Members of the MTG protein family function in the nucleus as transcriptional corepressors (Davis, 2003; Rossetti, 2004). Although a cytoplasmic function for Nervy, described in this section of The Interactive Fly, cannot be ruled out, it has been suggested that the axonal migration phenotypes observed in nervy mutant Drosophila embryos may be due to alterations in gene expression rather than a failure to anchor PKA to the plasma membrane (Ice, 2005: summarized below). Terman and Kolodkin (2005) respond to Ice (2005) by maintaining that the molecular, genetic, in situ hybridization, immunolocalization, and immunoprecipitation data support that Nervy can be found in the cytoplasm, interacts with the cytoplasmic domain of the plasma membrane receptor PlexA, and directly modulates PlexA signaling by functioning as an AKAP (Terman, 2005; full text of article).

Cyclic nucleotides regulate axonal responses to a number of guidance cues through unknown molecular events. Drosophila nervy, a member of the myeloid translocation gene family of A kinase anchoring proteins (AKAPs), regulates repulsive axon guidance by linking the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) to the Semaphorin 1a (Sema-1a) receptor Plexin A (PlexA). Nervy and PKA antagonize Sema-1a-PlexA-mediated repulsion, and the AKAP binding region of Nervy is critical for this effect. Thus, Nervy couples cAMP-PKA signaling to PlexA to regulate Sema-1a-mediated axonal repulsion, revealing a simple molecular mechanism that allows growing axons to integrate inputs from multiple guidance cues (Terman, 2004).

Many semaphorin proteins, including the Drosophila transmembrane semaphorin Sema-1a, are axonal repellents that signal through transmembrane plexin proteins. Nervy, a Drosophila homolog of the mammalian MTG8 proto-oncogene, was identified in a yeast interaction screen as a Plexin A-interacting protein. Nervy belongs to a phylogenetically conserved protein family consisting of Drosophila Nervy and three mammalian proteins: myeloid translocation gene (MTG) 8, MTG16, and MTGR1. Mammalian MTG proteins are tumorigenic, but the cellular processes they control are poorly defined. MTG8 and MTG16 were recently found to bind the regulatory subunit of type II cAMP-dependent protein kinase (PKA RIIalpha and function as PKA (or A kinase) anchoring proteins (AKAPs) in lymphocytes (Fukuyama, 2001; Schillace, 2002). AKAPs position type II PKA at defined locations to allow for the spatially and temporally specific phosphorylation of target proteins in response to local increases in cAMP. Drosophila Nervy shows considerable amino acid conservation with MTG proteins within a consensus PKA RII binding site, suggesting that Nervy may be an AKAP that couples cAMP-PKA signaling to semaphorin-plexin-mediated axon guidance. Cyclic nucleotides, including cAMP, are known to alter how axons respond to guidance cues, including semaphorins, but a direct molecular link coupling cAMP-PKA signaling to specific guidance cue signaling pathways has remained elusive (Terman, 2004).

Evidence is provided for cAMP-PKA modulation of Sema-1a–mediated repulsive guidance by the association of the AKAP nervy with the PlexA receptor. These results are consistent with in vitro observations describing a role for cAMP in regulating semaphorin repulsion in vertebrates. cAMP antagonists neutralize the effects of cyclic guanosine monophosphate (cGMP) on Sema3A repulsion, while Sema3A repulsion is increased by inhibiting PKA and neutralized by increasing cAMP. The conserved nature of semaphorin-plexin signaling suggests that vertebrate MTG proteins (Sacchi, 1998) may underlie these observations as well as observations that increased cAMP levels enhance axonal regeneration in the mammalian CNS. The observed interactions between plexin and Nervy are also consistent with roles described for AKAPs in other signaling pathways, allowing precisely localized PKA to be maintained close to its activators and substrates. What activates PKA bound to nervy? Anchored PKA is poorly dissociated by basal cAMP levels, so inputs from other signaling pathways, such as those activated by netrin or G protein–coupled receptors, may specifically regulate semaphorin signaling by locally increasing cAMP and activating PKA. PKA targets in the vicinity of PlexA may include L-type Ca²⁺ channels, or the Rho guanosine triphosphatase. Therefore, modulation of semaphorin signaling through the direct coupling of PKA to plexin by an AKAP allows for the integration of multiple guidance cues by the neuronal growth cone, enabling local, rapid, and directed responses to a complex extracellular environment (Terman, 2004).

