Grunge/Atrophin


REGULATION

Targets of Activity

A genetic screen designed to isolate regulators of teashirt expression identified one such regulator as Grunge, a gene that encodes a protein with motifs found in human arginine-glutamic acid dipeptide repeat, Metastasis-associated-like and Atrophin-1 proteins. Grunge is the only Atrophin-like protein in Drosophila, whereas several exist in humans. Evidence exists that Grunge is required for the proper regulation of teashirt but also has multiple activities in fly development. (1) Grunge is crucial for correct segmentation during embryogenesis via a failure in the repression of at least four segmentation genes known to regulate teashirt. (2) Grunge acts positively to regulate teashirt expression in proximoventral parts of the leg. Grunge has other regulatory functions in the leg, including the patterning of ventral parts along the entire proximodistal axis and the proper spacing of bristles in all regions (Erkner, 2002).

To understand how loss of Gug activity affects segmentation, the expression of hunchback (hb), Krüppel (Kr), knirps (kni) and fushi tarazu (ftz) was analyzed in embryos derived from Gug35 germline clones fertilized by Gug35 sperm. In wild-type embryos, the expression of these segmentation gene products localizes to discrete domains in the early embryo. In almost all of the expression domains, loss of Gug activity increases the number of cells expressing these segmentation genes, suggesting that Gug plays a role in their repression. Later the expression of ftz displays a more complex defective pattern with some stripes being broader, and others narrower, than wild type (Erkner, 2002).

Loss of Gug activity also affects the distribution of the Tsh protein. In wild-type embryos at the germ band retraction stage, Tsh is expressed evenly in trunk segments and not the head or tail. In Gug embryos, Tsh is expressed in the trunk but is lost from ventral regions and is expressed in a striped pattern in the dorsal part of the embryo. These results suggest that Gug is a regulator of the tsh gene during embryogenesis (Erkner, 2002).

To analyse the function of the Gug locus in the leg, clones of cells homozygous for Gug35 were induced at different stages of development. Mutant Gug clones were found in all parts of the leg with a frequency similar to that of control Gug+ clones, showing that Gug+ function is not required for cell viability. Mutant and control clones were always restricted to the anterior or posterior compartment, and never changed the overall segmental identity of the legs. Differential behaviour of Gug clones is observed along the dorsoventral axis of the legs. Mutant cells located in dorsal or lateral parts of the leg give rise to essentially wild-type patterns, although they exhibit a slight cell autonomous increase in bristle density, compared with wild type (Erkner, 2002).

By contrast, Gug clones, which occupy any ventral part of the leg, delete specific pattern elements and replace them with patterns that resemble those formed in more lateral distal regions of the leg. Gug clones delete ventral-specific patterns in both the anterior and the posterior compartments. For example, the large ventral bristles of the posterior compartment in the femur of the first leg are not produced. The apical bristle at the distal tip of the anterior tibia, the spur bristles at the tip of each tarsal segment, and the transverse row and sex comb bristles of leg 1 never develop in such clones. Ventrally located Gug clones in posterior or anterior compartments fuse the femur to the tibia, which reflects a defect in the leg-specific morphogenetic process that separates these segments during pupation (Erkner, 2002).

Large Gug clones located in the coxa, trochanter or proximal femur, irrespective of their provenance in the anterior or posterior compartment, lead to fusion of these segments. Pattern elements, which are associated with clones and in neighboring cells, were replaced with those found in more distal parts of the legs. Gug clones in these proximal parts generally bear bracts, as do bristles located more distally. Clones situated in dorsal regions do not affect proximal identity. However, proximal clones, which occupy a large region of both the dorsal and ventral domains, replace all patterns with more distal identities and cause a reversal of the polarity of bristles. These Gug clones have a non-autonomous effect on the polarity of more distally located, ventral bristles. Smaller clones affect patterning if they are located ventrally. Such clones lead to outgrowths forming a partial new axis. Although bristles in these outgrowths show a distal (bracted) identity, they never form a complete new leg. Outgrowths consist of Gug and Gug+ tissue, suggesting that Gug activity is crucial for normal cell communication (Erkner, 2002).

Since Gug+ activity is required for the identity of proximal cells of the leg, a test was made to determine whether the expression of Tsh and Dll is affected in Gug clones. Tsh and Dll are expressed respectively in proximal and distal domains of the wild-type leg. Gug clones were identified by the absence of Myc epitope tag and Tsh expression was simultaneously monitored in third instar leg imaginal discs. In proximoventral positions, Gug activity is required autonomously and non-autonomously for the expression of Tsh in the leg imaginal disc. In dorsal or lateral positions, Tsh expression is not affected by loss of Gug activity in clones. In the peripodial membrane, which corresponds to the future body wall, Tsh does not depend on Gug+ activity, even ventrally. In late third instar discs, Dll is expressed ectopically in such outgrowths, consistent with the observation that lack of Gug+ function replaces proximal with distal cellular identities. Abnormal patterns of Dll expression were not observed in other parts of the legs. These experiments show that tsh and Dll expression depends directly or indirectly on Gug+ activity in the proximoventral leg, confirming the crucial role of Gug in ventroproximal patterning of the leg (Erkner, 2002).

Gug clones lead to outgrowths in the ventroproximal region in a non-autonomous manner. Wg is known to act in the patterning of ventral cells and Dpp acts in the patterning of dorsal positions. Loss of Wg and gain of Dpp signaling in any part of the ventral leg produces outgrowths similar to those described for Gug, specifically in the proximal ventral leg. wg-lacZ and dpp-lacZ expression were examined in Gug clones in the leg discs. When Gug clones produce outgrowths in proximal ventral positions, wg-lacZ expression is diminished and dpp-lacZ is expressed ectopically. In more distal leg parts or in proximal clones that lack outgrowths, Gug clones have no effect on the expression of wg-lacZ or dpp-lacZ. Similarly, no effect of loss of Gug activity is observed on the expression of Wg signaling target genes H15 and Dll or on the expression of the Dpp signaling target gene omb. It is concluded that even though Gug+ activity acts in the patterning of ventral cells of the leg, this effect is not due to a deregulation of the wg or dpp genes, except in a proximoventral position (Erkner, 2002).

Thus Gug determines the global identity of the proximal leg and acts as a positive regulator of tsh specifically in ventroproximal cells. Additionally, Gug activity is required along the entire proximodistal leg axis especially in ventral leg cells. Tsh also acts in the trunk segments of the embryo. Gug activity is required for the normal repression of four segmentation genes known to be required for regulation of tsh during embryogenesis (Erkner, 2002).

Loss of Gug activity severely affects the process of segmentation and the expression of segmentation genes when missing from the female germ line. At the blastoderm stage, most of the expression domains of hb, Kr, kni and ftz genes are expanded compared with wild type. These observations indicate that maternal production of Gug is crucial for the repression of these genes to precise domains in the early embryo. Gap proteins, including Hb, Kr and Kni are known to be required to restrict each others domains of expression. It will be interesting to test if Gug acts with these proteins for these repression activities (Erkner, 2002).

Loss of gap gene products and especially the pair rule product ftz affects the normal regulation of tsh. Ftz acts as a positive and probably direct regulator of tsh. Loss of Gug activity does not effect the location of Tsh to the trunk segments of the embryo but Tsh expression is affected (Erkner, 2002).

One striking feature of Gug+ function is its role in the formation of proximal specific patterns of the leg. Loss of Gug+ activity in proximal ventral cells changes bristle polarity and replaces proximal with more distal cellular identities. Thus, patterns typical of the coxa, trochanter and proximal femur are replaced with leg tissue that partially resembles that found in more distal femur or tibia. These effects resemble those seen in clones of cells lacking Extradenticle or Homothorax activities. Since Gug+ activity is also crucial for ventral patterning of the leg, the proximal-to-distal change is never complete. Gug mutant clones also affect cell communication in the proximal leg, because they exhibit cellular non autonomy causing neighboring wild-type tissue to differentiate distal patterns in proximal positions (Erkner, 2002).

The role of Gug in patterning the proximal leg is shown at the molecular level, where tsh requires positive input from the Gug gene specifically in ventral proximal parts of the leg imaginal disc. Loss of Gug results in ectopic expression of Dll in this position. Since Gug is ubiquitously produced in the leg, proximodistal specificity of Gug function presumably derives from other proteins located in proximoventral parts. Recently, it has been shown that Dll and possibly Tsh act as mutual repressors only in cells where Wg is signaling. Gug may normally be required for this process (Erkner, 2002).

Gug activity is essential for patterning the ventral parts of the leg along the entire proximodistal leg axis. Loss of Gug in dorsal or lateral parts has no drastic effect on patterning, although the number of bristles is augmented in Gug mutant cells irrespective of dorsoventral position (Erkner, 2002).

Ventrally in the femur-tibia region, loss of ventral Gug activity causes the fusion of these leg segments. During early pupariation, a sack of cells is known to give rise to the femur and tibia. Ventrally situated cells then migrate to meet and separate the femur and tibia. If Gug is missing in these migrating groups of cells, the femur and tibia remain attached. Gug mutant clones also affect the process of segmentation of the tarsus. Similar defects on the morphogenesis of the femur-tibia and tarsus have been observed in clones lacking components of the Notch signaling pathway. The relationship between Gug and Notch signaling activities will be reported elsewhere (Erkner, 2002).

The normal ventral patterning of the legs is specifically under the control of the Wg signaling cascade of molecules; thus, there is a correlation between the domains where Wg signaling occurs and where Gug is active. Furthermore, both Gug and Wg signaling act in domains where wg is transcribed and where Wg is secreted (for example, in the posterior ventral part of the leg) (Erkner, 2002).

Although Wg and Gug act in the same domains of the leg with similar roles, they exhibit distinct functions. Gug seems to act in a fraction of Wg-dependent developmental events. Initially, loss of Wg signaling induces a novel axis in ventral leg parts, irrespective of proximodistal position. Gug, however, induces bifurcated legs only if its activity is removed from proximal ventral parts of the leg. Contrary to the loss of Wg signaling, Gug mosaics do not distalize bifurcated legs properly, presumably because Gug activity is required for this process. Finally, Gug replaces proximal tissue with distal patterns; loss of Wg signaling never produces such homeosis. These observations suggest that Gug functions are related to those controlled by Wg signaling but are more specialized. This specialization may reflect the fact that Gug controls the expression of tsh, which is required to modulate Wg signaling activity (Erkner, 2002).

The conserved microRNA miR-8 tunes atrophin levels to prevent neurodegeneration in Drosophila

microRNAs bind to specific messenger RNA targets to posttranscriptionally modulate their expression. Understanding the regulatory relationships between miRNAs and targets remains a major challenge. Many miRNAs reduce expression of their targets to inconsequential levels. It has also been proposed that miRNAs might adjust target expression to an optimal level. This study analyzes the consequences of mutating the conserved miRNA miR-8 in Drosophila. Mutant miR-8 was generated by homologous replacement. atrophin was identified as a direct target of miR-8. miR-8 mutant phenotypes are attributable to elevated atrophin activity, resulting in elevated apoptosis in the brain and in behavioral defects. Reduction of atrophin levels in miR-8-expressing cells to below the level generated by miR-8 regulation is detrimental, providing evidence for a 'tuning target' relationship between them. Drosophila atrophin is related to the atrophin family of mammalian transcriptional regulators, implicated in the neurodegenerative disorder DRPLA. The regulatory relationship between miR-8 and atrophin orthologs is conserved in mammals (Karres, 2007).

