Targets of Activity

An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila; charlatan is repressed by Ebi/SMRTER and promotes Dl expression

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. A mechanism is described by which the Ebi/SMRTER corepressor complex maintains Dl expression. charlatan (chn) is repressed by Ebi/SMRTER corepressor complex by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression (Tsuda, 2006).

The corepressor complex that includes Ebi, SMRTER and Su(H) is required for expression of Dl in Drosophila photoreceptor cells. To identify genetic loci that are transcriptionally repressed by the Ebi corepressor, a screen was set up using an ectopic gene expression system (Gene Search System). Insertion of a Gene Search (GS) vector, a modified P-element carrying the Gal4 upstream activating sequence (UASG) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UASG system. GS insertions into the chn locus were identified, whose overexpression phenotype in the eye using an eye-specific Gal4 driver (GMR-Gal4) was modified by reducing ebi activity. Thus the regulation of chn by Ebi-dependent transcriptional repression was studied (Tsuda, 2006).

In third instar larval-stage eye discs, the chn transcript is highly expressed in the morphogenetic furrow (MF), where photoreceptor differentiation initiates, but is downregulated in cells in the later stage photoreceptor development. In ebi mutant eye discs, however, chn expression becomes detectable in differentiating photoreceptor cells, and its expression in the MF is increased, suggesting that Su(H) in association with Ebi and SMRTER represses chn transcription in the eye disc (Tsuda, 2006).

To reveal the role of Su(H) as an activator, chn expression was examined when the level of Su(H) expression was reduced. Removing one copy of Su(H) suppresses the loss-of-Dl expression phenotype in ebi mutants. It was found that reducing one copy of Su(H) suppresses ectopic chn expression in ebi mutants, suggesting that ectopic expression of chn in ebi mutants is Su(H)-dependent. RT-PCR analysis of chn expression in ebi- eye discs differing in the dosage of Su(H) gene also supported these results. Strong reduction of Su(H) expression alone reduced expression of chn in the MF; this expression became weaker and was slightly broader. The phenotype of ebi, Su(H) double mutants is almost the same as Su(H) single mutants , suggesting that Su(H) acts as an activator in the absence of Ebi. This might be due to dual functions of Su(H) as an activator or repressor. Hence, reducing the amount of Ebi in the corepressor complex involving Su(H) might convert Su(H) to an activator by permitting the replacement of the corepressor complex with NICD (Tsuda, 2006).

To reveal the molecular nature of transcriptional regulation of chn by Su(H), Su(H) target sites were sought in the genomic region of chn. Since Su(H) binds slightly degenerate sequences, it was not easy to identify the functional Su(H) binding region from a simple genomic search. An alternative approach was taken to map the chn genomic region, which is regulated by Su(H) in the normal chromosomal context. Ebi-mediated repression involves SMRTER, a corepressor that recruits histone deacetylases and induces the formation of inactive chromatin, which spreads from the site where Su(H) recruits the corepressor complex. Promoters near the Su(H)-binding site are thus expected to be downregulated in an Ebi-dependent manner. Four insertion lines of the GS vector were identified in the chn promoter region. All these GS lines caused ectopic expression of chn with consequent abnormal eye morphology when they were crossed with GMR-Gal4. If the effect of the Ebi/SMRTER corepressor complex reaches the UASG in those insertions, reduction of Ebi activity will derepress UASG and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebiDN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebiDN strongly enhanced the overexpression phenotype of chnGS17605 and chnGS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebiDN failed to enhance the overexpression phenotype of other GS lines (chnGS2112 and chnGS17892) that were inserted downstream (+773 and +1040, respectively) of the first exon. From these results, it is concluded that Ebi-dependent transcriptional repression is targeted to the proximity of the transcriptional initiation site of the chn promoter (Tsuda, 2006).

Chn is a 1108-amino-acid protein with multiple C2H2-type zinc-finger motifs. Although no highly homologous gene within the mammalian genome could not be detected using BLAST, a small sequence of similarity between the N-terminal zinc-finger motif of Chn and the fifth zinc-finger of human NRSF/REST was found. Chn has several structural and functional similarities to human NRSF/REST, as follows. First, Chn and NRSF/REST each contain an N-terminal region with multiple zinc-finger motifs (five motifs in 264 residues in Chn and eight motifs in 251 residues in NRSF/REST), followed by a cluster of S/T-P motifs (serine or threonine followed by a proline) and a single zinc-finger motif at the C terminus. Second, the C-terminal region of NRSF/REST binds a corepressor, CoREST, which serves as an adaptor molecule to recruit a complex that imposes silencing activities. The Drosophila homolog of CoREST (dCoREST) (Andres, 1999; Dallman, 2004) can associate with the C-terminal half of Chn in cultured S2 cells. Finally, NRSF/REST binds to NRSE/RE1, a 21-bp sequence located in the promoter region of many types of neuron-restricted genes, via the N-terminal zinc-finger motifs. It was found that a recombinant protein containing the N-terminal zinc-fingers of Chn bound specifically to the NRSE/RE1 sequence in vitro. Thus, the structural similarity to NRSF/REST, binding to dCoREST and the DNA-binding specificity of Chn suggest that it is a candidate for a functional Drosophila homolog of NRSF/REST (Tsuda, 2006).