Comment on 'Nervy links protein kinase A to plexin-mediated semaphorin repulsion'

Terman and Kolodkin (2004) recently suggested that Nervy, a Drosophila homolog of the mammalian myeloid translocation gene (MTG) family, functions at the plasma membrane as a protein kinase (PKA) anchoring protein (AKAP). Their findings were surprising because mammalian MTGs localize to nuclei and function as transcriptional corepressors by recruiting histone deacetylases. The antiserum Terman and Kolodkin used to detect endogenous Drosophila Nervy in immunoblot analysis and to localize Nervy to the plasma membrane of neurons by immunostaining (1) was not raised against Nervy but was an antibody to ETO (MTG8) developed in the Heibert laboratory against the C terminus of ETO/MTG8 (marketed through EMD Biosciences as anti-ETO Ab-1). Although the human ETO/MTG family members are well conserved in the C-terminal region, this antibody modestly recognized MTG16 and only weakly recognized the third MTG family member, Mtgr1, which is 61% identical in the antigenic region (Ice, 2005).

To determine whether anti-ETO Ab-1 recognizes the less homologous Drosophila Nervy (which is only 29% homologous in this region), a Myc-epitope tagged form of Nervy (Myc-nvy) was obtained from Terman and Kolodkin. DNA sequence analysis confirmed the identity of the cDNA. A second form of Nervy was created containing both the Myc tag and the GAL4 DNA binding domain (Gal4-nvy). Using the cytomegalovirus (CMV) immediate early promoter, Gal4-Nvy and Myc-Nvy were expressed at very high levels in Cos-7 cells. In numerous experiments with several antibody concentrations, anti-ETO Ab-1 failed to detect either form of Nervy, even when expressed at very high levels and under conditions in which the ETO/MTG8 signal was rapidly overexposed. In contrast, antibodies directed to the epitope tags or to Nervy confirmed robust expression of GAL-Nvy and Myc-Nvy. Although anti-ETO Ab-1 was obtained from a single rabbit, two bleeds of antiserum were supplied to EMD Biosciences. Both bleeds were tested and it was found that neither recognized Nervy. Upon long exposures, however, several background bands were observed, with one migrating in the 75-kD range. Thus, anti-ETO Ab-1 did not recognize Nervy in immunoblot experiments, which suggests that the band observed previously by Terman and Kolodkin was a background band that was serendipitously reduced in the nervy mutant (Ice, 2005).

Immunolocalization results were compared in Drosophila embryos obtained with antibodies raised against Drosophila Nervy and anti-ETO Ab-1. In wild-type embryos, anti-ETO Ab-1 diffusely labeled the longitudinal axon tracts and several scattered cells, a pattern similar to that previously reported by Terman and Kolodkin. However, an identical pattern was observed in nervy loss-of-function mutant embryos, indicating that it is nonspecific staining. In contrast, an extensively characterized anti-Nvy labeled the nuclei of a subset of central nervous system (CNS) and peripheral nervous system (PNS) cells. The pattern of Nervy protein matched the nervy mRNA pattern and, in contrast to anti-ETO, anti-Nvy produced no detectable staining in nervy mutant embryos. Further, in contrast to the previous study, no maternal nervy expression was observed by in situ hybridization or by immunostaining. Anti-Nvy also failed to label axons, which were labeled by anti-Fasciclin II (FasII). Although these results do not exclude the possibility that there is some Nervy in axons, they demonstrate that Nervy is predominantly, and perhaps exclusively, a nuclear protein and that anti-ETO could not be used to detect Nervy in Drosophila embryos (Ice, 2005).

Although ETO/MTG8 and MTG16 have also been suggested to be AKAPs, these studies found the proteins in the Golgi, not the plasma membrane. The Golgi localization is surprising because MTGs lack membrane translocation signal sequences. The antisera used in some of these experiments detected several proteins approximately 40 kD larger than the predicted or observed molecular weights of MTG8 or MTG16, which raises the possibility that the antisera may cross-react with a Golgi protein or that these proteins correspond to novel isoforms of MTG family members (Ice, 2005).

