org Interactive Fly, Drosophila Grunge/Atrophin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Grunge

Synonyms - Atrophin, Atro

Cytological map position - 66D1

Function - Transcription co-factor

Keywords - segmentation, maternal, CNS

Symbol - Gug

FlyBase ID: FBgn0020427

Genetic map position -

Classification - homolog of mammalian Atrophin

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |
Recent literature
Yeung, K., Boija, A., Karlsson, E., Holmqvist, P. H., Tsatskis, Y., Nisoli, I., Yap, D. B., Lorzadeh, A., Moksa, M., Hirst, M., Aparicio, S., Fanto, M., Stenberg, P., Mannervik, M. and McNeill, H. (2017). Atrophin controls developmental signaling pathways via interactions with Trithorax-like. Elife 6. PubMed ID: 28327288
Summary:
Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro's critical role in development and disease, relatively little is known about Atro's binding partners and downstream targets. This study present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.
Kacsoh, B. Z., Greene, C. S. and Bosco, G. (2017). Machine learning analysis identifies Drosophila Grunge/Atrophin as an important learning and memory gene required for memory retention and social learning. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 28889104
Summary:
High throughput experiments are becoming increasingly common, and scientists must balance hypothesis driven experiments with genome wide data acquisition. This study sought to predict novel genes involved in Drosophila learning and long-term memory from existing public high-throughput data. An analysis was performed using PILGRM, which analyzes public gene expression compendia using machine learning. The top prediction was evaluated alongside genes involved in learning and memory in IMP, an interface for functional relationship networks. Grunge/Atrophin (Gug/Atro), a transcriptional repressor, histone deacetylase, was identified as the top candidate. It was found, through multiple, distinct assays, that Gug has an active role as a modulator of memory retention in the fly and its function is required in the adult mushroom body. Depletion of Gug specifically in neurons of the adult mushroom body, after cell division and neuronal development is complete, suggests that Gug function is important for memory retention through regulation of neuronal activity, and not by altering neurodevelopment. This study study providess a previously uncharacterized role for Gug as a possible regulator of neuronal plasticity at the interface of memory retention and memory extinction.
BIOLOGICAL OVERVIEW

A Drosophila gene encoding an Atrophin family protein has been identified. The gene is referred to in this essay as Atrophin, referring to its mammalian functional homolog, but its official FlyBase name, Grunge, refers to its role as a positive regulator of teashirt expression in the proximal parts of the imaginal discs (Erkner, 1997). Analysis of mutant phenotypes indicates that Atrophin/Grunge is required in diverse developmental processes, including early embryonic patterning. Atrophin genetically interacts with the transcription repressor even-skipped and is required for Eve's repressive function in vivo. Atrophin directly binds to Even-skipped in vitro. Furthermore, both human Atrophin-1 and Drosophila Atrophin repress transcription in vivo when tethered to DNA, and poly-Q expansion in Atrophin-1 reduces this repressive activity. It is proposed that Atrophin proteins function as a versatile transcriptional corepressor, and a model is offered that suggests that deregulation of transcription may contribute to the pathogenesis of neurodegeneration (Zhang, 2002).

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominantly inherited neuronal degenerative disease characterized by the variable combination of ataxia, choreoathetosis, myoclonus, epilepsy, and dementia. The disease is caused by the expansion of a polyglutamine tract (poly-Q) within the Atrophin-1 protein. In addition to DRPLA, expansion of poly-Q has been identified as the cause for a growing number of other neurodegenerative diseases including Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), and several spinocerebellar ataxias (SCA). One intriguing feature of these poly-Q diseases is that each appears to selectively affect only a specific subset of neurons, despite a widespread expression pattern of the disease gene products. For instance, in DRPLA patients, degeneration of the dentatofugal and pallidofugal systems in the brain is the most prominent neuropathological feature. This apparent selectivity suggests that the normal functions of the disease causative genes may provide a context for poly-Q to affect specific neurons (Zhang, 2002 and references therein).