If Chn acts as a regulator of neural-related functions, as suggested for NRSF/REST, then Chn would be expected to bind to a regulatory region common to many types of neural-related genes in Drosophila. Numerous sequences similar to NRSE/RE1 were identified in the Drosophila genome, and their binding to Chn was assessed by EMSA. Using these sequences, a consensus binding sequence for Chn (Chn-binding element (CBE), 5'-BBHASMVMMVCNGACVKNNCC-3') was derived. 26 CBEs were identified within 10 kb of annotated genes from the Drosophila genome. Binding to Chn was confirmed for 18 CBEs using EMSA competition assay. Genes containing the CBE include dopamine receptor 2 (DopR2) and the potassium channel, ether-a-go-go, for which the mammalian homologs are target genes of NRSF/REST. These results suggest that the CBE is a good indicator of Chn binding sites and that Chn regulates many types of neural-related genes, as is implicated for NRSF/REST. However, it was found that divergent forms of CBE adjacent to hairy and extramacrochaetae were bound specifically by Chn. Likewise, some of the CBE sites failed to bind to Chn. Thus, a further refinement will be necessary to predict a definitive set of Chn binding sites in the Drosophila genome (Tsuda, 2006).

Although it has been established that mammalian NRSF/REST is a key regulator of neuron-specific genes, attempts to isolate invertebrate homolog of NRSF/REST have so far failed to identify a true homologous factor in invertebrates. The properties of Chn, including the similarity in DNA-binding specificity, association with CoREST and transcriptional repressor activity, suggest that Chn is a strong candidate for a functional Drosophila homolog of NRSF/REST. chn was originally identified by its requirement in the development of the PNS. This study identified a number of candidate target genes of Chn, a large fraction of which is implicated in neural function and/or gene expression. It is expected that further analysis of these candidates will provide valuable information about chn function in vivo, which may be extended to the understanding of NRSF/REST (Tsuda, 2006).

The Chn mutation blocks eye development by preventing the initiation of MF, a process requiring Notch signaling. This phenotype is likely owing to a loss of Notch function, because elevated Dl expression is known to block Notch signaling. The function of Chn during the early stage of eye development might be to regulate Notch signaling at an appropriate level by downregulating Dl. It is possible that Chn-mediated modulation places a variety of Notch functions in eye under the influence of EGFR signaling and provides flexibility in its regulation (Tsuda, 2006).

Although chn is expressed in the MF, genetic analyses show that small clones of chn mutant cells permit progression of the MF and photoreceptor differentiation. It is speculated that the repressive effect of Chn is overcome by other signals in the MF, such as hedgehog signaling, which strongly induces Dl (Tsuda, 2006).

Developing photoreceptor cells are exposed to the EGFR ligand, Spitz, and the Notch ligand, Dl, and each cell must assess the level of the two signals and respond appropriately to perform each task of photoreceptor cell specification and induction of non-neural cone cells. This question was investigated by studying the expression of Dl in photoreceptor cells. chn was identified as a direct target of Ebi/SMRTER-dependent transcriptional repression and as a repressor of Dl expression. The abrogated expression of Dl in ebi mutants was recovered by reducing one copy of chn, suggesting that the negative regulation of chn by ebi is indeed prerequisite for photoreceptor cell development (Tsuda, 2006).

Genetic data suggest that Su(H) may activate or repress chn expression. This idea is supported by data showing that Ebi/SMRTER and NICD are recruited to the promoter region of chn. The Ebi/SMRTER complex formed in this region did not contain any detectable level of the intracellular domain of Notch (NICD), suggesting that the binding of Ebi/SMRTER and NICD to this region may be mutually exclusive, and therefore it is expected that a regulatory system controls the balance between the active and repressive states of Su(H). Taken together, these results suggest that chn is a key factor in the crosstalk between two major signal transduction pathways: the EGFR-dependent pathway and the Notch/Delta-dependent pathway (Tsuda, 2006).