These findings suggest that misregulation of atrophin is an important factor contributing to the defects associated with loss of miR-8 microRNA function. Atrophins are transcriptional corepressors, associated with histone deacetylase activity in Drosophila and in mammalian systems. Elevated expression of a transcriptional corepressor could be responsible for the extensive transcriptional changes observed in miR-8 mutants. Indeed, misregulation of transcription is thought to be a major cause of the neurodegeneration associated with trinucleotide repeat-expansion diseases, such as dentatorubral-pallidoluysian atrophy (DRPLA), which is caused by atrophin-1 (reviewed by Riley, 2006). RERE is the more similar of the two mammalian atrophins to Drosophila atrophin. RERE shares regulation by the miR-8-related miRNAs, miR-200b and miR-429, as well as the ability to recruit HDACs and serve as a transcriptional corepressor. In this context, it is of particular interest that HDAC inhibitors have been found to be effective against trinucleotide repeat-expansion cytotoxicity (Kariya, 2006; Karres, 2007 and references therein).

Vertebrates have multiple miR-8-related miRNAs: there are two in mammals and four in zebrafish. In zebrafish, two miR-8 family members are expressed in a subset of peripheral sensory organs, known as the lateral line. Overexpression of miR-200a or miR-200b results in impaired migration of the sense organ primordia and therefore fewer sense organs along the lateral line. One of the phenotypes observed in RERE mutant zebrafish is a reduced number of lateral-line sense organs (Asai, 2006). The resemblance between the consequences of overexpressing the miR-8 family members miR-200a and miR-200b and the RERE mutant phenotype is striking. It is therefore tempting to speculate that the regulatory relationship between atrophin and miR-8 might be conserved in zebrafish (Karres, 2007 and references therein).

miRNAs have been implicated in neurodegeneration caused by a polygutamine repeat-expansion disease model in Drosophila (Bilen, 2006). Loss of all miRNAs, using mutants that remove proteins required for miRNA biogenesis, caused increased sensitivity to polygutamine repeat-induced neurodegeneration. The findings of this study suggest that elevated expression of atrophin, resulting from loss of miR-8, is sufficient to cause CNS apoptosis and to impair performance in a behavioral assay. Although the climbing behavior seems deceptively simple; to perform it the fly must be able to determine which way is up, sense the position of its limbs, and coordinate its movements. Each of these depends on complex neural functions, and the flies' ability to perform them might be impaired as a consequence of the elevated CNS apoptosis. It is worth noting that reducing apoptosis suppresses the pupal eclosion defect of the miR-8 mutants, which may also have a motor coordination basis. It is tempting to interpret the age dependence of the impaired performance of miR-8 mutants in the climbing assay as a sign of progressive neurodegeneration resulting from to persistently elevated atrophin levels. It is noted that the effects in the mutant are likely concentrated in miR-8-expressing neurons and, so, do not affect all neural functions. Indeed, the mutant flies that survive beyond the first day after eclosion do not show the reduced lifespan associated with massive general neurodegeneration. The ability of mammalian miR-200b and miR-429 to downregulate RERE raises the possibility that they might limit HDAC activity. If so, mutations in miR-200b and miR-429 might be worth exploring as potential risk factors for DRPLA (Karres, 2007).

Recent computational and experimental studies have suggested that many miRNAs show essentially reciprocal patterns of expression to their target RNAs, with target RNA levels being naturally low in the miRNA-expressing cells or tissues. This has led to the proposal that the main role of many miRNAs is to reduce target-RNA expression to inconsequential levels. It has also been proposed that some miRNA-target relationships may be described as tuning, in which the miRNA buffers target expression levels. A critical distinction between the switch and tuning modes of regulation is that in switch mode the target is reduced to an inconsequential level, whereas in the tuning mode the remaining level of target expression must be required in the miRNA-expressing cell. This study has provided evidence that the relationship between miR-8 and atrophin constitutes an example of the latter type. Both atrophin and miR-8 are very broadly expressed during development, and the findings indicate that further reducing the expression of atrophin in miR-8-expressing cells causes developmental defects. Thus, the level of atrophin resulting from miR-8-mediated regulation can be seen as being optimal for these cells -- neither too high nor too low (Karres, 2007).

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, a very highly specific marker was used that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. The data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, it was shown that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, a targeted genetic screen was performed to identify players involved in ABCA specification. It was found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, it was shown that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third subesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification (Gabilondo, 2011).

The findings strongly suggest that the Capaergic abdominal ABCA neuron arises from NB 5-3. This conclusion is based on the expression in ABCA cells of gsb, wg and unpg, and the absence of the markers lbe(K) and hkb. However, even though gsb expression is known to be maintained specifically in the lineage of rows 5 and 6 NBs, whether expression of the other genetic markers used to identify NBs at stage 11 changes late in embryogenesis remains unanswered. Nonetheless, the specific combination of NB markers found in ABCA cells and their position in the hemineuromere are consistent with their birth from NB 5-3. Previous accounts showed that this NB gives rise to a lineage of 9–15 cells. Additionally, observations derived from studies in which PCD was blocked showed that NB 5-3 can potentially produce a large lineage (ranging from 19 to 27 cells), suggesting that it could generate 13 or 14 GMCs. The lack of a NB 5-3 specific-lineage marker prevented resolution of its complete lineage, and thus determining the birth order of the ABCA cell (Gabilondo, 2011).

Recent findings on the NB 5-6 and NB 5-5 demonstrate that cas and grh act together as critical temporal genes to specify peptidergic cell fates at the end of these lineages. cas mutants lack ABCA cells and Cas is expressed in these cells, while the normal pattern of ABCA cells is found in grh mutants, and Grh is not present in ABCA neurons. These data strongly support the birth of ABCA cells in a cas-only temporal window. This is different from the subesophageal Capaergic SECA cells, which while also arising from NB 5-3, show a reduction in cell number in both pdm and cas mutants, demonstrating birth at a mixed pdm/cas temporal window. Previous studies in other lineages have shown that when a temporal gene is mis-expressed, all progeny cells posterior to that temporal window can be transformed to the specific fate born at that particular temporal window. However, cas mis-expression failed at inducing ectopic ABCA cells, suggesting that cas in necessary but not sufficient to specify the ABCA fate (Gabilondo, 2011).

Programmed cell death (PCD) is a basic process in normal development. The results suggest that the ABCA and its sibling are equivalent cells committed to achieve the ABCA fate. First, it was shown that the ABCA-sibling cell dies by apoptosis, but produces an ABCA-like Capaergic neuron if PCD is inhibited. Second, when PCD is blocked, NB 5-3 also produces a GMC generating two ABCA-like Capaergic cells in the A5–A7 segments. These data indicate that a segment-specific mechanism prevents death of the ABCA cells in A2–A4 neuromeres. Segment specific cell death has been previously reported for the NB 5-3 lineage, and detailed studies on segment-specific apoptosis of other lineages have shown that this process is under homeotic control. In addition, the results show a different timing in the PCD undergone by the ABCA sibling and the ABCA cells born in A5–A7. This interpretation is based on the differential effect of p35 expression when cas-Gal4 or elav-Gal4 drivers were used. Although elav-Gal4 is transiently express in NBs and GMCs, robust and maintained driver expression commences in differentiating neurons. In contrast, cas expression starts in the NB and is maintained in the GMC and neuronal progeny. Therefore, the finding that death of the ABCA-sibling cell can be prevented by directing p35 with cas-Gal4, but not with elav-Gal4, suggests that the death of the ABCA sibling occurs earlier in development than the death undergone by the ABCA cells in A5–A7 segments (Gabilondo, 2011).

In the ABLK/LK peptidergic fate (derived from the NB 5-5), activation of Notch (N) signaling in the peptidergic cell prevents its death, while its sibling, NOFF cell undergoes apoptosis. On the contrary, in the EW3/Crz peptidergic fate (derived from the NB 7-3), silencing of N signaling is essential for the neuron survival, and therefore for it proper specification. The current results are in accordance with the last scenario, in which the ABCA cell is NOFF, and it sibling, which undergoes apoptosis, is NON. Therefore, Notch signaling must be switch off for the proper specification of the ABCA neuron (Gabilondo, 2011).

To search for genes involved in specification of the ABCA neural fate, a reduced set of mutants was screened of genes that are expressed in the embryonic CNS at stage 11, a time at which distinctly defined sublineages are being generated from all active NBs. Even though this method will certainly overlook important genes, the results reveal that it is in fact a very effective way to find genes involved in specification of a particular neural fate. Indeed, the ratio of success has been very satisfactory: 33.3% of the genes analyzed display a significant phenotype. Moreover, the set of identified genes could be further expanded by, for example, searching in interactome databases, and performing the subsequent screen on those putative interactors (Gabilondo, 2011).

It is assumed that the specification of a concrete cellular fate requires the combination of several transcription factors, namely a genetic combinatorial code. Recently, a detailed combinatorial code has been reported for three neuropeptidergic fates: ap4/FMRFa, ap1/Nplp1 and ABLK/Lk. However, very little is known about the specification of the rest of the 30 peptidergical fates. This study has identified several genes involved in the specification of the ABCA fate, which fit into three categories. First, genes were found whose loss-of-function produces a relevant increase of the number of ABCA cells. Most remarkable are the klu and rn phenotypes, which consist of duplications of the ABCA cells. These phenotypes suggest that these two transcription factors repress the ABCA fate in other neural cells (or/and NBs/GMCs). Interestingly, the normal phenotype of nab mutants indicates that, contrary to its mode of action in the wing, Rn does not work with the transcription cofactor Nab in this context (Gabilondo, 2011).

Second, genes were found whose loss-of-function produces a significant decrease of the number of ABCA neurons. In this category, the zhf2, ftz and grain phenotypes stand out. The effects of mutations on ftz are in agreement with its early role in segmentation: ftz is a pair-rule segmentation gene that defines even-numbered parasegments in the early embryo, and absence of ABCA cells was found in the A3 segment in ftz mutants. However, zfh2 and grain seem to be part of the specific combinatorial code of the ABCA cells. The Drosophila GATA transcription factor Grain has been reported to be involved in the specification of other cell fates, such as the aCC motoneuron fate. Based on its expression, the zinc finger homeodomain protein zfh2 has been proposed to mediate specification of the serotoninergic fate, but this has not been further demonstrated. Interestingly, during wing formation, zfh2 is required for establishing proximo-distal domains in the wing disc, and it does so partly by repressing gene activation by Rn. The opposite phenotypes that was observed in rn and zfh2 mutants suggest that similar interactions occur during ABCA specification. Analyses aimed to test this hypothesis are currently being performed (Gabilondo, 2011).

Third, two genes were found whose loss-of-function abolishes the ABCA fate: Grunge and hh. Grunge encodes a member of the Atrophin family of transcriptional co-repressors that plays multiple roles during Drosophila development. Taken together, studies from C. elegans to mammals suggest that Atrophin proteins function as transcriptional co-repressors that shuttle between nucleus and cytoplasm to transduce extracellular signals, and that they are part of a complex gene regulatory network that governs cell fate in various developmental contexts. Similarly, Hh is an extracellular signaling molecule essential for the proper patterning and development of tissues in metazoan organisms. It is noteworthy that two genes implicated in extracellular signaling pathways, Grunge and hh, are absolutely required for ABCA fate. Further studies will be needed to identify at which step/s they exert their actions, and to unravel possible interactions between them and with other players of the combinatorial code for ABCA specification (Gabilondo, 2011).