The normal function of Atrophin-1 is not well understood. Inspection of the Atrophin-1 protein sequence reveals a proline-rich region and several arginine-glutamic acid (RE) dipeptide repeats but no known functional domain. Atrophin-1 proteins are detected in both the cytoplasm and the nucleus, thus providing no insights into its function (Wood, 2000). Through yeast two-hybrid interaction screens, several potential regulators and effectors of Atrophin-1 have been isolated, including ETO/MTG8 protein (Wood, 2000). ETO/MTG8 functions in transcriptional regulation, and overexpression of Atrophin-1 can repress transcription of a luciferase reporter gene in tissue culture cells (Wood, 2000). However, the physiological relevance of these interactions remains to be demonstrated (Zhang, 2002 and references therein).

Drosophila has been a useful model system to investigate the functions of evolutionarily conserved genes. The FRT/FLP-based strategy has been used to identify mutants that exhibit phenotypes analogous to human diseases. From a mosaic genetic screen, mutations that affect a Drosophila gene encoding a member of the Atrophin protein family (Atro) have been isolated. Mosaic adults containing Atro mutant clones display defects in multiple developmental processes. Embryos lacking maternal Atro product exhibit complex patterning defects, including disruption of segmentation, dorsoventral patterning, and neurogenesis. The pleitropism of the Atro phenotype suggests that the Atro protein may function in a fundamental cellular process (Zhang, 2002).

To identify genes that regulate growth and patterning, the FRT/FLP system was used to screen for lethal mutations that cause adult phenotypes in mosaic animals. From a screen of 326 P element lines on chromosome 3L, one complementation group was found to be comprised of five P element lethal insertion alleles, l(3)J5A3, l(3)03928, l(3)01323, l(3)rO116, and l(3)rO154. A variety of adult mosaic phenotypes were found. Mutant clones in the adult wing cause mild overgrowth of vein tissue, ectopic wing vein, or notched wing. Clones of mutant cells in the intervein regions also alter the hair polarity of the surrounding wild-type cells. In the notum, mutant clones cause notal clefts. In the eye, mutant clones cause small and rough eyes. Tangential sections of the Atro-/- mosaic eyes reveal incorrect rotational direction of the wild-type ommatidia adjacent to the mutant clone. Furthermore, both ommatidia rotation and organization within the mutant eye clone become disorganized, with some ommatidia missing outer photoreceptor cells, while others contain extra cells or abnormally shaped rhabdomeres. Animals homozygous for these P element lines die at a late embryonic stage. These phenotypes are clearly caused by the P element insertions, since precise excision of the P elements revert the lethality and result in wild-type adults. Taken together, these observations suggest that the mutated gene has pleitropic effects and may be involved in a fundamental cellular process (Zhang, 2002).

As a dedicated repressor, the mechanism of transcriptional repression by eve has been extensively studied. Previous studies have shown that Eve can interact with the TATA binding protein (TBP) to prevent the binding of TBP to TATA elements, suggesting that it represses transcription by disrupting the formation of a preinitiation complex at the promoter (Li, 1998). However, TBP binding alone can not account for all of Eve's repressive activity, since Eve does not disrupt a preformed TFIID-TATA complex to prevent transcription (Li, 1998). Furthermore, Eve can inhibit transcription from a TATA-less promoter, which does not require the presence of TBP (Han, 1993). These findings suggest that Eve might utilize additional mechanisms to carry out its repressive function. In addition, Eve has been shown to interact with Rpd3 (Mannervik, 1999), a histone deacetylase, to regulate gene expression (Zhang, 2002).

The data suggests that Eve requires Atrophin/Grunge to repress transcription during embryogenesis. Atro is a ubiquitously expressed nuclear protein with strong maternal contribution. Strong dosage-sensitive genetic interactions are observed between eve and maternal Atro. The expression of eve's target genes wg and en is derepressed in the eve and Atro double heterozygous embryos with reduced Atro maternal contribution, a phenotype reminiscent of mutants with reduced eve activity. Using in vitro binding assays, it was found that Eve can interact with Atro protein via Eve's minimal repression domain. Finally, using the in vivo repression assay, it was shown that Atro can directly repress transcription when tethered to DNA via the Gal4 DNA binding domain. Together, these data support the notion that Atro/Grunge functions as a corepressor for Eve during embryogenesis (Zhang, 2002).