In the mammalian system, competition between SMRT and NICD for interaction with RBPJkappa determines the state of RBPJkappa-dependent transcriptional activity. Extracellular signaling may modulate this competition; diverse signaling pathways modulate the functions of N-CoR/SMRT. The current findings would prompt investigations of potential interaction of two repression systems of NRSF/REST and N-CoR/SMRT, and their regulation by Notch and EGF signaling in mammalian neuronal differentiation (Tsuda, 2006).

RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock

Transcription regulation of the Drosophila hsp70 gene is a complex process that involves regulation of multiple steps including establishment of paused Pol II and release of Pol II into elongation upon heat shock activation. While the major players involved in regulation of gene expression have been studied in detail, additional factors involved in this process continue to be discovered. To identify factors involved in hsp70 expression, a screen was developed that capitalizes on a visual assessment of heat shock activation using a hsp70-beta galactosidase reporter and publicly available RNAi fly lines to deplete candidate proteins. The screen was validated by showing that depletion of HSF, CycT, Cdk9, Nurf 301, or ELL prevented full induction of hsp70 by heat shock. The screen also identified the histone deacetylase HDAC3 and its associated protein SMRTER as positive regulators of hsp70 activation. Additionally it was shown that HDAC3 and SMRTER contribute to hsp70 gene expression at a step subsequent to HSF-mediated activation and release of the paused Pol II that resides at the promoter prior to heat shock induction (Achary, 2014).

Protein Interactions

Using Gal4-DBD fusions, a series of Smrter deletion and truncation constructs were generated to map receptor interaction domains. Their interaction with the Ecdysone receptor complex was measured in mammalian two-hybrid assays with EcR-vp16 and USP. The EcR harbors a VP16 activation domain, so that association with Smrter results in activation of a Gal4-responsive luciferase gene. Two independent ecdysone receptor-interacting domains (the ERID1 and the ERID2) were identified with this assay. ERID1, which maps to aa 1698-1924 of Smrter, confers a 17-fold induction of a reporter gene in the presence of EcR-vp16 and USP. ERID2 maps to aa 2951-3038 and, along with EcR-vp16 and USP, produces an 8-fold induction of the reporter gene. ERID2, but not ERID1, is located within the original E52 clone. The inclusion of regions flanking ERID1 (aa 1698-2063) and ERID2 (aa 2094-3040) or ERID2 (aa 2929-3181) increases the reporter activities by several-fold, although these additional regions possess no autonomous EcR-interacting activity. Both ERID1 and ERID2 display a dramatic preference to bind the EcR:USP heterodimer and to dissociate from the EcR:USP when ligand is added. Interestingly, vertebrate retinoic X receptor (RXR), the mammalian homolog of USP, fails to substitute for USP in potentiating the interaction of EcR with Smrter. This result further strengthens the notion that EcR:USP is a preferred binding complex for Smrter, since the EcR:RXR complex requires ligand to be stabilized while the formation of EcR:USP dimer is independent of ligand binding (Tsai, 1999).

Since cell culture results leave open the question of whether the interaction between Smrter and the EcR complex is direct, pull-down experiments were conducted with GST fusions of ERID1 and ERID2, mixed with either 35S-labeled EcR or 35S-labeled USP. GST-ERID1 (aa 1698-2063) and GST-ERID2 (aa 2951-3038), but not GST alone, both pull down labeled EcR, whereas little interaction is found between USP and any of the three GST proteins. In addition, the pull-down complex is disrupted by the addition of hormone when USP is present. These in vitro results establish that Smrter and EcR may interact directly. In vivo interference assays were used to assess whether ERID1 and ERID2 as well as SMRT bind similar regions within the EcR. In this experiment, Gal4 fusions encoding either ERID1 or ERID2 were transfected along with plasmids encoding EcR:USP heterodimers. A test was then carried out to see whether coexpression of excess nuclear-targeted ERID1, or ERID2, or c-SMRT interferes with or reduces reporter activity. Interaction between each Gal4-ERID fusion and EcR-vp16:USP is significantly decreased by both ERIDs and c-SMRT. Interestingly, a more prominent effect is observed in experiments when Gal4-ERID1 (aa 1698-2063) is challenged by ERID2, and, conversely, a more efficient competition is achieved by ERID1 to Gal4-ERID2 (aa 2094-3181). Together, these results suggest that ERID1, ERID2, and c-SMRT may bind similar or overlapping surface(s) in EcR (Tsai, 1999).

