in Ciechanover, 2000). The Slmb-containing SCF complex is also required for the degradation of Armadillo (Arm)/ß-catenin, allowing Wnt signaling, as well as for the degradation of IkappaB. Hyd is unlikely to act on Arm in the eye disc, since Arm accumulation would prevent the ectopic photoreceptor differentiation seen in hyd mutant clones; this may indicate another difference in the substrate specificity of Hyd and Slmb. However, it is possible that hyd affects Wg signaling in the wing disc, since hyd mutant clones can induce ectopic expression of the Wg target gene scute (Kazuhito Amanai, unpublished data cited in Lee, 2002). The HECT domain ligases Smurf1 and Smurf2 are important antagonists of BMP signaling, promoting downregulation of both Smads and receptors. Itch/Suppressor of deltex, another HECT domain ligase, ubiquitinates Notch. In addition, nuclear Notch is degraded by Sel-10, while the ligand Delta is the target of ubiquitination by Neuralized. Placement of hyd within the Hh pathway and upstream of hh expression expands this growing list of cases in which signaling pathways are regulated by ubiquitination (Lee, 2002).

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).

DEVELOPMENTAL BIOLOGY

Embryonic

Developmental Northern and Western analyses show coincident accumulation of the 9.5-kb transcript and the 280-kDa protein at all stages of development, with high levels at the embryonic and pupal stages (Mansfield, 1994).

EFFECTS OF MUTATION

The hyperplastic discs (hyd) gene (formerly called l(3)c43) is located at 85E1-10 on the third chromosome of Drosophila melanogaster. It was originally identified by a temperature-sensitive mutation that causes imaginal disc overgrowth in mutant larvae raised at a restrictive temperature. Twenty new alleles of hyperplastic discs have been recovered in gamma ray, ethyl methanesulfonate and hybrid dysgenesis screens, and the molecular lesions have been identified for several of the alleles. The null phenotype appears to be lethal at or before the second instar. Adults that can be obtained in crosses of temperature-sensitive alleles maintained at permissive temperatures are sterile with one exception and have defects in germ tissue morphology. The hyperplastic discs locus has been cloned by chromosome walking from the alpha-tubulin-2 gene and encodes a 9.5-kb messenger RNA, containing a 2897-amino-acid open reading frame. Sequence analysis of HYD reveals strong similarity to a portion of the C-terminus of poly(A) binding protein and to the rat 100-kDa protein, whose function is unknown. The 280-kDa HYD protein accumulates at decreased levels in mutant alleles and at restrictive temperatures in ts alleles. Examination of relative levels of Hyd protein in mutant animals supports the idea that less severe mutations are those that result in disc overgrowth, while more severe mutations result in variable disc growth phenotypes (Mansfield, 1994).

While hyd clones in third instar eye discs contain Elav-expressing photoreceptors, clones in the adult eye appear to form either scars in the eye or at the margin of the head cuticle. It was also noticed that hyd clones in the posterior of the eye disc are generally smaller than wild-type clones generated in a similar cross, and mutant clones contained reduced numbers of photoreceptors. The lack of mutant ommatidia in adult eyes could reflect either a loss of hyd mutant clones due to competition with the surrounding tissue, or a later requirement of hyd for viability. To further examine this question, hyd clones were made in a Minute genetic background, thereby reversing the growth disadvantage of hyd mutant tissue. These animals survive to pharate adult stages but can not eclose; they have greatly reduced eyes that nevertheless appear to contain properly formed hyd mutant ommatidia. This suggests that the lack of visible hyd clones in adults is due to their poor relative growth, leading to their elimination through competition with wild-type cells. Third instar eye discs containing hyd/Minute clones were also examined, and greatly reduced eye discs were found that appeared to contain full clusters of photoreceptors throughout the eye field. Since cells arrest division when they differentiate, premature differentiation could explain the small size of hyd/Minute eye discs. Ato is normally expressed in the morphogenetic furrow and in the youngest R8 photoreceptors; in early third instar hyd/Minute mutant discs the only remaining Ato staining is in single (presumably R8) cells, implying that the morphogenetic furrow has already reached the anterior margin. From this, it is inferred that differentiation in hyd/Minute discs still progresses from posterior to anterior, but at an accelerated rate (Lee, 2002).