Previous study has demonstrated that the CD region in Eve protein, a small alanine- and proline-rich region, functions as Eve's minimal repression domain, although the mechanism of repression mediated by this small domain is not clear (Han, 1993). The genetic and biochemical data suggest that the function of this minimal repression domain is to bind to the Atro protein. Furthermore, it seems likely that Eve can bind to multiple corepressors at the same time, because another region of Eve has been shown to interact with Gro. Thus, in addition to its ability to interfere with TBP function and interact with Rpd3, Eve might recruit a battery of corepressors to achieve its potent repressive power (Zhang, 2002).

One prominent feature displayed by the Atro mutants is the diversity of mutant phenotypes. For example, in embryos lacking maternal Atro, severe disruption of multiple patterning processes is observed, including dorsoventral patterning, segmentation, and neurogenesis. In mosaic adults, Atro mutant cells exhibit a variety of abnormalities in multiple tissues, including extra vein and notches in the wing, cleft notum, and cell nonautonomous defects of wing hair and ommatidium polarity. Since the data suggest that Atro functions as a corepressor, these phenotypes might result from the deregulation of Atro target genes (Zhang, 2002).

For example, Atro might affect the repressive activities of some gap genes, since the stripe boundaries of pair-rule gene expression are expanded in the Atromat- embryos. Furthermore, the evidence implies that Atro might also function as a corepressor for the Hkb repressor. Atro genetically interacts with hkb in a dosage-sensitive fashion and the Atro protein binds to Hkb in vitro. Moreover, pair-rule gene expression in Atromat- embryos is shifted posteriorly, a phenotype observed in hkb mutants (Zhang, 2002).

The activity of Atro may also regulate the output of other signaling cascades during development. Several defects observed in Atro mutants, such as polarity, neurogenic, and cleft notum phenotypes, are reminiscent of characteristic phenotypes associated with disruption of the frizzled, Notch, dpp, and JNK signaling pathways. Since the activation of these signaling pathways often leads to eventual transcriptional changes within the nucleus, it is conceivable that some downstream transcription factors in these signaling cascades function together with Atro (Zhang, 2002).

Atrophin-1 is the causative gene for DRPLA, a member in the family of progressive neurodegenerative diseases that are caused by poly-Q expansion. The data supports a growing body of evidence that links the functions of the poly-Q disease genes to transcriptional regulation. Besides Atrophin, two other members of the poly-Q disease genes also regulate transcription. Androgen receptor, the SBMA disease gene, encodes a transcription factor. Huntingtin (Htt), the HD gene product, has been shown to bind to transcription corepressor N-CoR and transcription coactivator CBP. Furthermore, Htt has been shown to regulate transcription of BDNF, a gene required for neuronal survival (Zhang, 2002 and references therein).

Data suggest that poly-Q expansion in Atrophin-1 reduces its normal repressive activity. Interestingly, poly-Q expansion in Htt also reduces HD's normal transcriptional activity, which in turn causes a diminished level of BDNF transcription. How can these observations explain the dominant feature of these diseases? It is hypothesized that poly-Q expansion might alter the normal activity of these transcriptional regulators, which would in turn lead to aberrant gene expression. In a heterozygous patient, such a quantitative change in gene expression could lead to cumulative damages in long-lived neurons, providing one possible explanation for the progressive, dominant pathogenic effects of these diseases. Indeed, several recent findings support this hypothesis. For example, specific downregulation of multiple neuronal genes is observed in the SCA1 mouse long before obvious pathologic changes. Poly-Q-expanded Htt inhibits the function of several transcriptional coactivators, including CBP, and modulating such inhibition by administering histone deacetylase inhibitors can arrest poly-Q-dependent neurodegeneration in fly. Together, these data suggest that deregulation of transcription contributes to the pathogenesis of neurodegeneration (Zhang, 2002 and references therein).

Deregulating the activities of specific transcription regulators could also explain the specificity of the affected tissues observed in each of the poly-Q diseases, despite the ubiquitous expression of the disease genes. The data show that although fly Atro is widely expressed, it controls the activities of specific transcription factors (e.g., eve and hkb) that regulate gene expression in a spatially restricted fashion. Interestingly, Hkb and Eve homologs are expressed in the developing mouse and zebrafish brains. Human Eve homologs are expressed in the adult brain, including the regions that are affected in the DRPLA patients. These data suggest that Atrophin-1 may also interact with Eve and Hkb homologs in humans; this could contribute to the pathogenesis of the DRPLA disease (Zhang, 2002 and references therein).