The corepressor Atrophin specifies odorant receptor expression in Drosophila

In both insects and vertebrates, each olfactory sensory neuron (OSN) expresses one odorant receptor (OR) from a large genomic repertoire. How a receptor is specified is a tantalizing question addressing fundamental aspects of cell differentiation. This study demonstrates that the corepressor Atrophin (Atro) segregates OR gene expression between OSN classes in Drosophila. The knockdown of Atro results in either loss or gain of a broad set of ORs. Each OR phenotypic group correlated with one of two opposing Notch fates, Notch responding, Nba (Non), and nonresponding, Nab (Noff) OSNs. The data show that Atro segregates ORs expressed in the Nba OSN classes and helps establish the Nab fate during OSN development. Consistent with a role in recruiting histone deacetylates, immunohistochemistry revealed that Atro regulates global histone 3 acetylation (H3ac) in OSNs and requires Hdac3 to segregate OR gene expression. It was further found that Nba OSN classes exhibit variable but higher H3ac levels than the Nab OSNs. Together, these data suggest that Atro determines the level of H3ac, which ensures correct OR gene expression within the Nba OSNs. A mechanism is proposed by which a single corepressor can specify a large number of neuron classes (Alkhorn, 2013).

Protein Interactions

Since the genetic data indicates that Grunge/Atro functions closely with eve and hkb, tests were performed for possible physical interactions using a GST pull-down assay. It was found that the full-length radiolabeled Atro can bind to a full-length Eve (GST-Eve) or Hkb (GST-Hkb), but not to GST alone (Zhang, 2002).

To map the domains within Atro responsible for these interactions, a series of Atro deletions were generated and the pull-down assay was performed. The results suggest that the C-terminal region of Atro (aa 1324-1985) is responsible for its binding to Eve, and further deletion in this region diminishes its binding ability. Interestingly, this C terminus Eve binding domain is highly conserved in the Atrophin family proteins, suggesting that this interaction might be evolutionarily conserved (Zhang, 2002).

To define the region in Eve responsible for its interaction with Atro, a series of GST-Eve deletions were generated. Previous work has divided the Eve protein into six regions (regions A-F). It was found that neither Eve's homeodomain (region B) nor the EF region shows significant interaction with Atro. Instead, only the CD region of Eve binds to the Atro protein. Further deletion of either region C or D significantly reduces its binding ability to Atro. This CD region has been previously defined as Eve's minimal repressor domain (Han, 1993), suggesting that the minimal repression domain in Eve functions to bind Atro (Zhang, 2002).

The binding data, along with the genetic data, suggest a possible mechanism in which Atro functions as a corepressor for site-specific repressors like Eve and Hkb. One function of Eve and Hkb might be to recruit Atro to the promoter site where Atro can exert its repressive activities. This hypothesis would predict that Atro can directly repress transcription when it is tethered to DNA via a heterologous DNA binding domain. To test this, an Atro-GAL4 fusion was generated and its function was examined in the Kreggy/NEE-LacZ system. Briefly, full-length Atro was fused to the C terminus of the Gal4 DNA binding domain (Gal4DB::Atro), and the chimeric gene was placed under the control of the Kruppel promoter (Kr-Gal4DB::Atro), which drives gene expression in a broad band in the central region of the blastoderm-stage embryo. The LacZ reporter gene (UAS-NEE)-LacZ, which is driven by a modified rhomboid NEE enhancer that contains three UAS sites for Gal4 binding, is normally expressed in the ventral side of the same stage embryos. However, when the (UAS-NEE)-LacZ flies are crossed with the Kr-Gal4DB::Atro transgenic animals, their progeny show a repressed LacZ transcription in the central region where the Gal4DB::Atro fusion protein is expressed. This result suggests that the full-length Atro protein can behave as a transcriptional corepressor in vivo (Zhang, 2002).

Given the sequence similarity between Atro and human Atrophin-1, it is possible that human Atrophin-1 also functions as a transcriptional corepressor in vivo. Thus, this possibility was tested using the Kreggy/NEE-LacZ system in fly embryos. Interestingly, when tethered to Gal4 DNA binding domain, human Atrophin-1 can repress LacZ transcription in (UAS-NEE)-LacZ reporter embryos, since more than 75% of the examined embryos exhibited a repressed LacZ transcription, suggesting that the function of Atrophin family proteins are evolutionarily conserved. Human Atrophin-1 was further tested with a poly-Q expansion in the same system and it was found that only about 18% of the examined embryos displayed a repressed LacZ transcription, suggesting that poly-Q expansion alters Atrophin's transcriptional repressive activity (Zhang, 2002).

The tumor-suppressor and cell adhesion molecule Fat controls planar polarity via physical interactions with Atrophin, a transcriptional co-repressor

Fat is an atypical cadherin that controls both cell growth and planar polarity. Atrophin (Grunge) is a nuclear co-repressor that is also essential for planar polarity; however, it is not known what genes Atrophin controls in planar polarity, or how Atrophin activity is regulated during the establishment of planar polarity. Atrophin is shown to bind to the cytoplasmic domain of Fat and Atrophin mutants show strong genetic interactions with fat. Both Atrophin and fat clones in the eye have non-autonomous disruptions in planar polarity that are restricted to the polar border of clones and that there is rescue of planar polarity defects on the equatorial border of these clones. Both fat and Atrophin are required to control four-jointed expression. In addition mosaic analysis demonstrates an enhanced requirement for Atrophin in the R3 photoreceptor. These data suggest a model in which fat and Atrophin act twice in the determination of planar polarity in the eye: first in setting up positional information through the production of a planar polarity diffusible signal, and later in R3 fate determination (Fanto, 2003).

Mosaic analysis of ft mutant clones demonstrates a strong bias for the cell that retains ft function to become the R3 cell. This has been interpreted to indicate that Fat directly biases the cell to become an R3 cell. Atro clones also show a bias for the R3 cell to retain Atro function, supporting the model that Atro, like Fat, works in R3 fate determination (Fanto, 2003).

However, an extensive mosaic analysis found that all anterior cells (R1, R2 and R3) tend to be ft+, and all posterior cells (R4, R5 and R6) tend to be ft-. It has been suggested that this bias is due to spatial considerations, cells that are polar in the precluster, undergo a 90° rotation, leaving them in a posterior position in the adult. At the polar border of ft clones, ommatidia rotate in the opposite direction to wild type, therefore the bias is reversed, leading to an increase in ft- anterior cells. Therefore, it as been concluded that additional data are required to show that ft function is specifically needed in R3. To determine if ft and Atro are specifically required in the R3 photoreceptor, mosaic analysis of Atro, ft and wild-type clones has been undertaken. In wild-type clones, marked only by white, ommatidia at the polar border of the clone show a weak preference for posterior photoreceptors to be wild-type. This bias is strictly due to the spatial constraints of recruitment in clones. In ft clones, it has been found that at the equatorial border (where polarity is unaltered) there is no discernable difference between anterior photoreceptors subclass types; 85% of all photoreceptors that retain ft are anterior class. The increase from ~61% to 85% is probably due to the adhesive properties of ft, which result in smooth edged clones (Fanto, 2003).

By contrast, at the polar border, which is where planar polarity is altered, there is a marked tendency for ft function to be retained specifically in the R3 photoreceptor; 100% of R3 cells retained ft, whereas only 83% of all anterior photoreceptors retained ft. Mosaic analysis of Atro clones shows that the bias introduced by planar polarity (PP) alterations at the polar border of clones (similar to ft) introduces a general bias for anterior photoreceptors. However, again, the bias is stronger in the R3 cell than for other anterior photoreceptors. This increased bias in the R3 photoreceptor over the other anterior class member suggests that ft and Atro are important in R3 fate (Fanto, 2003).

The conclusion that Atro function is important for the R3 cell fate is also strongly supported by the observation that loss of Atro often results in symmetric ommatidia with two R4 cells. This is reflected by the increase in R4/R4 ommatidia seen in Atro clones in the eye disc marked by expression of the R4 marker, md-lacZ. In addition the overexpression of Atro in R3 and R4 generates symmetric ommatidia with two R3 cells. Together, these data support the proposal that Atro is needed for the R3 fate (Fanto, 2003).

The non-autonomous nature of the PP defects associated with ft and Atro mutant clones could have presented some problems to mosaic analysis. It might be expected that non-autonomous alterations in polarity would equally affect all photoreceptors, yet the data clearly show enhanced requirements for ft and Atro function in the R3 photoreceptor over other photoreceptors. In addition, the proposal that Atro is needed for the R3 cell fate is supported by analysis of the R4 marker in eye discs. Interestingly, the tendency to lose the R3 cell fate in Atro clones is seen throughout the clone, and does not appear to participate in the phenomena of equatorial rescue or polar nonautonomy (Fanto, 2003).

Because Frizzled (Fz) is also needed for R3 fate decisions, it has been suggested that Fat positively affects Fz signaling. The observation that Atro acts with Fat and also biases towards the R3 fate suggests that the regulation of Fz by Fat may not be direct. It is proposed instead that Atro is necessary for the ft-dependent bias to an R3 cell fate and for the production of a diffusible PP molecule that controls Fz activity (Fanto, 2003).

The proposal that Fat increases Fz activity, and thereby biases a cell towards the R3 fate, does not explain the non-autonomous disruptions of wild-type tissue on the polar side of ft and Atro clones, or the rescue of ft and Atro mutant tissue from wild-type tissue on the equatorial side of the clone. There are several models that could explain the non-autonomous disruptions of planar polarity. One model suggests that planar polarity is established through a 'domino effect'. This model is suggested by the striking accumulation of planar polarity components, such as Fz and Dsh on the distal edge of every cell in the wing. This observation, coupled with genetic data that suggest that high Fz activity on one side of the cell forces low Fz activity on the other side, leads to a model in which accumulation or loss of polarity in a cell leads to templating of that state onto the next cell, non-autonomously propogating PP defects. However in the eye, Fz and Dsh show differential distribution on only a subset of ommatidial precursor cells, and, importantly, intervening cells show no altered accumulation. These data argue against a simple templating model for PP in the eye (Fanto, 2003).

An alternative model suggests that the juxtapositioning of ft+ and ft- tissue contributes to midline determination and emphasizes the role of Fat in inhibiting DV signaling away from the equator. This inhibition would be relieved at the equator by an unidentified molecule that would inhibit Fat function. If this model were correct, a small ft clone should mimic the situation at the equator, where Fat function is predicted to be locally inhibited. One would therefore be expected to find an ectopic equator in the middle of the clone. Instead, however, the opposite phenotype is found, since the ommatidia on the two sides of the clone point toward the middle of the clone, rather than away from it (Fanto, 2003).