Experiments indicating that EcR A483T disrupts the interaction with SMRT, led to experiments to see whether this mutation also severs its association with ERID1 and 2. Gal4 fusions harboring either ERID1 or ERID2 were examined for their interaction with wild-type EcR or with EcR A483T in the presence of vp16-USP. In both cases, no significant induction of reporter was observed in cells transfected with the EcR mutation, A483T, in either the presence or absence of ligand, confirming that A483 of EcR represents a common target for corepressor binding (Tsai, 1999).

An EGFR/Ebi/Sno pathway promotes Delta expression by inactivating Su(H)/SMRTER repression during inductive Notch signaling

The Notch and Epidermal growth factor receptor (Egfr) pathways both regulate proliferation and differentiation, and the cellular response to each is often influenced by the other. A mechanism is described that links them in a sequential fashion, in the developing compound eye of Drosophila. Egfr activation induces photoreceptor (R cell) differentiation and promotes R cell expression of Delta. This Notch ligand then induces neighboring cells to become nonneuronal cone cells. ebi and strawberry notch (sno) regulate Egfr-dependent Delta transcription by antagonizing a repressor function of Suppressor of Hairless [Su(H)]. Sno binds to Su(H), and Ebi, an F-box/WD40 protein, forms a complex with Su(H) and the corepressor Smrter. Egfr-activated transcriptional derepression requires ebi and sno, is proteasome-dependent, and correlates with the translocation of Smrter to the cytoplasm (Tsuda, 2002).

The Notch signaling pathway plays multiple roles in eye development. At the morphogenetic furrow, the proneural protein Atonal facilitates the expression of Dl in the R8 cell. The first step of ommatidial assembly involves lateral inhibition between equivalent cells, but successive steps are inductive, arising from an already differentiated cell to its uncommitted neighbors. The Notch pathway is involved in the regulation of both of these processes. Similarly, the Egfr ligand, Spi, expressed in R8, activates the receptor in neighbors allowing them to assume their respective R1–R7 cell fates. Subsequently, these R cells express Spi, and as described in this study, they also express Dl in response to Egfr activation. The cone cells receive an Egfr signal and a Notch signal from the R cells and this combination is critical for the assumption of their fate. Later, after their fate is determined, these cone cells, too, will express Delta, which is important for pigment cell induction. Presumably, the level of the Egfr signal rises in the cone cells with time, and as a threshold of Egfr activation is surpassed, the proteasome mediated arm of the pathway becomes effective causing derepression of Su(H) and expression of functional levels of Dl sufficient for pigment cell development. Thus, a temporally and spatially positioned combination of parallel and sequential Egfr/Notch signals is important for the successive induction of cell types in the eye (Tsuda, 2002).

Evidence from mammalian systems has suggested that CBF1, the mammalian homolog of Su(H), is a component of a large repressor complex. The activation function of CBF1 results from a displacement of repressive components (such as HDAC) by the intracellular domain of Notch which converts Su(H) into a transcriptional activator. Genetic analysis of the embryonic midline and the pupal bristle complexes in Drosophila have also supported a switch from Su(H)-mediated repression to activation. A second mechanism for relieving Su(H) mediated repression is through Sno, Ebi, and the Egfr pathway. In response to the Egfr signal, Ebi, an F-box protein, presumably causes a proteasome-mediated degradation of an unknown component of the Su(H) inhibitory complex. Mammalian TBL1 (Ebi) can function downstream of the tumor suppressor gene, p53, in the degradation of the ß-catenin protein in a novel ubiquitin-dependent degradation pathway involving Siah, the mammalian homolog of the Drosophila Sina protein. Similarly, Drosophila Ebi can also act in combination with Sina to degrade protein targets. More generally, phosphorylation by MAPK downstream of RTK pathways is known to trigger proteasome-mediated degradation of target proteins. In addition to Ebi, a core component of the proteasome, encoded by l(3)73Ai gene, is also important for expression of Dl. The simplest model is that in response to Egfr signaling, one or more of the many components in the large Su(H)/SMRTER repression complex becomes a target of a proteasome-mediated degradation process (Tsuda, 2002).

The studies presented here also show that the corepressor SMRTER is redistributed from the nucleus to the cytoplasm in an Egfr/Sno/Ebi dependent manner. These results are in complete agreement with the role of the corresponding mammalian protein SMRT in its function as a repressor. Like Su(H), nuclear hormone receptors such as retinoic acid receptor and thyroid hormone receptor can function as both repressors and activators. SMRT has been shown to be phosphorylated in response to an RTK signal. This leads to translocation of SMRT out of the nucleus. Thus, steroid hormone receptors lose their ability to repress but not activate transcription. In an in vivo example, the Egfr/Sno/Ebi pathway promotes the dissociation of the Su(H)/SMRTER repressor complex and causes the nuclear export of SMRTER. As a result, target genes such as Dl are derepressed (Tsuda, 2002).