The F-box protein Slmb has been shown to promote processing of Ci to Ci₇₅ as a component of an SCF ubiquitin ligase complex. Therefore the effects were compared of slmb and hyd mutations on Ci levels in the wing disc. Ci₁₅₅ is much more dramatically increased in slmb clones than in hyd clones. An interesting difference was also observed between hyd and slmb in the regulation of dpp. dpp expression is increased in hyd mutant clones close to the AP border, but is very rarely activated outside this domain. In contrast, slmb mutant clones activated dpp expression only when they lay outside the wing pouch, perhaps because of activation of Wg signaling, which represses dpp expression, within the wing pouch. Consistent with these third instar phenotypes, anterior duplications like those resulting from loss of slmb are not observed in adult wings containing hyd mutant clones, although outgrowths did arise from internal regions of the wing. Such duplications would require dpp to be misexpressed at a distance from its normal domain of expression. Ptc expression, which requires activation of the full-length form of Ci, was not altered in either hyd or slmb mutant clones. Slmb and Hyd thus appear to have distinct effects on Ci protein accumulation and activity, suggesting that they have either different substrates or different effects on the same substrate (Lee, 2002).

EVOLUTIONARY HOMOLOGS

The poly(A)-binding protein (PABP) recognizes the 3' mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein-protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the Hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four alpha-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners (Deo, 2001).

Use of the differential display technique to isolate progestin-regulated genes in T-47D human breast cancer cells led to identification of a novel gene, EDD. The cDNA sequence contains a 2799 amino acid open reading frame sharing 40% identity with the predicted 2894 amino acid product of the Drosophila melanogaster tumor suppressor gene hyperplastic discs, while the carboxy-terminal 889 amino acids show 96% identity to a rat 100 kDa HECT domain protein. EDD mRNA is progestin-induced in T-47D cells and is highly abundant in testes and expressed at moderately high levels in other tissues, suggesting a broad role for EDD. Anti-EDD antibodies immunoprecipitated an approximately 300 kDa protein from T-47D cell lysates. HECT family proteins function as E3 ubiquitin-protein ligases, targeting specific proteins for ubiquitin-mediated proteolysis. EDD is likely to function as an E3 since in vitro translated protein binda ubiquitin reversibly through a conserved HECT domain cysteine residue. EDD was localized by FISH to chromosome 8q22, a locus disrupted in a variety of cancers. Given the homology between EDD and the Hyperplastic discs protein, which is required for control of imaginal disc growth in Drosophila, EDD potentially has a role in regulation of cell proliferation or differentiation (Callaghan, 2002).

The ubiquitin-protein ligase EDD encodes an ortholog of the hyperplastic discs tumor suppressor gene, which has a critical role in Drosophila development. Frequent allelic imbalance at the EDD chromosomal locus in human cancers suggests a role in tumorigenesis. In addition to a HECT (homologous to E6-AP carboxyl terminus) domain, the EDD protein contains a UBR1 zinc finger motif and ubiquitin-associated domain, each of which indicates involvement in ubiquitinylation pathways. This study shows that EDD interacts with importin alpha 5 through consensus basic nuclear localization signals and is localized in cell nuclei. EDD also binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation. This activity is comparable with that of the coactivator SRC-1, but, in contrast, the interaction between EDD and PR does not appear to involve an LXXLL receptor-binding motif. EDD also binds calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein, a potential target of ubiquitin-mediated proteolysis, and an altered association is found between EDD and calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein in response to DNA damage. The data presented here demonstrate a role for EDD in PR signaling but also suggest a link to cancer through DNA damage response pathways (Henderson, 2002).

The human homolog of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen has shown that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacts with hHYD. Endogenous hHYD binds the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transfer ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 is usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminishes the ubiquitination of TopBP1, probably via phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks. These results demonstrate that hHYD coordinates TopBP1 in the DNA damage response (Honda, 2002).