In summary, the results described here are consistent with previous findings showing that members of the MTG protein family function in the nucleus as transcriptional corepressors (Davis, 2003; Rossetti, 2004). Although a cytoplasmic function for Nervy cannot be ruled out, it is suggested that the axonal migration phenotypes observed in nervy mutant Drosophila embryos may be due to alterations in gene expression rather than a failure to anchor PKA to the plasma membrane (Ice, 2005).

Evidence that nervy promotes mechanosensory organ development by enhancing Notch signaling

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. This study describes the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. A model is proposed in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP (Wildonger, 2005).

These results suggest that Nvy plays a role, albeit subtle, in the SOP's ability to send a strong Dl signal to neighboring cells. Although the data demonstrate that nvy is not required for the SOP fate, it is suggested that the ability of Nervy to increase the Dl signal sent by the SOP helps to reinforce the SOP fate. When nvy is ectopically expressed it completely inhibits the formation of mechanosensory organs. Using reagents that mark the PNC and SOP, it was found that ectopic Nvy blocks the formation of the SOP, but not the PNC. In contrast, elevating Nvy levels specifically within the SOP (using neur-Gal4) does not affect sensory organ development, indicating that ectopic Nvy blocks the formation of the SOP but does not inhibit its development once it is specified. Furthermore, ectopic Nvy does not block mechanosensory organ formation when Sens is also over-expressed, suggesting that ectopic Nvy blocks SOP formation before there are high levels of Sens in the nascent SOP. Consistent with this idea, no Sens expression is observed in the pnr domain of pnr-Gal4 UAS-nvy wing discs or in clones that ectopically express Nvy. These data suggest that ectopic Nvy interferes with SOP formation at a stage before Sens is expressed, which corresponds to when the SOP is initially specified (Wildonger, 2005).

nvy is normally expressed in the SOP shortly after Ac and Sc levels increase. Given the expression of endogenous nvy within the SOP, the following two possibilities werre considered to explain the ectopic Nvy phenotype and to gain some clues about wild type function of nvy. (1) It is possible that ectopic Nvy blocks SOP formation cell autonomously by inhibiting the expression of ac, sc, or their downstream targets (such as sens) that are necessary for SOP formation. (2) It is possible that ectopic Nvy acts cell non-autonomously by enhancing Dl signaling, resulting in the 'mutual inhibition' of cells expressing precociously high levels of nvy. A closer examination of clones that ectopically express Nvy revealed that SOPs were significantly less likely to form near the borders of Nvy expressing clones than control clones. These results suggest that Nvy is acting, at least in part, cell non-autonomously, perhaps by increasing the strength of the Dl signal (we discuss the possibility that Nvy may also act cell autonomously in the following section). As a test of this idea, Nvy was ectopically expressed in clones lacking nic, which encodes a transmembrane protein required for cleaving and activating N in response to ligand binding. Ectopic Nvy was unable to block SOP formation in nic mutant clones, demonstrating that Nvy's ability to block SOP formation requires the N signaling pathway to be intact. This finding is therefore consistent with the idea that Nvy normally enhances the level of active Dl in the SOP. Importantly, loss-of-function nvy experiments are also consistent with this proposed role for Nvy. Using two different methods to remove nvy (expressing nvy RNAi or generating clones of a nvy deficiency), it was found that PNC cells that neighbor nvy⁻ clones are more likely to adopt the SOP fate than PNC cells that neighbor wild type clones. This result is similar to what was observed when the relative amount of Dl differs between neighboring PNC cells: PNC cells that neighbor cells with less Dl are more likely to differentiate as SOPs. In contrast to the Dl experiments, however, the complete absence of nvy did not cause all PNCs to become SOPs. Keeping in mind that nvy expression is restricted to the SOP (nvy is not detectably expressed in the PNC), these data suggest that nvy is not a general regulator of Dl signaling throughout the PNC, but that nvy enhances Dl activity in the SOP when it is forming (Wildonger, 2005).