GENE STRUCTURE

cDNA clone length - 7327

Bases in 5' UTR - 463

Exons - 14

Bases in 3' UTR - 963


PROTEIN STRUCTURE

Amino Acids - listed as 1966 and 1985 in various resources

Structural Domains

BLAST search analysis has shown that the predicted protein is a Drosophila member of the Atrophin protein family. While there are at least two known Atrophin family proteins in humans (Atrophin-1 and a related protein, Atrophin-2), only one Atrophin homolog was found in the Drosophila genome and no Atrophin homolog was found in Caenorhabditis elegans or yeast. The identified gene was thus named Drosophila Atrophin (Atro) (Zhang, 2002).

By sequence comparison, the Atro protein can be subdivided into four regions. (1) The C-terminal one-third of the Atro protein (amino acids [aa] 1387-1985), a region rich with charged amino acids and several highly conserved arginine-glutamic acid (RE) dipeptide repeats, shares high levels of sequence identity with the corresponding region in human Atrophin-1 and Atrophin-2 (33% and 27% identity, respectively). (2) Similar to Atrophin-1 and Atrophin-2, the middle region of Atro (aa 723-1386) contains a high percentage of proline and other noncharged amino acids. (3) The more N-terminal region of Atro (aa 206-722) contains multiple stretches of isolated sequences that are conserved in both Atrophin-1 and Atrophin-2, which have been named ANR (Atrophin-1 N-terminal region related. (4) Interestingly, the very N terminus sequence of Atro (aa 1-205) is not present in Atrophin-1, but homologous sequences are found at the N terminus of Atrophin-2 (aa 1-206) and human MTA2 related proteins. There are also two glutamine repeats (Q11 and Q14) and two putative nuclear localization signals (NLS) in Atro (Zhang, 2002).

Sequence analysis of two overlapping cDNAs reveals an open reading frame encoding for a putative protein of 1966 amino acids. The putative Gug protein has closest similarity to human arginine-glutamic acid dipeptide repeat (RERE) protein, an Atrophin-1-related protein. Distinct domains of this protein also show similarity with vertebrate Atrophin-1-related and with the Metastasis-associated (Mta)-like proteins. Atrophin-1 and Atrophin-1-related proteins are found in mice, rats and humans. Human Atrophin-1 contains a poly-glutamine repeat, which is expanded in individuals with a dentatorubral-pallidoluysian atrophy (DRPLA), resulting in neuronal apoptosis. The normal function of Atrophin-1 is not known. Atrophin-1 and the human Atrophin-1-related (RERE) protein are similar in the C-terminal half of each protein (60% identity), but RERE has no poly-glutamine stretch. Gug contains two poly-glutamine stretches and has a conserved C-terminal box found in Atrophin-1 and Atrophin-1-like proteins. Human RERE exhibits weaker identity in a second region of Gug, extending from amino acid 334-513. This domain is also conserved in vertebrate Atrophin-1 proteins but is less extensive. Another weak region of homology is found between Gug and mouse Atrophin-1 (30% identity, 43% homology) and rat Atrophin-1-related (22% identity; 30% similarity); this domain is not found in human RERE (Erkner, 2002).

An N-terminal region of Gug shows homology with the C. elegans protein EGL-27, which is similar to vertebrate Mta1. This domain includes a putative DNA-binding domain called SANT (or Myb) preceded by an ELM2 homology region. RERE, EGL-27 and Mta1 possess a GATA-like domain, but Gug does not, and RERE has a BAH (bromo adjacent homology) domain, unlike Gug, at the extreme N-terminal end. Mta1 is thought to be required for normal chromatin structure because it associates with components possessing histone deacetylase and nucleosome remodelling activities (Xue, 1998). EGL-27 is a nuclear protein and is required with Hox and Wnt signaling components for normal cell migration and polarity. EGL-27, like Gug, has polyglutamine repeat regions (Ch’ng and Kenyon, 1999; Herman, 1999; Solari, 1999). Finally, Gug possesses three putative nuclear localization signals, one of which overlaps the SANT domain. These observations suggest that Gug plays a role in the nucleus (Erkner, 2002).


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

date revised: 2 February 2002

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