Conjugation of ubiquitin to proteins is activated during spermatogenesis. Ubiquitination is mediated by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (UBCs or E2s), and ubiquitin protein ligases (E3s). Since activated ubiquitination is UBC4 dependent, Rat100, a UBC4-dependent E3 expressed in the testis, was characterized. Analysis of expressed sequence tag sequences and immunoblotting have shown that Rat100 is actually a 300-kDa protein expressed mainly in the brain and testis and is similar to the human E3 identified by differential display (EDD) protein and the Drosophila hyperplastic discs gene, mutants of which cause a defect in spermatogenesis. Rat100 is induced during postnatal development of the rat testis, peaking at d 25. It is localized only in germ cells and is highly expressed in spermatocytes, moderately in round and slightly in elongating spermatids. In contrast to UBC4 whose removal from a testis extract abrogates much of the conjugation activity, immmunodepletion of Rat100 from the extracts has little effect. Rat100 therefore has a limited subset of substrates, some of which appear associated with the E3, since the immunoprecipitate containing Rat100 supports incorporation of (125)I-ubiquitin into high molecular weight proteins. Thus, Rat100 is the homolog of human EDD and likely of Drosophila Hyperplastic discs. This homology suggests that induction of this E3 results in ubiquitination of specific substrates, some of which are important in male germ cell development (Oughtred, 2002).

REFERENCES

Search PubMed for articles about Drosophila hyperplastic discs

Callaghan, M. J., Russell, A. J., Woollatt, E., Sutherland, G. R., Sutherland, R. L. and Watts, C. K. (1998). Identification of a human HECT family protein with homology to the Drosophila tumor suppressor gene hyperplastic discs. Oncogene 17: 3479-3491. 10030672

Chambers, M., Turki-Judeh, W., Kim, M. W., Chen, K., Gallaher, S. D. and Courey, A. J. (2017). Mechanisms of Groucho-mediated repression revealed by genome-wide analysis of Groucho binding and activity. BMC Genomics 18(1): 215. PubMed ID: 28245789

Ciechanover, A., Orian, A. and Schwartz, A. L. (2000). Ubiquitin-mediated proteolysis: biological regulation via destruction. BioEssays 22: 442-451. 10797484

Craig, A. W., Haghighat, A., Yu, A. T. and Sonenberg, N. (1998). Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature 392: 520-523. 9548260

Deo, R. C., Sonenberg, N. and Burley, S. K. (2001). X-ray structure of the human hyperplastic discs protein: an ortholog of the C-terminal domain of poly(A)-binding protein. Proc. Natl. Acad. Sci. USA 98: 4414-4419. 11287654

Flack, J. E., Mieszczanek, J., Novcic, N. and Bienz, M. (2017). Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5. Mol Cell 67(2): 181-193.e185. PubMed ID: 28689657

Henderson, M. J., et al. (2002). EDD, the human hyperplastic discs protein, has a role in progesterone receptor coactivation and potential involvement in DNA damage response. J. Biol. Chem. 277(29): 26468-78. 12011095

Honda, Y., Tojo, M., Matsuzaki, K., Anan, T., Matsumoto, M., Ando, M., Saya, H. and Nakao, M. (2002). Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response. J. Biol. Chem. 277: 3599-3605. 11714696

Kozlov, G., Trempe, J. F., Khaleghpour, K., Kahvejian, A., Ekiel, I. and Gehring, K. (2001). Structure and function of the C-terminal PABC domain of human poly(A)-binding protein. Proc. Natl. Acad. Sci. 98: 4409-4413. PubMed ID: 11287632

Lee, J. D., Amanai, K., Shearn, A. and Treisman, J. E. (2002). The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms. Development 129: 5697-5706. 12421709

Mansfield, E., Hersperger, E., Biggs, J. and Shearn, A. (1994). Genetic and molecular analysis of hyperplastic discs, a gene whose product is required for regulation of cell proliferation in Drosophila melanogaster imaginal discs and germ cells. Dev. Biol. 165: 507-526. 7958417

Martin, P., Martin, A. and Shearn, A. (1977). Studies of 1(3)c43hs1 a polyphasic, temperature-sensitive mutant of Drosophila melanogaster with a variety of imaginal disc defects. Dev. Biol. 55: 213-232. PubMed ID: 20604743

Oughtred, R., et al. (2002). Characterization of rat100, a 300-kilodalton ubiquitin-protein ligase induced in germ cells of the rat testis and similar to the Drosophila hyperplastic discs gene. Endocrinology 143(10): 3740-7. 12239083

van Tienen, L. M., Mieszczanek, J., Fiedler, M., Rutherford, T. J. and Bienz, M. (2017). Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9. Elife 6. PubMed ID: 28296634