Although these experiments are consistent with the idea that nvy enhances Dl signaling in the SOP, no changes in Dl protein levels were directly detected in either nvy loss- or gain-of-function situations. There are several possible explanations for this negative result: (1) it is possible that nvy does affect Dl expression levels, but that the change is too slight or brief to distinguish with the available anti-Dl antibody; (2) nvy might not affect Dl expression, but affect its localization and/or signaling ability in a manner that cannot be detected in these experiments; (3) it is also possible that nvy does not affect Dl at all, but interacts with other factors to produce the phenotypes observed. It is suggested that experiments using VP16-Nvy help to distinguish between these possibilities. Expressing VP16-Nvy produces results opposite to those resulting from expressing Nvy: VP16-Nvy enhances E-lacZ expression, which ectopic Nvy represses, and its expression results in ectopic Sens⁺ SOPs. Based on these data and the evidence that ETO, the mammalian homolog of Nvy, acts as a transcriptional repressor, it is suggested that VP16-Nvy acts as a transcriptional activator of targets that wild type Nvy normally represses. When expressed in a PNC, VP16-Nvy strongly reduces the amount of Dl observed at the cell surface and in intracellular vesicles. This result suggests that wild type Nvy has the potential to affect Dl, although the result does not distinguish an effect on expression from an effect on protein stability or trafficking. That ectopic Nvy does not inhibit the expression of Dl-lacZ suggests that Nvy may be more likely to transcriptionally regulate a factor is involved in Dl stability or trafficking. Regardless of the mechanism, the finding that VP16-Nvy reduces Dl levels suggests that wild type Nvy has the potential to increase Dl levels, a proposal that is consistent with loss- and gain-of-function experiments (Wildonger, 2005).

The VP16-Nvy results, while consistent with the idea that Nvy affects Dl, do not explain why no change in Dl levels were detected in nvy loss- and gain-of-function experiments. Thus, it is thought that Nvy causes a small and/or transient increase in Dl activity (by affecting its expression, stability or localization). Nevertheless, no change in the amount or localization of Dl in wild type SOPs has been observed, despite genetic evidence that Dl signaling is a critical step in SOP fate determination. The lack of an observable change in Dl during wild type development, in combination with the current findings, lead to a proposal that the presumptive SOP may send a transient pulse of increased Dl signal that is sufficient to bias cell fates within the PNC. Nvy may, therefore, contribute to this transient pulse of Dl (Wildonger, 2005).

The experiments described here shed some light on the molecular activities Nvy has in the SOP. (1) Based on its ability to repress well-defined lacZ reporter genes, Nvy appears to be a transcriptional repressor, as is its mammalian homolog ETO. (2) This study shows that ectopic Nvy appears to interfere with the function (as opposed to the expression) of Ac and Sc because re-supplying Ac and Sc in pnr-Gal4 UAS-nvy flies was unable to rescue the bald phenotype. In contrast, expression of Da, a bHLH DNA binding partner for Ac and Sc, was able to partially rescue the bald phenotype of pnr-Gal4 UAS-nvy flies. Moreover, nvy and da were found to genetically interact (e.g., reducing nvy levels enhanced a da gain-of-function phenotype), and Nvy and Da were found to physically interact. These findings are consistent with a recent report showing that ETO directly interacts with HEB, a bHLH factor in the same class as Da. The domain through which ETO interacts with HEB (and other mammalian class I bHLH transcription factors) is conserved in Nvy, and HEB's ETO interaction domain is found in Da. These data lead to a proposal that Nvy, a presumptive transcriptional repressor, has the ability to function with Ac/Da and Sc/Da heterodimers to repress the transcription of some target genes. In the absence of Nvy, such as in the non-SOP cells of a PNC, Ac/Da and Sc/Da may have the potential to activate these same target genes. However, these experiments also suggest that the interaction between Nvy and Da may not be required for all of Nvy's functions because VP16-Nvy is able to lower Dl levels even in da mutant clones. One potential explanation for this Da-independent function is that Nvy may be able to directly interact with DNA. In summary, it is speculated that the Nvy–Da interaction is only required for the regulation of a subset of target genes (Wildonger, 2005).