This study highlights the function of two unusual proteins, Sno and Ebi, in controlling the expression of Dl. Mammalian Ebi (TBL1) interacts with a SMRT/HDAC complex as also supported by this study in Drosophila. There are two human and three mouse genes similar to Sno identified by genome projects. The function of the mammalian Sno proteins is unknown. Whether the mammalian proteins also function upstream of the Notch pathway, as they do in Drosophila, remains to be established. Given the conservation of developmental pathways between Drosophila and mammals, this may not be an unreasonable expectation (Tsuda, 2002).

The SIN3/RPD3 deacetylase complex is essential for G(2) phase cell cycle progression and regulation of SMRTER corepressor levels

The SIN3 corepressor and RPD3 histone deacetylase are components of the evolutionarily conserved SIN3/RPD3 transcriptional repression complex. The SIN3/RPD3 complex and the corepressor SMRTER are required for Drosophila G2 phase cell cycle progression. Loss of the SIN3, but not the p55, SAP18, or SAP30, component of the SIN3/RPD3 complex by RNA interference (RNAi) causes a cell cycle delay prior to initiation of mitosis. Loss of RPD3 reduces the growth rate of cells but does not cause a distinct cell cycle defect, suggesting that cells are delayed in multiple phases of the cell cycle, including G2. Thus, the role of the SIN3/RPD3 complex in G2 phase progression appears to be independent of p55, SAP18, and SAP30. SMRTER protein levels are reduced in SIN3 and RPD3 RNAi cells, and loss of SMRTER by RNAi is sufficient to cause a G(2) phase delay, demonstrating that regulation of SMRTER protein levels by the SIN3/RPD3 complex is a vital component of the transcriptional repression mechanism. Loss of SIN3 does not affect global acetylation of histones H3 and H4, suggesting that the G2 phase delay is due not to global changes in genome integrity but rather to derepression of SIN3 target genes (Pile, 2002).

Boat, an AXH domain protein, suppresses the cytotoxicity of mutant ataxin-1; Both proteins interact with the transcriptional corepressor SMRT and its Drosophila homolog SMRTER

Ataxin-1 is a neurodegenerative disorder protein whose glutamine-repeat expanded form causes spinocerebellar ataxia type 1 (SCA1) in humans and exerts cytotoxicity in Drosophila and mouse. The cytotoxicity caused by ataxin-1 is modulated by association with a related protein, Brother of ataxin-1 (Boat). Boat and ataxin-1 share a conserved AXH (ataxin-1 and HMG-box protein 1) domain, which is essential for both proteins' interactions with the transcriptional corepressor SMRT and its Drosophila homolog, SMRTER. The Boat-ataxin-1 interaction is mediated through multiple regions in both proteins, including a newly identified NBA (N-terminal region of Boat and ataxin-1) domain. The physiological relevance of the Boat-ataxin-1 interaction was investigated in Drosophila; it was discovered that a mutant ataxin-1-mediated eye defect is suppressed by ataxin-1's association with Boat. Correspondingly, in transgenic SCA1 mouse, Boat expression is greatly reduced in Purkinje cells, the primary targets of SCA1. This study thus establishes that Boat is an in vivo binding partner of ataxin-1 whose altered expression in Purkinje cells may contribute to their degeneration in SCA1 animals (Mizutani, 2005).

Ataxin-1 is a polyglutamine disease protein whose glutamine-repeat expanded form is involved in SCA1. Inspired by a recent finding that ataxin-1 associates with the transcriptional corepressors SMRT and SMRTER, this study investigated how these associations are established. It was shown that the ataxin-1-SMRT interaction is mediated through a conserved AXH domain (11024, 800Figure 1AFigure1. Extending from this initial finding, the possibility was examined whether any other AXH domain proteins also interact with SMRT in a similar manner. Indeed, a novel human AXH domain protein, Boat, was identified whose interaction with SMRT also depends on its AXH domain. The Boat-SMRT interaction was further confirmed in human cultured cells, in which Boat-enriched nuclear foci recruit endogenous SMRT. Boat also shares additional properties with ataxin-1, including interaction with other SMRT-related factors, such as N-CoR and SMRTER, forming complexes with HDAC3, and functioning as a potent transcriptional repressor. The resemblances between ataxin-1 and Boat suggest that the two proteins are likely involved in overlapping transcriptional regulation pathways in vertebrate cells (Mizutani, 2005).