The model that best explains both the equatorial rescue and polar nonautonomy of ft and Atro clones is that Fat and Atro together control expression of a planar polarity morphogen, here called 'factor X'. It is imagined that factor X is in a gradient with high levels at the equator and low levels at the poles, thus all ommatidia will appear to 'point' down this gradient. If Fat and Atro are essential to the production of factor X, then the ft/Atro mutant tissue will be void of factor X, producing a sink in the gradient. The gradient will still be pointing in the same direction initially, explaining the wild-type polarity of ommatidia at the equatorial side of the clone and 'equatorial rescue' seen in ft and Atro clones. For ommatidia at the polar edge of the clone, the gradient will be reversed, and ommatidia will point in the opposite direction. The gradient will also be disrupted outside of the clone, leading to inversions of the polarity of wild-type tissue on the polar side of the clone and 'polar nonautonomy' seen in these mutant clones. In large clones, there will be a region in the center of the clone where there is no detectable factor X, and as a result polarity will be randomized. All of these predictions are met in ft and Atro clones. Loss of Ds, which inhibits Fat function, should increase factor X. As predicted by this model, ds clones show disruptions in wild-type tissue on the equatorial side of the clone, and rescue of mutant tissue on the polar side of the clone. Without ft or ds function there would be no gradient and, consistent with this prediction, complete loss of planar polarity is seen in eyes that are homozygous for strong alleles of ft or ds (Fanto, 2003).

A gradient of Wg protein (which is high at the poles and low at the equator) initially establishes a gradient of Ds protein over the eye field. This gradient of Ds protein in turn produces a gradient of Fat activity, which, it is believed, creates a gradient of Atro activity. It is proposed that each cell will produce factor X at a level that is proportionate to the level of Atro activity, which varies according to the position of that cell in the ds and ft activity gradients. This model assumes that Factor X is a short-range diffusible molecule, which provides polarity information to ommatidial preclusters to direct their rotation. Since Fat has been shown to be upstream of Fz, it is speculated that the Atro-dependant Factor X is a ligand for Fz (Fanto, 2003).

Both ft and Atro also act in other, apparently unrelated, pathways. One of the prominent features of ft mutant larvae is the loss of growth control, which leads to dramatically overgrown discs and mutant clones that are markedly larger than their sister twin spots. However, Atro- clones do not display overgrowth in the eye, suggesting that ft restricts growth via an Atro-independent pathway. In addition, in the adult eye Atro clones (unlike ft clones) show severe defects in photoreceptor number and type, suggesting Atro has additional roles in photoreceptor specification and/or survival that are not shared by Fat. One particularly surprising result was the finding that Atro- clones are markedly smooth before the furrow, and that this smoothness is lost after the furrow passes. This suggests that Atro may function in a cell adhesion process that is lost upon cell differentiation (Fanto, 2003).

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominantly inherited neuronal degenerative disease characterized by the variable combination of ataxia, choreoathetosis, myoclonus, epilepsy and dementia. This disease is caused by the expansion of a polyglutamine tract within the Atrophin 1 protein. Atro is the sole fly homolog of human atrophins. Atro has been shown to act as a transcriptional co-repressor in vivo in Drosophila. Atro interacts genetically with even skipped, a transcriptional repressor, and is required for the in vivo repressive activity of even skipped. The transcriptional repressor activity of Atro has been localized to the highly conserved C-terminal region of Atro. This C-terminal region can bind to Even skipped in vitro and interacts with the minimal repression domain of Even skipped (Fanto, 2003).

This study has shown that the intracellular domain of Fat binds the C-terminal domain of Atro. The cytoplasmic expression of Atro and its interaction with Fat raises the possibility that instead of acting as a simple co-repressor, Atro functions in a more complex manner. Other transcriptional co-repressors are known to be converted to transcriptional activators upon cell signaling, and future work will determine if the interaction of Fat with Ds alters the transcriptional activity of Atro (Fanto, 2003).

Owing to the fact that Atro binds the cytoplasmic domain of Fat, a model is favored in which Atro acts downstream of Fat, possibly relaying a Fat-dependant signal to the nucleus. However, the similarity of the ft and atro loss-of-function phenotypes makes classical epistasis experiments difficult, therefore a model in which Atro acts upstream of ft cannot be excluded. Examination of the amount or subcellular distributions of Fat and Atro, suggest that Atro does not control Fat expression or localization, nor does ft control the levels or subcellular localization of Atro (Fanto, 2003).

Histone deacetylase-associating Atrophin proteins are nuclear receptor corepressors: Repression of knirps by Tll involves Atrophin

Drosophila Tailless (Tll) is an orphan nuclear receptor involved in embryonic segmentation and neurogenesis. Although Tll exerts potent transcriptional repressive effects, the underlying molecular mechanisms have not been determined. Using the established regulation of knirps by tll as a paradigm, it is reported that repression of knirps by Tll involves Atrophin, which is related to vertebrate Atrophin-1 and Atrophin-2. Atrophin interacts with Tll physically and genetically, and both proteins localize to the same knirps promoter region. Because Atrophin proteins interact with additional nuclear receptors and Atrophin-2 selectively binds histone deacetylase 1/2 (HDAC1/2) through its ELM2 (EGL-27 and MTA1 homology 2)/SANT (SWI3/ADA2/N-CoR/TFIII-B) domains, this study establishes that Atrophin proteins represent a novel class of nuclear receptor corepressors (Wang, 2006).

Since SMRT is a transcriptional corepressor for many NRs and SMRTER is the Drosophila cognate of SMRT, the first step in identifying Tll/Tlx-interacting corepressors was to test whether Tll and Tlx interact with SMRT and SMRTER. Using yeast two-hybrid assays, it was found that, whereas EcR, TR, and RAR interact with both SMRTER and SMRT, both Tll and Tlx fail to interact with SMRTER or SMRT (Wang, 2006).

To find potential corepressors of Tll/Tlx, a yeast two-hybrid screen was used, in which a Tll-expressing bait construct was deployed against a Drosophila embryonic library. A positive clone was identified, whose insert codes for the (1301-1966) region of Atro. This clone was selected for further investigation for several reasons: (1) In yeast, this clone also interacts strongly with chick and human Tlx, but not with RAR or TR; (2) Atro encodes a SANT domain, a RERE stretch, and an ELM2 domain; (3) Atro is a transcriptional corepressor of the Drosophila segmentation gene even-skipped; (4) two Atro-related proteins, Atr1 and Atr2, exist in vertebrates; and (5) Atr2 interacts with HDAC1 in mouse embryos. These properties of Atro proteins highlight the possibility that they are corepressors for Tll and Tlx (Wang, 2006).

To determine which region in Tll is required for Atro association, a series of truncated Tll expression constructs was prepared and their interactions with Atro were tested in yeast. The (192-452) region of Tll was found to be sufficient to mediate its interaction with Atro. Since this region of Tll harbors its ligand-binding domain (LBD), it suggested that an intact LBD is required for Tll to bind Atro. Indeed, no association between Tll variants lacking an intact LBD [e.g., Tll(33-161) or Tll(132-352)] and Atro could be detected (Wang, 2006).

A LBD-dependent interaction between Tll and Atro was further confirmed in human cells by using an immunostaining approach. CFP-tagged Atro (CFP-Atro) localizes to subnuclear regions when expressed in cells. This nuclear focal pattern of Atro resembles the nuclear pattern known for Atr2. Expressing Atro with Tll or Tlx in the same cells alters the nuclear distribution of Tll and Tlx: Both Tll and Tlx shift from their evenly distributed nuclear patterns to punctate nuclear patterns virtually identical to that displayed by CFP-Atro. Deleting the LBD from Tll and Tlx abrogates their localization to Atro-positive nuclear foci, confirming that Atro-Tll/Tlx interactions are mediated through the LBD of Tll and Tlx (Wang, 2006).

The regions in Atro responsible for Tll or Tlx interaction were mapped, using serial deletion Atro constructs. Two regions in Atro were found to mediate its interaction with Tll: Atro(965-1511) interacts weakly with Tll, whereas Atro(1711-1966) interacts strongly with both Tll and Tlx. The latter finding is of great interest, since the 1711-1966 region of Atro contains sequences conserved in the C-terminal regions of vertebrate Atr1 and Atr2. This correlation prompted an investigation of whether Atr1 and Atr2 interact with Tll or Tlx (Wang, 2006). Accordingly, two constructs expressing the C-terminal regions of Atr1 and Atr2 were generated and tested individually against Tll- or Tlx-expressing plasmids. As expected, both Atr1(846-1191) and Atr2(1224-1566), like Atro(1711-1966), interact strongly with Tll and Tlx in yeast, confirming that all Atro proteins are commonly targeted by Tll/Tlx (Wang, 2006).

Because the mapped Tll/Tlx-interacting regions in Atro, Atr1, and Atr2 share a stretch of highly conserved residues, whether mutations created within this region, which is called in this study the Atrobox, affect Tll/Tlx interaction was examined. Indeed, substitution of two leucine residues with alanine abolishes the interaction between Atro(1711-1966) and Tll or Tlx in yeast. Atro-Tll/Tlx interactions are, therefore, in part mediated through the Atro-box (Wang, 2006).

Tll/Tlx belong to the NR2 subfamily of the NR superfamily. The similarity shared by members of the NR2 subfamily suggests that additional NR2 proteins may interact with Atro proteins as well. This possibility was tested first with GST pull-down assays, in which several 35S-methionine-labeled NR2 and NR1 proteins were tested for their interactions with GST or GST-Atro fusion proteins. Atro proteins specifically bind Tll, Tlx, human chicken ovalbumin upstream promoter-transcription factor (COUP-TF), and Seven-Up1 (SVP1) (the Drosophila COUP-TF homolog), but not TRß and Ultraspiracle (USP). A similar interacting profile was observed between Atro proteins and COUP-TF or SVP1 in yeast. Therefore, Atro proteins do not interact with all NRs; rather, they preferentially bind a subset of NR2, including Tll, Tlx, SVP1, and COUP-TF (Wang, 2006).

Having demonstrated that Atro physically interacts with various NRs, the biological relevance of these interactions was examined. In this study, focus was placed on the in vivo relationship between Atro and Tll in flies by exploiting the known role of Tll in the segmentation process during Drosophila early embryogenesis. At this stage, Atro is expressed as a nuclear protein throughout the embryos. Consistent with previous observations that tll represses kni expression at the posterior end of the embryo, in situ hybridization for tll1 embryos shows a posterior expansion of kni stripe, especially in the ventral region of the embryos. Removal of zygotic Atro alone, as in the P-element excision line Atro35, does not cause such expansion, due to the presence of maternally deposited Atro. When maternal alone or both maternal and zygotic Atro are depleted using the dominant female sterile-FLP method, however, kni expression expands posteriorly in embryos. Because mutations of Atro and tll alter kni expression similarly, these in vivo observations suggest that Atro and tll are involved in overlapping transcriptional pathways (Wang, 2006).

To address the genetic interaction between tll and Atro further, advantage was taken of the hypomorphic nature of the tll1 allele, and it was asked whether the tll1-mediated phenotype is aggravated by additional Atro mutation. Specifically, whether the observed posterior expansion of kni stripe in tll1 embryos becomes more prominent when zygotic Atro is removed was investigated. Accordingly, a tll1, Atro35 double-mutant fly line was generated, in which both tll1 and Atro35 alleles were recombined to the same chromosome, and kni expression was tested in the resulting homozygous mutant embryos. Indeed, a further posterior expansion of kni stripe was observed in tll1, Atro35 double-mutant embryos, mimicking that found in tlle embryos. Therefore it is concluded that Atro is required for Tll to repress kni (Wang, 2006).