The proposal that Nvy works with Ac/Da and Sc/Da to repress target genes may on the surface seem at odds with the suggestion that Nvy can transiently increase the levels of Dl, because it is thought that Ac/Da and Sc/Da heterodimers activate Dl expression in the SOP. However, it is not known if Dl levels are in fact directly increased by Ac/Sc. It is stressed that the timing of expression of these genes is critical to understanding how they function in vivo. Based on the wild type timing of its expression, nvy is likely to be a target of Ac/Sc in the presumptive SOP. Accordingly, there will be a window of time when Ac/Sc levels are high and Nvy levels are low in the presumptive SOP. This window of time may be sufficient for Ac/Sc to affect Dl expression and initiate the bias in favor of the SOP fate. Once Nvy levels increase, it may then work with Ac/Sc to repress the expression of some target genes, some of which may cause a further increase in Dl signaling. However, it is hypothesized that nvy's role in this process is after the bias has already been initiated (Wildonger, 2005).

In summary, it is suggested that Nvy plays a subtle but observable role in the establishment of the SOP fate. Although it is not essential for the SOP fate, it may be that Nvy helps the SOP/non-SOP bias by increasing the strength of the Dl signal sent by the SOP. Because nvy is evolutionarily conserved, both in its protein sequence and nervous system expression, it is suggested that this role, although subtle, is important for the stereotyped uniformity of mechanosensory organ development. In addition, nvy may also play a role in later stages of neurogenesis, in particular axon pathfinding. Because of Nvy's role as a transcriptional repressor, it is further suggested that Nvy increases the Dl signal indirectly, by repressing a gene (factor X) that normally inhibits Dl activity. Based on Nvy's ability to interact with Da, this hypothetical target may be repressed by Nvy in combination with Ac/Da and Sc/Da heterodimers. Interestingly, it follows that in non-SOP cells of the PNC, which express ac and sc but not nvy, this hypothetical target may continue to be expressed, helping to downregulate Dl activity in these cells and thereby further increase the SOP/non-SOP bias. Clearly, the test of this proposal requires the identification of factor X as well as a more detailed understanding of how Dl levels and activity are modulated in the SOP (Wildonger, 2005).

PROTEIN STRUCTURE

Amino Acids - 743

Structural Domains

Protein structure of Nervy is described at Ensembl. The mammalian homolog of Nervy, ETO, shows two unusual, putative zinc-fingers, three proline-rich regions, a PEST domain and several phosphorylation sites.

EVOLUTIONARY HOMOLOGS

Structure and expression of Nervy homologs

The t(8;21) translocation associated with acute myeloid leukemia (AML) disrupts two genes, the AML1 gene also known as the core binding factor A2 (CBFA2) on chromosome 21, and a gene on chromosome 8, hereafter referred to as MTG8, but also known as CDR and ETO. Extensive information is available on AML1, a member of the CBF family of transcription factors, containing a highly conserved domain, the runt box, of the Drosophila segmentation gene runt. This gene is essential for the hematopoietic development and is found disrupted in several leukemias. In contrast, the function of the MTG8 gene is poorly understood. The predicted protein sequence shows two unusual, putative zinc-fingers, three proline-rich regions, a PEST domain and several phosphorylation sites. In addition, a region was found encompassing aa 443-514 that was predicted to have a significant propensity to form coiled coil structures. MTG8 displays a high degree of similarity with nervy, a homeotic target gene of Drosophila, expressed in the nervous system. Human and mouse wild-type MTG8 are also highly expressed in brain relative to other tissues. For these reasons, the expression and subcellular localization of the MTG8 protein in neural cells was investigated. Immunohistochemical experiments in a 12.5-day-old mouse embryo clearly show that the protein was expressed in the neural cells of the developing brain and the spinal cord. In primary cultures of hippocampal neurons of 2-3 day-old mice, MTG8 is found in the nucleus, in the cytoplasm and as fine granules in the neurites. Cytoplasmic localization of the protein is observed in Purkinje cells of both human and mouse cerebellum. The molecular mass of MTG8 in total human and mouse brain was analysed by immunoblotting and determined to be between 70 and 90 kDa. Isoforms with the same molecular mass were demonstrated in synaptosomes isolated from mouse forebrain. The evidence of MTG8 in the nucleus and cytoplasm of neural cells suggests a specific mechanism regulating the subcellular localization of the protein (Sacchi, 1998).