Significantly, it was further discovered that Boat also interacts directly with ataxin-1. Their physical interaction was observed not only in yeast and human cultured cells, but also in Drosophila cells. In Drosophila, it was possible to demonstrate the physiological relevance of the Boat-ataxin-1(82Q) interaction by showing that ataxin-1(82Q)-mediated cytotoxicity is suppressed when ataxin-1(82Q) interacts with Boat. In mouse, Boat and ataxin-1 share strikingly similar expression patterns in the brain, including in Purkinje cells. Most importantly, it was found that Boat expression is dramatically decreased in the Purkinje cells of SCA1 mouse as early as 3 weeks old. This research therefore establishes that Boat is an in vivo binding partner of ataxin-1, whose absence in Purkinje cells may contribute to their degeneration in SCA1 mouse (Mizutani, 2005).

Boat was discovered because it shares an AXH domain with ataxin-1. This 114-amino-acid domain was first identified because it is common to ataxin-1, HBP1, and the Drosophila CG4547 and C. elegans K04F10.1 proteins. Although the protein structure of the AXH domain has been solved, very little is known about the exact functions of this conserved domain. This study linked the functions of the AXH domain to SMRT association and to transcriptional repression. The connection between the AXH domain and transcriptional repression correlates with an earlier report that a region encompassing the AXH domain of HBP1, an HMG-box chromatin remodeling transcription factor, is responsible for its transcriptional repressive effects. Based on this correlation, it is possible that a common feature of the AXH domain is transcriptional repression, and that additional AXH domain proteins may also associate with SMRT/SMRTER (Mizutani, 2005).

The AXH domain of ataxin-1 may also possess other functional properties. For example, structural analysis of the AXH domain indicates that it forms an oligomer-binding fold structure -- a motif found in many oligonucleotide-binding proteins. Therefore, the ability of ataxin-1 to bind RNA could be mediated through its AXH domain. In addition, the mapped regions in ataxin-1 responsible for LANP interaction, USP7 ubiquitin-protease binding, and p80 coilin association also encompass the AXH domain. These various studies suggest that the AXH domain is a molecular scaffold domain engaged in multiple protein-protein interactions and in RNA binding. The close resemblance between the AXH domains in ataxin-1 and Boat raises the possibility that some of the known features of ataxin-1 may be shared by Boat as well (Mizutani, 2005).

Although substantial similarities were found between ataxin-1 and Boat, these two AXH domain proteins also encode distinct properties. Their respective N-terminal regions, which was called the NBA domain, may be in part responsible for their functional differences. Notably, it was found that whereas the NBA domain of Boat is able to interact with the 1526-1833 region of SMRT and with itself, these two properties are absent from the NBA domain of ataxin-1. Therefore, even though the NBA domains of ataxin-1 and Boat show sequence resemblance, their divergence is sufficient to enable Boat to adopt a different structural configuration for self-association and for SMRT interaction (Mizutani, 2005).

The finding that ataxin-1 self-associates by means of its NBA domain is of particular significance, since it has long been presumed that ataxin-1 self-association is dictated by a single self-association motif. The results, on the contrary, reveal that ataxin-1 self-association depends on complex interactions among at least three domains in the protein. In addition to a previously mapped SAR domain, the NBA domain and a third domain targeted by the NBA domain are also involved. Based on these mapped domains, it is proposed that ataxin-1 self-association can be achieved not only through intermolecular interaction between separate ataxin-1 molecules, but also through intramolecular interaction between the NBA domain and the third region of the same ataxin-1 molecule (Mizutani, 2005).

Significantly, both the NBA and SAR domains of ataxin-1 are also targeted by Boat: the SAR domain of ataxin-1 is a target of Boat's NBA domain, whereas the NBA domain of ataxin-1 is a target of full-length Boat. The coincidence between the domains for ataxin-1 self-association and ataxin-1-Boat interaction therefore provides a conceptual framework to explain why Boat and Boat(NBA) can suppress ataxin-1(82Q)-mediated phenotypes (cytotoxicity) in Drosophila eyes: the formation of ataxin-1-Boat heterodimer takes place at the expense of self-association of mutant ataxin-1. Various studies in fly on different polyglutamine disease proteins have shown that the levels of protein expression correlate with their toxicity. Therefore, a reduced level of self-associated mutant ataxin-1, as a result of its dimerization with Boat, may be a reason for its reduced toxicity (Mizutani, 2005).