Since Atro is a binding factor of another terminal gap gene product, Huckebein (Hkb), the expression of kni was examined in hkb2 mutant embryos. No significant posterior expansion of kni was observed, therefore indicating that the repression of kni in the posterior-terminal region primarily results from the combined effect of Tll and Atro (Wang, 2006).

The genetic interaction between tll and Atro was further assessed by monitoring the expression of the pair-rule gene fushi tarazu (ftz) in the posterior region of the mutant embryos described above. In wild-type and in Atro35 zygotic mutant embryos, ftz is expressed as seven stripes in the central region. In tll1 embryos, however, the posterior stripes of ftz (mostly the fifth, sixth, and seventh stripes) shift toward the posterior end. In the most severely affected tll1 embryos, the seventh stripe of ftz is lost. This altered ftz pattern is known to be the consequence of cell fate changes, partly owing to the posterior expansion of kni, when tll is mutated. In tll1, Atro35 double-mutant embryos and in tlle embryos, additional loss of the sixth stripe of ftz was observed. Because the cell fate change is more pronounced in tll1, Atro35 double mutants than in tll1 or Atro35 embryos, it is concluded that Atro participates with Tll in determining posterior-terminal cell fates in early Drosophila embryos (Wang, 2006).

To verify the involvement of Atro in the regulation of kni by Tll at the chromatin level, chromatin immunoprecipitation (ChIP) assays were carried out for 0- to 4-h-old Drosophila embryos using Atro antibody, Tll antibody, and control IgG, respectively. The immunoprecipitated (IP) chromatin was subjected to PCR using primers corresponding to two separate regions, P1 and P2, in the kni gene, and a region in a randomly selected control (CG11562) promoter. In the kni promoter, P1 resides 2.5 kb upstream of the transcription initiation site and has a defined Tll-binding site. P2 corresponds to the 3' untranslated region of the kni gene, where no Tll-binding site is found (Wang, 2006).

In vivo ChIP assays revealed that both Atro and Tll antibodies, but not the control IgG, specifically precipitated chromatin that harbors the P1 site, but not chromatin containing P2 or the CG11562 promoter. These results establish that Atro, by forming protein complexes with Tll, is present naturally on the kni promoter (Wang, 2006).

Many transcriptional corepressors, including SMRT and N-CoR, are associated with HDAC activity. Because the results indicate that Atro proteins are corepressors of Tll/Tlx, the following was further investigated: (1) whether Atro proteins also show HDAC activity; (2) whether Atro proteins bind selected HDACs; and, if so, (3) which regions/domains in Atro proteins mediate their HDAC binding. To address these interconnected questions, fluorometric HDAC assays and Western blot analysis were performed on protein complexes immunoprecipitated by Flag-tagged Atro, Atr1, Atr2, or truncated Atr2 variants expressed in HEK293 cells. In parallel experiments, Flag and Flag-SMRT were used as a negative and a positive control, respectively. The expression of tested Flag fusion proteins was first examined using Western blot analysis (Wang, 2006).

As expected, Flag-SMRT is associated with potent HDAC activity that is sensitive to trichostatin A (TSA), an HDAC inhibitor. Robust levels of TSA-sensitive HDAC activity were also observed for both Atro and Atr2, confirming that both proteins' properties involve HDACs. surprisingly, Atr1 displays no prominent HDAC activity. Since Atr1 lacks the conserved ELM2 and SANT domains found in the N-terminal regions of Atr2 and Atro, it is suspected that the missing N-terminal region in Atr1 might be important for the HDAC activity of Atro proteins (Wang, 2006).

To determine whether the HDAC activity of Atro or Atr2 depends on its N-terminal region, the BAH (Bromo adjacent homology), the ELM2, and the SANT domains in this region of Atr2 were deleted sequentially. Note that the BAH domain is absent in Atro. Whereas Atr2DeltaBAH still exerts a robust level of HDAC activity, a dramatic reduction of HDAC activity was observed with Atr2DeltaBAH-ELM2. A further deletion of the SANT domain, Atr2DeltaBAH-ELM2-SANT, causes a complete loss of HDAC activity, indicating that both the ELM2 and SANT domains are central to Atr2's HDAC activity (Wang, 2006).

Next, which HDACs Atro proteins interact with was investigated, and whether Atr2's association with HDACs involves its ELM2/SANT domains. Protein complexes immunoprecipitated by Flag-tagged Atro proteins and Atr2 variants were examined by Western blot for a panel of potential associating proteins, including HDAC1, HDAC2, HDAC3, and Sin3A. Sin3A was not detected in any of the IP complexes. In contrast, a significant level of HDAC3 was precipitated along with SMRT. Although SMRT also interacts with HDAC1 or HDAC2, these interactions are considerably weaker. Conversely, abundant HDAC1 and HDAC2 (but only minimal HDAC3) are present in the protein complexes associated with Atr2. Similarly, Atro, but not Atr1, also precipitates HDAC1/2 specifically, indicating that Atro-family (except Atr1) and SMRT-family proteins display distinct preferences for different HDACs (Wang, 2006).

Consistent with HDAC assay results, removing the ELM2 domain or both the ELM2 and SANT domains from Atr2DeltaBAH impairs or disrupts its ability to associate with HDAC1/2. Given the fact that similar results were also obtained when the distribution of endogenous HDAC1 was examined in cells expressing different CFP-Atr2 variants, it is therefore concluded that the ability of Atr2 to exert HDAC activity and to recruit HDAC1/2 depends on its ELM2 and SANT domains (Wang, 2006).

In many respects, the transcriptional properties that discover in this study for Atro proteins parallel those found for SMRT, N-CoR, and SMRTER. (1) These two classes of corepressors share a SANT domain and RERE stretch; (2) they are conserved in vertebrates and in flies; (3) they bind NRs, albeit selectively, and (4) they associate with HDACs, also selectively. Additionally, Atr1, like SMRT and N-CoR, also interacts with ETO/MTG8, which is known to be a transcriptional repressor involved in acute myeloid leukemia. Considering that SMRT and N-CoR interact with a large number of NRs, with a wide variety of transcriptional factors, and also with type II HDACs, it is predicted that Atro proteins may have similar qualities as well. Therefore, more Atro-interacting factors still await discovery (Wang, 2006).

In the context of human diseases, it is known that polyglutamine expansion in human Atr1 causes DRPLA. It has been shown that Atr1 lacks HDAC activity, yet it binds Atr2 through their RERE stretches, and it associates with both Tlx and COUP-TF, two known NRs with key roles in CNS development and functioning. It is therefore proposed that mutant Atr1 may cause its pathological effects by interfering with the normal transcriptional properties of Atr2 and its associated nuclear receptors (Wang, 2006).

Drosophila Brakeless interacts with Atrophin and is required for tailless-mediated transcriptional repression in early embryos

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. Mutations were isolated in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, an expanded expression pattern was noted of the Krüppel (Kr) and knirps (kni) genes. Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. Both Drosophila and human Brakeless and Atrophin interact in vitro, and it is proposed that they act together as a co-repressor complex in many developmental contexts. The possibility is discussed that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA) (Haecker, 2007).

Repression plays a pivotal role in establishing correct gene expression patterns that is necessary for cell fate specification during embryo development. For example, in the early Drosophila embryo, repression by gap and pair-rule proteins is essential for specifying the positions of the 14 segments of the animal. The mechanisms by which transcriptional repressors delimit gene expression borders are not well understood. However, many repressors require co-repressors for function. In the Drosophila embryo, the CtBP and Groucho co-repressors are required for activity of many repressors. Atrophin has been identified as a co-repressor for Even-skipped and Tll. Still, co-regulators for several important transcription factors in the early embryo have not yet been identified. Therefore a screen was performed for novel maternal factors that are required for establishing correct gene expression patterns in the early embryo (Haecker, 2007).

From this screen, mutations were identified in the bks gene that cause severe phenotypes on gap gene expression and embryo segmentation. The Bks protein is evolutionarily conserved between insects and deuterostomes, but has not been characterized in any species except Drosophila, in which it has been shown to repress runt expression in photoreceptor cells and thickveins expression in wing imaginal discs. However, the molecular function of Bks has been unknown. This study shows that Bks interacts with the transcriptional repressor Tll, is recruited to target gene CRMs, and will repress transcription when targeted to DNA (Haecker, 2007).

Tll has been shown to utilize Atrophin as a co-repressor. Atrophin genetically interacts with Tll and physically interacts with its ligand binding domain. Atrophin binding is conserved in nuclear receptors within the same subfamily, such as Seven-Up in Drosophila as well as Tlx and COUP-TF in mammals. When expressed in mammalian cells, Drosophila Atrophin and mouse Atrophin-2 interact with the histone deacetylases HDAC1 and HDAC2. Histone deacetylation may therefore be part of the mechanism by which Atrophin functions as a co-repressor. Another recent report described genetic interactions among bks and atrophin mutants in the formation of interocellar bristles in adult flies. Furthermore, it was shown that atrophin mutants have virtually identical phenotypes as bks mutants, including de-repression of runt expression in the eye, thickveins expression in the wing, and Kr and kni expression in the embryo (Haecker, 2007).

Both proteins are recruited to the kni CRM, a Tll-regulated target gene, in the embryo. Importantly, Atrophin and Bks interact in vitro and that they can be co-immunoprecipitated from S2 cells. It is proposed that Bks and Atrophin function together as a co-repressor complex, and based on the similar bks and atrophin mutant phenotypes at several developmental stages, the complex may function throughout development. These results are compatible with the existence of a tripartite complex consisting of Tll, Bks, and Atrophin. Bks binding to Tll is enhanced by the Tll DNA binding domain, whereas the interaction of Tll with Atrophin is mediated through the C-terminal ligand binding domain. Tll may therefore simultaneously interact with Bks and Atrophin. Alternatively, Tll interacts separately with Bks and Atrophin on the kni CRM. In either case, both Bks and Atrophin are required for full Tll activity. However, at high enough Tll concentration, Bks activity is dispensable. Some bks embryos misexpressing Tll still repress kni expression, and overexpressing Tll from a heat-shock promoter can repress the posterior kni stripe in both wt and bks mutant embryos. For this reason, it is believed that Bks and Atrophin are cooperating as Tll co-repressors, so that Tll function is only partially impaired by the absence of either one. It was found that Tet-Bks-mediated repression in cells is insensitive to the deacetylase inhibitor trichostatin A (TSA). It is possible, therefore, that whereas Atrophin-mediated repression may involve histone deacetylation, Bks could repress transcription through a separate mechanism (Haecker, 2007).

These results have not revealed any differences between the molecular functions of the two Bks isoforms. Both Bks-A and Bks-B repress transcription when tethered to DNA, and the sequences that mediated binding to Tll and Atrophin are shared between the two isoforms. However, the bks339 allele that selectively affects the Bks-B isoform causes a weaker, but comparable phenotype to the stronger bks alleles that disrupt both isoforms. Therefore, the C-terminus of Bks-B provides a function that is indispensable for embryo development and regulation of kni expression. This part of Bks-B contains two regions (D3 and D4) that are highly conserved in insects and loosely conserved in deuterostome Bks sequences, but does not resemble any sequence with known function. The only sequence similarity to domains found in other proteins is a single zinc-finger motif in Bks-B. Preliminary results indicate that the zinc finger in isolation or together with the conserved D2 domain does not exhibit sequence-specific DNA binding activity. Indeed, multiple zinc fingers are generally required to achieve DNA binding specificity. Instead, Bks is likely brought to DNA through interactions with Tll and other transcription factors (Haecker, 2007).