MTG8 (HGMW-approved symbol: CBFA2T1) was originally identified as one of the loci involved in the t(8;21)(q22;q22) of acute myeloid leukemia. Two human MTG8-related genes, MTGR1 and MTGR2 (HGMW-approved symbols: CBFA2T2 and CBFA2T3) have been characterized. The former is duplicated in mouse, one locus possibly being a retroposon. Multiple MTG8-related sequences are found in several vertebrate species, from fish to mammals, albeit not in a urodele. MTGR2 maps to 16q24 and, like MTG8 and MTGR1, is close to one of three loci encoding a syntrophin (dystrophin-associated proteins). Moreover, an alternative MTGR1 promoter/5' exon is contained within the alpha1-syntrophin locus. Thus, the two classes of genes may define novel paralogous groups. MTGR1 is expressed mainly in brain, while MTGR2 is expressed in the thymus and possibly in monocytes. Like MTG8, MTGR1 is transcribed into a number of isoforms due to alternative splicing of different 5' exons onto a common splice acceptor site. Comparison of the three predicted human MTG8-related polypeptides to their Drosophila counterpart (Nervy) highlights four separate regions of sequence conservation that may correspond to distinct domains. The most NH2-terminal of these is proportionately more conserved among the human polypeptides, presumably due to specific structural/functional constraints (Calabi, 1998).

Nervy homologs serve as an AKAP by interacting with a subunit of PKA

MTG8 interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RIIalpha). The binding site of MTG8 is NHR3 domain, and that of RIIalpha is the N-terminus for interacting with PKA anchoring proteins (AKAPs). NHR3 contains a putative alpha-amphipathic helix which is characteristic in binding of AKAPs with RII. Indirect immunofluorescence microscopy showed that MTG8 and RIIalpha overlap at the centrosome-Golgi area in lymphocytes. These findings suggest that MTG8 may function as an AKAP at the centrosome-Golgi area in lymphocytes (Fukuyama, 2001).

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatoryresponse, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, -- AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes (Schillace, 2002).

Nervy homologs interact with a repressor complex

The t(8;21) translocation between two genes known as AML1 and ETO is seen in approximately 12%-15% of all acute myeloid leukemia (AML) and is the second-most-frequently observed nonrandom genetic alteration associated with AML. AML1 up-regulates a number of target genes critical to normal hematopoiesis, whereas the AML1/ETO fusion interferes with this trans-activation. The fusion partner ETO binds to the human homolog of the murine nuclear receptor corepressor (N-CoR). The interaction is mediated by two unusual zinc finger motifs present at the carboxyl terminus of ETO. Human N-CoR (HuN-CoR), which was cloned and sequenced in its entirety, encodes a 2,440-amino acid polypeptide and has a central domain that binds ETO. N-CoR, mammalian Sin3 (mSin3A and B), and histone deacetylase 1 (HDAC1) form a complex that alters chromatin structure and mediates transcriptional repression by nuclear receptors and by a number of oncoregulatory proteins. ETO, through its interaction with the N-CoR/mSin3/HDAC1 complex, is also a potent repressor of transcription. This observation provides a mechanism for how the AML1/ETO fusion may inhibit expression of AML1-responsive target genes and disturb normal hematopoiesis (Wang, 1998).

Nuclear receptor corepressor (CoR)-histone deacetylase (HDAC) complex recruitment is indispensable for the biological activities of the retinoic acid receptor fusion proteins of acute promyelocytic leukemias. ETO (eight-twenty-one or MTG8), which is fused to the acute myelogenous leukemia 1 (AML1) transcription factor in t(8;21) AML, interacts via its zinc finger region with a conserved domain of the corepressors N-CoR and SMRT and recruits HDAC in vivo. The fusion protein AML1-ETO retains the ability of ETO to form stable complexes with N-CoR/SMRT and HDAC. Deletion of the ETO C terminus abolishes CoR binding and HDAC recruitment and severely impairs the ability of AML1-ETO to inhibit differentiation of hematopoietic precursors. These data indicate that formation of a stable complex with CoR-HDAC is crucial to the activation of the leukemogenic potential of AML1 by ETO and suggest that aberrant recruitment of corepressor complexes is a general mechanism of leukemogenesis (Gelmetti, 1998).

ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription (Lutterbach, 1998).