The finding that Boat, ataxin-1, and SMRT are all expressed in many NeuN-positive cells in the mouse brain demonstrates that these three proteins can in fact encounter one another and modulate one another's functions in such neuronal cells. Because Boat and SMRT are both expressed in Purkinje cells (the primary targets of SCA1) it is likely that these two ataxin-1-associating factors are relevant to SCA1 pathogenesis as well. Most revealing is that the expression level of Boat in the Purkinje cells of 3-week-old SCA1 mouse is significantly reduced. This reduction in Boat expression takes place prior to the appearance of nuclear inclusions and dendritic thinning of Purkinje cells, therefore representing an early event in SCA1 pathogenesis. This initial loss of Boat protein could result in increased levels of self-associated mutant ataxin-1 protein in Purkinje cells, hence aggravating the mutant protein's toxicity. This in turn might set off a feed-forward mechanism for increasing damage, since increased ataxin-1 self-association would cause further reduction of Boat expression (Mizutani, 2005).

Reduced Boat expression in SCA1 is regulated at the post-transcriptional level, since Boat transcripts are still abundantly present in the Purkinje cells of SCA1 mouse. Intriguingly, this phenomenon appears to be unique to Purkinje cells, since Boat expression is unaffected by ataxin-1(82Q) in HEK293 cells and in Drosophila cells. Therefore, future studies on how Boat is downregulated by ataxin-1(82Q) in the Purkinje cells of SCA1 mice may yield critical information on how tissue specificity is established for SCA1 (Mizutani, 2005).

The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors

In Drosophila a large zinc finger protein, Schnurri, functions as a Smad cofactor required for repression of brinker and other negative targets in response to signaling by the transforming growth factor beta ligand, Decapentaplegic. Schnurri binds to the silencer-bound Smads through a cluster of zinc fingers located near its carboxy-terminus and silences via a separate repression domain adjacent to this zinc-finger cluster. This study shows that this repression domain functions through interaction with two corepressors, CtBP and Sin3A, and that either interaction is sufficient for repression. Schnurri contains additional repression domains that function through interaction with CtBP, Groucho, Sin3A and SMRTER. By testing for the ability to rescue a shn RNAi phenotype evidence is provided that these diverse repression domains are both cooperative and partially redundant. In addition Shn harbors a region capable of transcriptional activation, consistent with evidence that Schnurri can function as an activator as well as a repressor (Cai, 2009).


Smrter antibodies were prepared in order to examine its cytological and chromosomal localization patterns. Consistent with its action as a corepressor of EcR, Smrter was localized to nuclei of salivary glands and of fat bodies, as well as to nuclei of eye, wing, and leg imaginal discs isolated from the third instar larvae. Whether Smrter is associated with the EcR:Usp complex on chromosomes was examined. The fact that Usp and EcR colocalize with each other on polytene chromosomes, allowed the use of the Usp staining pattern as an index for EcR's presence on chromosomes. Chromosomal spreads prepared from the salivary glands of wandering third instar larvae (prior to pupariation) were subjected to simultaneous immunological staining with antibodies against Smrter and Usp. Indirect immunofluorescence staining reveals that Smrter is a chromosome-bound protein and colocalizes with Usp at a majority of chromosomal sites. The strongest Smrter staining is primarily associated with the boundary between band and interband regions as well as within the interband regions of chromosomes counterstained with DAPI. This result confirms that, as an EcR-associating factor, Smrter is recruited by the EcR:Usp heterodimers to their specific target chromosomal loci. Interestingly, Smrter staining can still be detected in puffed regions, such as the 2B puff. Since the polytene chromosomes consist of a parallel arrangement of several hundred to two thousand copies of the euchromatic portions of the chromosomes, an individual binding protein like Smrter may be cycling on and off, resulting in a steady state of signals detected in the broader chromatin regions. Whether or not Smrter levels actually change prior to or after the peak of ecdysone pulses remains to be established (Tsai, 1999).

Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). This study characterized global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Beadex, also know as LIM-only. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and this study identified different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, it was found that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. These results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs (Hirano, 2016).

This study has identified Bx and Smr as LTM maintenance genes and has characterize a shift in transcription between CREB/CRTC-dependent maintenance (1-4 days) to Bx-dependent maintenance (4-7 days). In addition, a biological consequence of this shift was identified in defining a time window during which LTM can be modified, β-Spec was identified as being required for memory extinction (Hirano, 2016).

LTM maintenance mechanisms change dynamically during storage. In particular, CRTC, which is not required during memory formation, becomes necessary during 4-day LTM maintenance and then becomes dispensable again. Consistent with this, CRTC translocates from the cytoplasm to the nucleus of MB neurons during 4-day LTM maintenance and returns to the cytoplasm within 7 days. On the other hand, Bx expression is increased at both phases, suggesting that transcriptional regulation of memory maintenance genes may change between these two phases. Supporting this idea, it was found that Bx expression requires CRTC during 4-day LTM maintenance but becomes independent of CRTC 7 days after training. It is proposed that CREB/CRTC activity induces Bx expression, which subsequently activates a feedback loop where Bx maintains its own expression and that of other memory maintenance genes (Hirano, 2016).