Atrophins are required for embryo development in C. elegans, Drosophila, zebrafish, and mice. In vertebrates, two atrophin genes are present. Atrophin-1 is dispensable for embryonic development in mice, and lacks the N-terminal MTA-2 homologous domain that interacts with histone deacetylases . However, the homologous C-termini of Atrophin-1 and Atrophin-2 can interact, and it was found that this domain can also bind to the human Bks homolog ZNF608. Atrophin-1 interacts with another co-repressor-associated protein as well, ETO/MTG8, and can repress transcription when tethered to DNA. These data are consistent with the emerging view that deregulated transcription may be an important mechanism for the pathogenesis of polyglutamine diseases. Recent evidence indicates that interactions with the normal binding partners may cause toxicity of polyglutamine-expanded proteins such as Ataxin-1 . It will be interesting to investigate whether the interaction between human Bks homologs and Atrophin-1 is important for the neuronal toxicity of polyglutamine-expanded Atrophin-1 (Haecker, 2007).

Atrophin-Rpd3 complex represses Hedgehog signaling by acting as a corepressor of CiR

The evolutionarily conserved Hedgehog (Hh) signaling pathway is transduced by the Cubitus interruptus (Ci)/Gli family of transcription factors that exist in two distinct repressor (CiR/GliR) and activator (CiA/GliA) forms. Aberrant activation of Hh signaling is associated with various human cancers, but the mechanism through which CiRGliR properly represses target gene expression is poorly understood. This study used Drosophila and zebrafish models to define a repressor function of Atrophin (Atro) in Hh signaling. Atro directly binds to Ci through its C terminus. The N terminus of Atro interacts with a histone deacetylase, Rpd3, to recruit it to a Ci-binding site at the decapentaplegic (dpp) locus and reduce dpp transcription through histone acetylation regulation. The repressor function of Atro in Hh signaling is dependent on Ci. Furthermore, Rerea, a homologue of Atro in zebrafish, represses the expression of Hh-responsive genes. It is proposed that the Atro-Rpd3 complex plays a conserved role to function as a CiR corepressor (Zhang, 2013).


DEVELOPMENTAL BIOLOGY

Embryonic

RNA in situ and Northern analysis have revealed that a single Atro transcript is expressed throughout embryonic development, including 0- to 2-hr-old embryos, indicating a maternal contribution. Immunostainings with anti-Atro antibodies reveal that in early-stage embryos, Atro protein was ubiquitously expressed in every cell, while in later-stage embryos the protein was expressed at a higher level in the nervous system. Atro is also expressed in all larval tissues examined, including brain and imaginal discs. Interestingly, the Atro protein was mainly located within the nucleus in a punctuate form (zhang, 2002). .

Effects of Mutation or Deletion

Animals homozygous for the P element insertions or e46-2 alleles died at a late embryonic stage with no obvious morphological defects. Since defects caused by Atro could be masked by its maternal contribution, mutant embryos lacking maternal Atro products (Atromat-) were generated using the FLP-DFS technique. Among the embryos produced from Atromat- mothers and Atro-/+ fathers, those lacking both maternal and zygotic Atro gene product did not develop, whereas those maternally mutant but zygotically rescued embryos displayed a range of patterning defects. To understand the function of Atro in a greater detail, the role of Atro was further investigated in early embryonic patterning where many molecular markers are available (Zhang, 2002).

Cuticle preparations showed that some Atromat- embryos have widened ventral denticle belts in the dorsal/ventral direction. Using an antibody against Twist, a protein expressed in the ventral-most 20 cells of stage 5 wild-type embryos, it was found that Twist expression becomes broader and covers most of the ventral cells in Atromat- embryos. These data indicate that Atromat- embryos are ventralized and suggest a requirement for Atro in dorsoventral patterning (Zhang, 2002). .

In addition, cuticle preparations also showed that many of these embryos have holes or patches of naked cuticle. Such phenotypes have been observed in animals mutant for neurogenic genes in which the hyperplasia of neuronal tissues at the expense of epidermis leaves an insufficient amount of epidermal cells to cover the whole embryo. To test whether Atromat- embryos exhibit neurogenic defects, the mutant embryos were stained with antibodies against Elav, which marks both the central and peripheral nervous system. The number of Elav-positive cells is dramatically increased in the Atromat- embryos, suggesting a role for Atro in the neurogenic process as well (Zhang, 2002).

Anti-Elav staining also revealed that the number of metameric neuronal units in the Atromat- embryos is reduced, suggestive of a simultaneous segmentation defect. Indeed, even in mutant embryos with less severe neurogenic defect, the missing and misshaped ventral denticle belt phenotype is evident. To further study the segmentation phenotype, the expression of engrailed (en), a segment-polarity gene, which in wild-type embryo is expressed as 14 stripes at the anterior boundary of each parasegment, was examined. In Atromat- embryos, the number of en stripes is reduced, and several of those remaining en stripes are incomplete or fused together. Such a segmentation defect was also confirmed by examining the expression of another segment-polarity gene, wg. Taken together, these results indicate that maternal Atro is essential for proper embryonic pattern formation (Zhang, 2002).

Because the expression of segment-polarity genes is defined by the pair-rule genes, it was possible that the abnormal en and wg expression patterns are a result of aberrant pair-rule gene regulation caused by the Atro mutations. Therefore, the expression patterns of pair-rule genes were examined in Atro mutant embryos. In wild-type embryos, the pair-rule genes (such as eve, hairy, runt, and ftz) are expressed as seven precisely defined stripes. However, in Atromat- embryos, these stripes are expanded and their boundaries become less defined. Of special note is the shift in expression of the pair-rule genes into the posterior region in the Atromat- embryos, resembling the phenotype observed in embryos with mutations in the terminal gap gene hkb (Zhang, 2002).

Since the stripe boundaries of the pair-rule genes are restricted by the repressive activities of gap genes, gap gene expression was further examined in the Atromat- embryos. The expression patterns of the four gap genes examined, including hb, kni, kr, and giant, are nearly normal as compared to the wild-type embryos. This result suggests that the repressive activities of the gap genes, but not their expression, might be compromised in the Atromat- embryos. Taken together, these findings suggest that Atro might be required for the repressive activities of multiple transcription factors during embryonic segmentation (Zhang, 2002).

The above results suggest that the maternal Atro is essential for proper embryo pattern formation. To further understand the function of Atro in embryonic development, possible genetic interactions were sought between Atro and genes involved in early embryogenesis. Heterozygous Atro-/+ flies were crossed to flies carrying heterozygous mutations affecting early embryogenesis, and the number of transheterozygous progeny from these crosses was scored. While most mutants tested did not display obvious genetic interactions, mutations affecting the eve and hkb transcriptional repressors showed a dosage-sensitive interaction with maternal Atro. From crosses of heterozygous Atro-/+ females and eve-/+ males, almost all of the eve-/+ progeny (both eve-/+; Atro-/+ and eve-/+; Atro+/+) were absent. In contrast, in the reciprocal crosses where Atro-/+ males were mated with eve-/+ females, a normal percentage of eve-/+ progeny was observed. These data showed that the lethality is caused by a reduction of maternal Atro dosage. Similar interactions were also observed with hkb. These findings suggested that Atro might mediate the functions of eve and hkb repressors in vivo (Zhang, 2002).

The eve-/+; Atro-/+ double heterozygous embryos produced by Atro-/+ mothers die at a late embryonic stage. These embryos lose some or all of the even-numbered ventral denticle belts, mimicking the phenotype of hypomorphic eve mutant embryos. However, staining of these embryos with an Eve antibody reveal that the expression of Eve is normal, indicating that it is the activity of eve that is affected by the reduced Atro dosage. To confirm this, the expression patterns of two eve target genes, wg and en were analyzed. In wild-type embryos, the wg expression stripe is mostly one-cell wide, with only the even-numbered wg expression restricted by eve. In the eve-/+; Atro-/+ double heterozygous embryos, while the odd-numbered wg stripes maintain their one-cell width, there is considerable anterior expansion of the even-numbered wg stripes, indicating that the repressive activity of eve is compromised. Similarly, in wild-type embryos, en is expressed as 14 evenly spaced stripes in the embryo trunk region. Although both the odd- and the even-numbered en expression stripes are regulated by eve, a higher level of eve's repressive activity is required to define the odd-numbered en stripes, while a low level of eve's activity is sufficient for the even-numbered ones. In the eve-/+; Atro-/+ double heterozygous embryos, the odd-numbered en stripes are expanded posteriorly, resembling phenotypes observed in homozygous hypomorphic eve mutant embryos. Taken together, these results demonstrate that Atro is important for the repressive activity of eve and suggest that these two genes might function closely in the segmentation pathway (Zhang, 2002).

Grunge is required for patterning of the ventral proximal leg; in mutant cells proximal patterns are replaced with more distal ones. Gug is also required in all ventrally located cells along the entire proximal-distal axis of the leg for specific ventral identities or processes (Erkner, 1997).

A screen for genes that interact with the Drosophila pair-rule segmentation gene fushi tarazu identifies Atrophin/Grunge

fushi tarazu is expressed at the blastoderm stage in seven stripes that serve to define the even-numbered parasegments. ftz encodes a DNA-binding homeodomain protein and is known to regulate genes of the segment polarity, homeotic, and pair-rule classes. Despite intensive analysis in a number of laboratories, how ftz is regulated and how it controls its targets are still poorly understood. To help understand these processes, a screen was conducted to identify dominant mutations that enhance the lethality of a ftz temperature-sensitive mutant. Twenty-six enhancers were isolated, that define 21 genes. All but one of the mutations recovered show a maternal effect in their interaction with ftz. Three of the enhancers proved to be alleles of the known ftz protein cofactor gene ftz-f1, demonstrating the efficacy of the screen. Four enhancers are alleles of Atrophin (Atro), the Drosophila homolog of the human gene responsible for the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Embryos from Atro mutant germ-line mothers lack the even-numbered (ftz-dependent) engrailed stripes and show strong ftz-like segmentation defects. These defects likely result from a reduction in Even-skipped (Eve) repression ability, since Atro has been shown to function as a corepressor for Eve. In this study, evidence is presented that Atro is also a member of the trithorax group (trxG) of Hox gene regulators. Atro appears to be particularly closely related in function to the trxG gene osa, which encodes a component of the brahma chromatin remodeling complex. One additional gene was identified that causes pair-rule segmentation defects in embryos from homozygous mutant germ-line mothers. The single allele of this gene, called bek, also causes nuclear abnormalities similar to those caused by alleles of the Trithorax-like gene, which encodes the GAGA factor (Kankel, 2004).