Distinct domains of ETO contact the mSin3A and N-CoR corepressors; two binding sites within ETO are described for each of these corepressors. Of eight histone deacetylases (HDACs) tested, only the class I HDACs HDAC-1, HDAC-2, and HDAC-3 bind ETO. However, these HDACs bind ETO through different domains. The murine homologue of MTG16, ETO-2, is also a transcriptional corepressor that works through a similar but distinct mechanism. Like ETO, ETO-2 interacts with N-CoR, but ETO-2 fails to bind mSin3A. Furthermore, ETO-2 binds HDAC-1, HDAC-2, and HDAC-3 but also interacts with HDAC-6 and HDAC-8. Expression of AML-1-ETO causes disruption of the cell cycle in the G(1) phase. Disruption of the cell cycle requires the ability of AML-1-ETO to repress transcription because a mutant of AML-1-ETO, Delta469, which removes the majority of the corepressor binding sites, has no phenotype. Moreover, treatment of AML-1-ETO-expressing cells with trichostatin A, an HDAC inhibitor, restores cell cycle control. Thus, AML-1-ETO makes distinct contacts with multiple HDACs and an HDAC inhibitor biologically inactivates this fusion protein (Amann, 2001).

Nervy homologs as transcriptional co-repressors

The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which binds to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO is completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase (Melnick, 2000).

DRPLA is one of the family of neurodegenerative diseases caused by expansion of a polyglutamine tract. The drpla gene product, atrophin-1 (Drosophila homolog: Grunge), is widely expressed, has no known function or activity, and is found in both the nuclear and cytoplasmic compartments of neurons. Truncated fragments of atrophin-1 accumulate in neuronal nuclei in a transgenic mouse model of DRPLA, and may underlie the disease phenotype. Using the yeast two-hybrid system, studies have identified ETO/MTG8, a component of nuclear receptor corepressor complexes, as an atrophin-1-interacting protein. When cotransfected into Neuro-2a cells, atrophin-1 and ETO/MTG8 colocalize in discrete nuclear structures that contain endogenous mSin3A and histone deacetylases. These structures are sodium dodecyl sulfate-soluble and are associated with the nuclear matrix. Cotransfection of ETO/MTG8 with atrophin-1 recruits atrophin-1 to the nuclear matrix, while atrophin-1 and ETO/MTG8 cofractionate in nuclear matrix preparations from brains of DRPLA transgenic mice. Furthermore, in a cell transfection-based assay, atrophin-1 represses transcription. Together, these results suggest that atrophin-1 associates with nuclear receptor corepressor complexes and is involved in transcriptional regulation. Emerging links between disease-associated polyglutamine proteins, nuclear receptors, translocation-leukemia proteins, and the nuclear matrix may have important repercussions for the pathobiology of this family of neurodegenerative disorders (Wood, 2000).

Nearly 40% of cases of acute myelogenous leukemia (AML) of the M2 subtype are due to a chromosomal translocation that combines a sequence-specific DNA binding protein, AML1, with a potent transcriptional repressor, ETO. ETO interacts with nuclear receptor corepressors SMRT and N-CoR, which recruit histone deacetylase to the AML1-ETO oncoprotein. SMRT-N-CoR interaction requires each of two zinc fingers contained in C-terminal Nervy homology region 4 (NHR4) of ETO. However, polypeptides containing NHR4 are insufficient for interaction with SMRT. NHR2 is also required for SMRT interaction and repression by ETO, as well as for inhibition of hematopoietic differentiation by AML1-ETO. NHR2 mediates oligomerization of ETO as well as AML1-ETO. Fusion of NHR4 polypeptide to a heterologous dimerization domain allows strong interaction with SMRT in vitro. These data support a model in which NHR2 and NHR4 have complementary functions in repression by ETO. NHR2 functions as an oligomerization domain bringing together NHR4 polypeptides that together form the surface required for high-affinity interaction with corepressors. Since nuclear receptors also interact with corepressors as dimers, oligomerization may be a common mechanism regulating corepressor interactions (Zhang, 2001).

E protein silencing by the leukemogenic AML1-ETO fusion protein

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 15% of acute myeloid leukemia (AML) cases. This study shows that AML1-ETO, as well as ETO (Drosophila homolog: Nervy), inhibits transcriptional activation by E proteins (e.g. Drosophila Daughterless) through stable interactions that preclude recruitment of p300/CREB-binding protein (CBP) coactivators. These interactions are mediated by a conserved ETO TAF4 homology domain and a 17-amino acid p300/CBP and ETO target motif within AD1 activation domains of E proteins. In t(8;21) leukemic cells, very stable interactions between AML1-ETO and E proteins underlie a t(8;21) translocation-specific silencing of E protein function through an aberrant cofactor exchange mechanism. These studies identify E proteins as AML1-ETO targets whose dysregulation may be important for t(8;21) leukemogenesis, as well as an E protein silencing mechanism that is distinct from that associated with differentiation-inhibitory proteins (Zhang, 2004).