Although it is proposed that the shifts in transcriptional regulation that were observed occur temporally in the same cells, the possibility cannot be discounted that LTM lasting 7 days is maintained in different cells from LTM lasting 4 days. MB Kenyon cells can be separated into different cell types, which exert differential effects on learning, short-term memory and LTM. Thus, it is possible that LTM itself consists of different types of memory that can be separated anatomically. In this case, CRTC in one cell type may exert non-direct effects on another cell type to activate downstream genes including Bx and Smr. However, as that CRTC binds to the Bx gene locus to promote Bx expression and both CRTC and Bx are required in the same α/β subtype of Kenyon cells, it is likely that the shift from CRTC-dependent to Bx-dependent transcription occurs within the α/β neurons (Hirano, 2016).

Currently, it is proposed that the alterations in histone acetylation and transcription that were uncovered are required for memory maintenance. However, it is noted that decreases in memory after formation could be caused by defects in retrieval and maintenance. Thus, it remains formally possible that the epigenetic and transcriptional changes reported in this study are required for recall, but not maintenance. However, this is unlikely, as inhibition of CRTC from 4 to 7 days after memory formation does not affect 7 day memory, whereas inhibition from 1 to 4 days does. This suggests that at least one function of CRTC is to maintain memory for later recall (Hirano, 2016).

Consistent with a previous study in mice, which suggests distinct transcriptional regulations in LTM formation and maintenance (Halder, 2016), the data indicate that memory formation and maintenance are distinct processes. Although the HAT, CBP, is required for formation but dispensable for maintenance, other HATs, GCN5 and Tip60, are required for maintenance but dispensable for formation. Through ChIP-seq analyses, those downstream genes, Smr and Bx, were identified as LTM maintenance genes and these are not required for LTM formation. Collectively, these results suggest differential requirements of histone modifications between LTM formation and maintenance. Although other histone modifiers besides GCN5 and Tip60 were identified in the screen, knockdown of these histone modifiers did not affect LTM maintenance. There are ~50 histone modifiers encoded in the fly genome, raising the possibility that the lack of phenotype in some knockdown lines is due to compensation by other modifiers (Hirano, 2016).

The results indicate some correlation of increase in CRTC binding with histone acetylation and gene expression. Interestingly, DNA methylation shows higher correlation to gene expression in comparison with histone acetylation in mice. Notably, flies lack several key DNA methylases and lack detectable DNA methylation patterns. Hence, histone acetylation rather than DNA methylation may have a higher correlation with transcription in flies. Reduction in histone acetylation was detected, overlapping with increase in CRTC binding. Those reductions could be due to CRTC interacting with a repressor isoform of CREB, CREB2b or other transcriptional repressor that binds near CREB/CRTC sites. These interactions would decrease histone acetylation and gene expression, and may be related to LTM maintenance. Although this study focused on the upregulation of gene expression through CREB/CRTC, downregulation of gene expression by transcriptional repressors may also be important in understanding the transcriptional regulation in LTM maintenance. The results demonstrate the importance of HATs for LTM maintenance; however, the data do not conclude that histone acetylation is a determinant for gene expression, but rather it might be a passive mark of gene expression. HATs also target non-histone proteins and also interact with various proteins, both of which could support gene expression in LTM maintenance (Hirano, 2016).

Similar to traumatic fear memory in rodents, this study found that aversive LTM in flies can be extinguished by exposing them to an extinction protocol specifically during 4-day LTM maintenance. These observations suggest the time-limited activation of molecules that allows LTM extinction only during the early storage. Supporting this concept, it was found that CRTC is activated during the extinguishable phase of LTM maintenance and prolonging CRTC activity extends the time window for extinction. Thus, CRTC is the time-limited activated factor determining the time window for LTM extinction in flies. In cultured rodent hippocampal neurons, CRTC nuclear translocation is not sustained, suggesting that other transcription factors may function in mammals to restrict LTM extinction (Hirano, 2016).

This work demonstrates that LTM formation and maintenance are distinct, and involve a shifting array of transcription factors, coactivators and HATs. A key factor in this shift is CRTC, which shows a sustained but time-limited translocation to the nucleus after spaced training. Thus, MB neurons recruit different transcriptional programmes that enable LTM to be formed, maintained and extinguished (Hirano, 2016).


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Smrter: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology

date revised: 10 April 2017

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