Four of the ftz enhancers isolated in the screen proved to be alleles of Atrophin (Atro). Polyglutamine tract expansion within one of the human homologs of Atro (Atrophin-1) causes the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Humans possess at least one additional Atrophin family member, Atrophin-2, which encodes a protein that can heterodimerize with Atr1. The functions of the mammalian Atrophin proteins are not well characterized. However, a role in gene repression seems likely, because Atr1 binds Eto1, a corepressor complex component, and overexpression of Atr1 can repress transcription of a reporter gene in tissue culture cells. In addition, Atr2 has been shown to interact with the histone deacetylase Hdac1. Compelling evidence has been presented that Atro also functions as a corepressor in Drosophila. eve mutations show strong dominant lethality when crossed to mothers heterozygous for Atro alleles. In the eve/+; Atro/+ embryos produced in this cross, odd-numbered en stripes are expanded, suggesting a weakening in the ability of Eve to repress paired, runt, or sloppy-paired (other pair-rule genes involved in specifying these stripes). Atro binds to the minimal repression domain of Eve, and artificial recruitment of Atro to a target gene can cause repression in vivo. A failure in the repressive activity of Eve may account for the absence of even-numbered en stripes described for embryos from Atro mutant germ-line mothers. In normal development, the even-numbered en stripes form as a result of differential repression of ftz and odd-skipped (odd) by Eve. Ftz is an activator of en, whereas Odd is a repressor. The even-numbered en stripes form where odd, but not ftz, has been repressed by Eve. If there were a failure of Eve to repress odd, zones expressing ftz but not odd would not form, and the even-numbered en stripes would not be established. Exactly this mechanism appears to be responsible for a reduction in even-numbered en stripes in mutants for the Rpd3 histone deacetylase. However, it is also possible that the even-numbered en stripes fail to appear in Atro- embryos because of a defect in the ability of Ftz to activate en. It is important to note that the odd-numbered en stripes are established almost normally in Atro mutant embryos (although they are wider than normal). These stripes are thought to be defined by differential repression of sloppy-paired, runt, and paired by Eve; the presence of these stripes in Atro- embryos indicates that Atro is not required for all repressive activities of Eve (Kankel, 2004).

Although the atrophin proteins have largely been viewed as dedicated corepressors, the current results indicate that Atro also functions in a positive fashion. Atro is a member of the trxG of Hox gene positive regulators. Mutations in trxG genes enhance the phenotypes of loss-of-function alleles of the Hox genes and suppress the Hox gain-of-function phenotypes caused by mutations in Polycomb group genes. In otherwise wild-type backgrounds, trxG mutations also often cause weak transformations similar to those caused by Hox gene loss of function. Consistent with these effects, Atro mutations enhance haltere-to-wing transformations in Ubx heterozygotes, anteriorly directed transformations of the posterior abdominal segments in BX-C deficiency [Df(3R)P9] heterozygotes, and T1-to-T2 leg transformations in Df(3R)Scr heterozygotes. They also suppress the effects of the PcG gene Pcl and enhance the effects of the trxG mutations brm and trx. Finally, somatic clones homozygous for Atro alleles show partial transformations of the fifth and sixth abdominal segments to the anterior, transformations that are likely caused by loss of expression of the Abd-B gene of the BX-C (Kankel, 2004).

The genes of the trxG play diverse roles in promoting the transcription of the Hox genes and other loci. Several encode components of chromatin-remodeling complexes that function to render genes more accessible to activators or to facilitate their transcription. One of the best-characterized trxG genes is brahma (brm), which encodes a Drosophila homolog of the yeast SWI2/SNF2 protein. SWI2/SNF2 is the catalytic DNA-stimulated ATPase subunit of a large multiprotein complex that functions in chromatin remodeling. Brm is also part of a large protein complex, which consists of a core of 10 tightly associated proteins as well as several loosely associated factors. This complex can function in vitro to alter nucleosome spacing and to enhance transcription. Three of the Brm complex proteins are encoded by trxG genes (brm, osa, and mor) and four (Brm, Snr1, BAP155, and BAP60) are conserved in the yeast remodeling complexes SWI/SNF and RSC. The proteins of the Brm complex show a surprising degree of functional specialization: mutations in some show strong trxG phenotypes, whereas others (e.g., snr1 show no clear homeotic phenotypes. Although the Brm complex is primarily involved in gene activation, it also functions in repression, particularly for genes that are targets of wingless signaling. At least two other trxG protein complexes are known, one containing Ash1 and the other Ash2. Ash1 functions as a multifunctional histone methyl transferase whose activity may recruit Brm complexes to target genes. The function of Ash2 is not yet known (Kankel, 2004 and references therein).

Atro appears particularly closely related to the Brm complex in function. The parallels between Atro and the Brm complex component Osa are the most striking, as both proteins appear to be intimately involved in regulating wingless targets. osa encodes a subunit present in some, but not all, Brm complexes. Osa-containing brahma chromatin-remodeling complexes are required for the normal expression of several wg targets, including dpp, Dll, nubbin, en, and the UbxB midgut enhancer. In each case, loss of osa function causes ectopic expression of the target, indicating that osa is required for the normal repression of these targets. brahma (brm) and moira (mor), which encode other components of the Brm complex, are also required for this repression, at least for nubbin and the UbxB enhancer. Although Atro has not been as well studied, the current results suggest that Atro is also involved in wg target regulation. In the leg, wg specifies ventral characteristics. Atro- clones located ventrally in the leg show numerous pattern deletions, whereas clones located dorsally develop almost normally. In the proximo-ventral portion of the leg, some Atro- clones show ectopic expression of dpp or Dll, both targets of wg signaling that also require osa for their normal repression. The EGL-27 protein, which shows sequence similarity with the N-terminal portion of Atro, may function similarly, as it is required for normal Wnt signaling in C. elegans (Kankel, 2004 and references therein).

There are additional similarities between Atro alleles and Brm complex mutations. Atro alleles cause ectopic wing venation and bristle defects in homozygous clones; similar phenotypes are caused by loss-of-function of the Brm complex genes brm, osa, and snr1. Atro alleles suppress the antenna-to-leg transformation caused by AntpNs, in which expression is driven by the endogenous Antp P2 promoter, but not Antp73b, in which expression is driven by the promoter of a non-Hox gene. The same promoter specificity is shown by alleles of brm, mor, and osa. Although no clear pair-rule modulation has been described for Brm complex members, severe segmentation defects are seen in embryos from osa homozygous germ-line mothers and from mothers heterozygous for two partially complementing alleles of brm. For embryos from osa homozygous germ-line mothers, expression of gap gene proteins is normal, but the pair-rule stripes of eve are abnormal. Gap gene expression also appears normal in embryos from Atro homozygous germ-line mothers, but expression of eve and other pair-rule genes is abnormal (Kankel, 2004).

The similar effects of Atro alleles and of mutations affecting Brm complex subunits suggest a close functional relationship. However, Atro could be one of the high-molecular-weight Brm complex components or a core component of another Brm-like complex that has yet to be characterized. Consistent with the latter possibility, the N-terminal portion of Atro shares homology with the N-terminal portion of mammalian metastasis-associated protein 2 (Mta2), a component of the NURD chromatin-remodeling complex. NURD has nucleosome remodeling and histone deacetylase activity. The N-terminal regions of Atr2 and EGL-27 also contain Mta2 homologous regions, and in Atr2 this region has been shown to interact with Hdac1, suggesting that Atr2 may be part of a novel histone deacetylase complex. These observations suggest that Atro might also be part of a chromatin-modifying complex (Kankel, 2004).

Although Atro has been considered only as a transcriptional repressor, the finding that Atro is a member of the trithorax group indicates that it also plays a positive role. In previous reports, defects in the ftz striping pattern in Atro- embryos have been interpreted as resulting from a failure in the repression ability of segmentation gene products. However, the current results suggest these defects could result from a failure in stripe activation. The ftz striping pattern in Atro-mutant embryos bears a striking resemblance to the ftz pattern in embryos lacking the pair-rule gene runt (run) or the maternal gene caudal (cad). In run- embryos ftz stripes 2-6 look essentially the same as in Atro mutant embryos. However, stripe 1 is less well developed and stripe 7 is better developed in run- embryos than in Atro-mutant embryos. In cad- embryos, stripes 1-4 are very similar to these stripes in Atro-mutant embryos, but stripes 5-7 are less well developed. Both run+ and cad+ are known to function as activators of the ftz zebra element. These observations suggest that Atro might serve as a coactivator for Run and/or Cad, perhaps by mediating recruitment of the Brm complex or other chromatin remodeling complexes to the ftz zebra element (Kankel, 2004).

Atrophin contributes to the negative regulation of epidermal growth factor receptor signaling in Drosophila

Dentato-rubral and pallido-luysian atrophy (DRPLA) is a dominant, progressive neurodegenerative disease caused by the expansion of polyglutamine repeats within the human Atrophin-1 protein. Drosophila Atrophin and its human orthologue are thought to function as transcriptional co-repressors. Drosophila Atrophin participates in the negative regulation of Epidermal Growth Factor Receptor (EGFR) signaling both in the wing and the eye imaginal discs. In the wing pouch, Atrophin loss of function clones induces cell autonomous expression of the EGFR target gene Delta, and the formation of extra vein tissue, while overexpression of Atrophin inhibits EGFR-dependent vein formation. In the eye, Atrophin cooperates with other negative regulators of the EGFR signaling to prevent the differentiation of surplus photoreceptor cells and to repress Delta expression. Overexpression of Atrophin in the eye reduces the EGFR-dependent recruitment of cone cells. In both the eye and wing, epistasis tests show that Atrophin acts downstream or in parallel to the MAP kinase rolled to modulate EGFR signaling outputs. Atrophin genetically cooperates with the nuclear repressor Yan to inhibit the EGFR signaling activity. Finally, it was found that expression of pathogenic or normal forms of human Atrophin-1 in the wing promotes wing vein differentiation and these forms act as dominant negative proteins inhibiting endogenous fly Atrophin activity (Charroux, 2006).

Four observations serve as evidence that Atro contributes to the negative regulation of EGFR signaling: (1) clones mutant for Atro display phenotypes characteristic of overactive EGFR signaling and express high levels of the known EGFR target gene Dl. These effects are enhanced when negative regulators of EGFR signaling, such as Argos, are simultaneously removed in Atro clones. (2) Increased amounts of Atro reduce the activity of EGFR signaling; (3) ectopic expression of Atro enhances the effects of decreased EGFR signaling, whereas reduced Atro enhances the effects of ectopic signaling and (4) Atro genetically interacts with yan suggesting that both repressors may cooperate to block EGFR signaling output (Charroux, 2006).

The likely C. elegans orthologue of Atrophin, Egl27, has been shown to inhibit vulval development induced by the Ras signal transduction pathway. Thus, the role of Atro as a negative regulator of the RTK/EGFR pathway may have been conserved during evolution. Egl27 is a component of a repressor complex, the nucleosome remodeling and histone deacetylase (NURD) complex, which is composed of HDAC-1, HDAC-2, two proteins of the Mi-2/CHD family, and MTA1 or MTA2. During vulval induction, the NURD complex is proposed to interact with the sequence-specific transcription factors LIN-31, an Ets-related transcription factor and LIN-1, a winged-helix molecule. LIN-1 and LIN-31 are repressors of vulval development that are negatively regulated upon phosphorylation by the MAPK mpk1/sur-1 (Charroux, 2006).