Nervy homologs and leukemia

Leukemic disease can be linked to aberrant gene expression. This often is the result of molecular alterations in transcription factors that lead to their misrouting within the nucleus. The acute myelogenous leukemia-related fusion protein AML1ETO is a striking example. It originates from a gene rearrangement t(8;21) that fuses the N-terminal part of the key hematopoietic regulatory factor AML1 (RUNX1) to the ETO (MTG8) repressor protein. AML1ETO lacks the intranuclear targeting signal of the wild-type AML1 and is directed by the ETO component to alternate nuclear matrix-associated sites. To understand this aberrant subnuclear trafficking of AML1ETO, a series of mutations in the ETO protein was created. These were characterized biochemically by immunoblotting and in situ by immunofluorescence microscopy. Two independent subnuclear targeting signals were identified in the N- and C-terminal regions of ETO that together direct ETO to the same binding sites occupied by AML1ETO. However, each segment alone is targeted to a different intranuclear location. The N-terminal segment contains a nuclear localization signal and the conserved hydrophobic heptad repeat domain responsible for protein dimerization and interaction with the mSin3A transcriptional repressor. The C-terminal segment spans the nervy domain and the zinc finger region, which together support interactions with the corepressors N-CoR and HDACs. These findings provide a molecular basis for aberrant subnuclear targeting of the AML1ETO protein, which is a principal defect in t(8;21)-related acute myelogenous leukemia (Barseguian, 2002).

Developmental roles of Nervy homologs

The interaction between early proneural genes and lateral inhibition determines the number of primary neurons. The mechanism for regulating the size of the proneural domain, however, has not been clarified. This study shows that inhibition of the function of XETOR in Xenopus, a homolog of human oncoprotein ETO/MTG8, leads to a neurogenic phenotype of expanded proneural domain without alteration in the density of primary neurons. This result suggests that XETOR is a prerequisite for regulating the size of the proneural domain. Such a regulation is accomplished by establishing a negative feedback loop between XETOR and proneural genes except Xngnr-1, as well as by antagonism between XETOR and lateral inhibition (Cao, 2002).

The putative transcriptional corepressor ETO/MTG8 has been extensively studied due to its involvement in a chromosomal translocation causing the t(8;21) form of acute myeloid leukemia. Despite this, the role of ETO in normal physiology has remained obscure. ETO is highly expressed in preadipocytes and acts as an inhibitor of C/EBPbeta during early adipogenesis, contributing to its characteristically delayed activation. ETO prevents both the transcriptional activation of the C/EBPalpha promoter by C/EBPbeta and its concurrent accumulation in centromeric sites during early adipogenesis. ETO expression rapidly reduces after the initiation of adipogenesis, and this is essential to the normal induction of adipogenic gene expression. These findings define, for the first time, a molecular role for ETO in normal physiology as an inhibitor of C/EBPbeta and a novel regulator of early adipogenesis (Rochford, 2004).

A TAF4-homology domain from the corepressor ETO is a docking platform for positive and negative regulators of transcription

ETO, is implicated in 12%-15% of acute human leukemias as part of a gene fusion with RUNX1. Of the four ETO domains related to Drosophila Nervy, only two are required to induce spontaneous myeloid leukemia upon transplantation into the mouse. One of these domains is related in sequence to TAF4, a component of TFIID. The structure of this domain, ETO-TAFH, is similar to yeast Rpb4 and to Escherichia coli sigma(70); it is the first TAF-related protein with structural similarity to the multisubunit RNA polymerases. Overlapping surfaces of ETO-TAFH interact with an autonomous repression domain of the nuclear receptor corepressor N-CoR and with a conserved activation domain from the E-box family of transcription factors. Thus, ETO-TAFH acts as a structural platform that can interchange negative and positive coregulatory proteins to control transcription (Wei, 2007).

date revised: 3 August 2005

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