MAPK-dependent phosphorylation of the ETS transcription factor Pnt is necessary for the activation of the EGFR target genes in third instar eye imaginal discs and in embryos. Yan and Atro show synergistic genetic interaction, suggesting that both are required for the repression of EGFR signaling function. Thus, by analogy with EGL-27 and LIN-31 from C. elegans, a model is proposed where Yan cooperates with Atro in order to achieve tight repression. How does EGFR signaling counteract Atro-mediated repression? Localized downregulation (such as nuclear export and/or protein degradation) of specific repressors is a common mechanism for the activation of target genes by the EGFR pathway. Two observations argue against this mechanism for the co-repressor Atro: (1) in cells with high levels of EGFR activity, such as either side of the dorso-ventral boundary in the wing pouch, or later in prospective veins of pupal wings, Atro protein is detected ubiquitously and at invariant levels in all nuclei and (2) when EGFR signaling is overactivated in clones (by expressing the constitutive form of EGFR, EGFRACT), the amount and/or subcellular localization of the co-expressed Atro protein is unchanged (Charroux, 2006).

Several lines of evidence show that, in the late phases of imaginal disc patterning, Atro plays a specific role for EGFR repression. It was found that Atro does not contribute to other signaling pathways during imaginal disc development. For instance, expression of both Distal-less and the vestigial quadrant enhancer (vgQE), two known wingless (wg) target genes, is not affected in Atro clones located in the wing pouch. Plus, it was found that signaling from the Notch (N) receptor does not require Atro activity since Atro clones expressing the constitutively active, intra-cellular fragment of the N receptor (Nintra) display identical phenotypes to Nintra control clones, when located in the wing pouch (Charroux, 2006).

Other signaling pathways are known to affect vein differentiation such as Decapentaplegic (DPP), which promotes vein differentiation in late pupae, and N whose activity is necessary to restrict vein territories. However, the idea is favored that Atro contributes mainly to EGFR signaling since Atro acts in third instar larvae and is dispensable for N activity in the wing (Charroux, 2006).

Despite the strong correlation of Atro repression of EGFR target genes in the imaginal discs, Atro is required for patterning where EGFR has not been implicated. For example, Atro is required for normal segmentation of the Drosophila embryo. However, it is noted that both EGFR signaling and Atro are required for cell survival during embryogenesis. Additionally, Atro is not required for all EGFR-dependent events. For example, Atro is not involved in the function of the EGFR defining the identity of the proximal wing disc. These observations indicate that variable mechanisms of control are implicated in the negative regulation of EGFR signaling in the nucleus (Charroux, 2006).

This notion is supported even in different imaginal tissues. EGFR signals via Strawberry notch (Sno) and Ebi, to inhibit the repressor activity of a Su(H)/SMRTER complex, leading to activation of Dl expression. Clones of cells mutant for the Su(H)SF8 hypomorphic allele cause high level expression of Dl in PR cells, but not in the wing pouch. It was found that clones of cells mutant for the Su(H)del47 null allele similarly do not show ectopic expression of Dl in the wing pouch. As expected, Su(H)del47 cells located at the D/V border abolish the expression of Cut. Thus, Su(H), unlike Atro, is dispensable for Dl repression in the wing pouch. The reverse is true in the eye, where Su(H) activity is absolutely required to repress Dl expression whereas Atro is less important. This is in agreement with the weak phenotype caused by the Atro clones in the eye (i.e. no ectopic PRs, few extra cone cells), and indicates a redundancy with other negative regulators of EGFR signaling. This distinction between the relative requirements in different tissues for different regulators of EGFR signaling provides an interesting insight into tissue-specific control of ubiquitous signaling pathways. Regulators such as Atro, with functions restricted to some tissues, may contribute to the diverse outcomes of signaling through these common pathways (Charroux, 2006).

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominant, hereditary malady typified by the degeneration of specific neurons in the brain. Although DRPLA has been mimicked in a mouse model, the molecular and cellular mechanisms leading to the disease remain obscure. The data point to the role of Atro in the repression of EGFR signaling. It was found that expression of human N917Atrophin-1 in the wing mimics the loss of Atro activity; this raises the possibility that N917Atrophin-1 is acting as a dominant negative. Additionally, this phenotype is independent of polyQ expansion and is sensitive to the dose of EGFR signaling components. Such effects are not seen following expression of polyQ repeats alone or the exon 1 of Huntingtin with expanded polyQ (93Q) in the wing, indicating that human N917Atrophin-1 has specific effects on this pathway. This mechanistic insight into the role of the fly gene may have broader implications concerning Atrophin function in other organisms (Charroux, 2006).

Genetic interactions among scribbler, Atrophin and groucho in Drosophila uncover links in transcriptional repression

In eukaryotes, the ability of DNA-binding proteins to act as transcriptional repressors often requires that they recruit accessory proteins, known as corepressors, which provide the activity responsible for silencing transcription. Several of these factors have been identified, including the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Strong genetic interactions are seen between gro and Atro and also with mutations in a third gene, scribbler (sbb), which encodes a nuclear protein of unknown function. Mutations in Atro and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs, which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro and Atro/sbb mutant phenotypes suggests that they do not function together, but instead that they may interact with the same transcription factors, including Engrailed and C15, to provide these proteins with maximal repressive activity (Wehn, 2006; full text of article).

Previous studies demonstrated that Atro acts as a corepressor in Drosophila, the most convincing of these being the demonstration that fusion of Atro to a heterologous DNA-binding domain confers repressive activity to the chimera. Atro has been shown to interact directly with two transcription factors, Even-Skipped (Eve) and Huckebein, and the repressive ability of Eve is compromised in Atro mutants, probably accounting for the loss of en expression in even-numbered parasegments in Atro mutant embryos (Wehn, 2006).

These studies here are consistent with Atro acting as a corepressor since it was shown that several genes, including run, tkv, al, and B, are completely or partially derepressed in Atro mutant clones in imaginal discs, suggesting that transcriptional repressors required to silence these genes recruit Atro. Atro-dependent repression of Bar (B) in the center of the leg disc is very likely due to interaction with the transcription factor C15, which is expressed in the center of the leg and is required for repression of B. Similarly, Atro-dependent repression of al in the posterior of the wing is very likely due to interaction with En, which is expressed in the posterior and required to exclude al from this compartment. At present it is unclear which transcription factors recruit Atro to repress run in the eye or tkv in the wing, although a strong candidate for run would be the Rough homeodomain protein, which is expressed in the same cells, R2 and R5, that exhibit ectopic run expression in Atro mutant clones. Whether Atro can, in fact, bind directly to C15, En, and possibly Rough, needs to be tested biochemically, since previous studies with Eve and Hkb did not identify a possible interaction motif for Atro nor do sequence comparisons among C15, En, Eve, and Hkb suggest a common motif (Wehn, 2006).

The sbb gene encodes a nuclear protein with unknown function. sbb mutations have many different phenotypes affecting multiple tissues. sbb and Atro interact very strongly genetically and that many of the phenotypes of sbb mutants are very similar to those of Atro mutants, including derepession of run, tkv, al, and B in imaginal discs. Thus, Atro is unable to silence these genes in the absence of Sbb, suggesting that it is required for Atro activity either to recruit Atro to transcription factors or possibly to assist binding of these factors to DNA. Since these transcription factors appear to function normally in some respects in the absence of Sbb (or Atro), it appears more likely that Sbb and Atro function together in a corepressor complex (Wehn, 2006).

One problem with the proposal that Atro activity is dependent upon Sbb is that the phenotypes of Atro and sbb mutants are not identical. For example, embryos lacking both maternal and zygotic Atro have a very severe, almost uncharacterizable phenotype, while embryos lacking both maternal and zygotic Sbb have a much less severe phenotype, characterized by a reduced number of abdominal segments, that is similar to that of embryos lacking only maternal Atro. This could be explained if Atro is partially active in the absence of Sbb, or if it is dependent upon Sbb for repression of some genes but not others. Alternatively, the difference between Atro and sbb mutant phenotypes could be related to Atro having functions other than that of a corepressor. It is has been implicated in positive regulation of Hox gene expression, and it also functions in the cytoplasm to control planar cell polarity. This analysis of sbb mutants does not reveal any potential involvement of Hox gene expression or planar cell polarity and, consequently, if Sbb is required only for Atro to act as a corepressor, then it is not surprising that Atro and sbb mutant phenotypes are not identical. Further experiments are required to determine the nature of the Atro dependence on Sbb for transcriptional repression and how direct any interactions might be (Wehn, 2006).

Mutations in sbb and Atro were originally uncovered in a genetic screen for enhancers of al. It is likely that they act as enhancers because they are utilized by the C15 transcription factor to repress genes such as Bar; C15 is expressed in the same cells as Al and it is thought that they bind together to regulate gene expression. Strong genetic interactions were uncovered among sbb, Atro, and en mutations, that could be explained if En also recruits Atro/Sbb (Wehn, 2006).

Curiously, genetic studies also revealed strong interactions among gro, sbb, and Atro. This could be explained if Gro was also required for Atro activity; i.e., all three proteins may form a corepressor complex. However, this appears to be unlikely because, in contrast to the similar phenotypes of sbb and Atro mutants, there are several distinct differences among the phenotypes of gro mutants and those of sbb and Atro mutants. For example, repression of tkv in the anterior of the wing is dependent on both Sbb and Atro but not on Gro, while repression of run in the antennal disc is dependent upon Gro but not upon Atro or Sbb. This suggests that a specific transcription factor recruits Atro/Sbb to repress tkv in the wing and another transcription factor recruits Gro to repress run in the antenna. The identity of these transcription factors remains to be uncovered (Wehn, 2006).

In some cases gro mutants do have a similar phenotype to those of Atro and sbb; this includes partial derepression of al expression in the posterior of the wing and Bar in the center of the leg. This can be explained if C15 (expressed in the center of the leg) and En (expressed in the posterior of the wing) recruit both Gro and Atro/Sbb and if each imparts some but not all the repressive activity to these transcription factors. Consistent with this, both C15 and En possess eh1-type Gro-interaction motifs and previous studies have revealed that En can repress in the absence of Gro. Further biochemical studies are required to determine if C15 and En can indeed recruit Atro (Wehn, 2006).

At present it is unclear whether Atro and Gro provide all the repressive activity to C15 and En; this will await the generation of Atro gro double-mutant clones. sbb gro double-mutant clones have been analyzed and these reveal that some targets of C15 and En are still at least partially repressed, although En activity appears to be somewhat compromised following the simultaneous loss of Sbb and Gro, in comparison to loss of one of these alone. Either Atro has some activity in the absence of Sbb or C15 and En can use mechanisms other than recruitment of Gro and Atro to repress transcription. Many transcription factors have been shown to have the ability to repress by several mechanisms; for example, although Brk recruits both CtBP and Gro, it can repress some genes in the absence of both, using additional repression domains (Wehn, 2006).

Why do C15 and En need to recruit both Gro and Atro? En can repress some genes completely in the absence of either Gro or Atro, for example, ci and dpp in the wing. However, for repression of al, the activity of En is clearly reduced in the absence of either, indicating that it needs to recruit both to completely repress this gene. This would suggest a quantitative explanation; i.e., En recruits both Gro and Atro to increase its activity, rather than to allow it to repress specific genes repressed more efficiently by one or the other. This is consistent with the suggestion that both corepressors function via a similar mechanism: both Gro and a vertebrate homolog of Atro have been shown to recruit a histone deacetylase. The recruitment of both may decrease histone acetylation to a level that cannot be achieved with either alone (Wehn, 2006).


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Grunge/Atrophin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 July 2014

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