BIOLOGICAL OVERVIEW

Mammalian JUN is a transcription factor and oncogene. It is activated through RAS/MAPK-mediated phosphorylation. In mammalian cells JUN interacts with FOS, another basic leucine zipper protein (See Drosophila Fos-related antigen). AP-1 is a FOS-JUN heterodimer that serve to activate genes involved in neural function and the immune response. Multiple FOS and JUN proteins are found in mammals. Most of the genes encoding AP-1 components behave as "immediate early" genes, that is, genes whose transcription is rapidly induced following cell stimuation, independent of de novo protein synthesis. JUN transcriptional activity is regulated by phosphorylation carried out by JUN kinase, which in turn is activated by phosphorylation cascades initiated by growth factor signals (Karin, 1995).

In the Drosophila eye, Jun-related antigen is activated through the Boss-Sevenless pathway. The ligand Boss is secreted by R8 photoreceptor cells and triggers a phosphorylation cascade through the receptor tyrosine kinase Sevenless. Sevenless is found on the surface of cells for only a limited time restricting the number of cells that can turn into photoreceptors (Treier, 1995). Similarly, Jun is upregulated on either side of the morphogenetic furrow (Bohmann, 1994), and thus acts in only a restricted number of cells. Sevenless signaling activates the RAS-MAPK pathway. Rolled MAPK in turn activates JRA, again by means of phosphorylation.

Jun-related antigen cooperates with the ETS domain protein Pointed to induce R7 fate in the developing eye. Jun-related antigen and Pointed are presumed to act on common targets to promote the R7 photoreceptor fate. After a decade of research, a role for Jun-related antigen has been found in only one phase of differentiation, the determination of the R7 photoreceptor. Where else is JUN involved in cell differentiation? The next decade may reveal more targets, following clues provided by an examination of the Jra promoter.

As in mammalian systems, Jun-related antigen is activated by AP-1, or said another way, it is subject to autoregulation. In addition, a cyclic AMP response element (CRE) is found, suggesting that Jra is responsive to regulation by CREB, the cyclic AMP response element binding protein. In fact, deletion of the CRE reduces by half the Jra promoter activity. The involvement of cAMP and CREB in Jra transcription suggests Jra is involved in memory. An additional three ecdysone response elements are found in an upstream region. Since ecdysone regulates molting this suggests that Jra is regulated during molting (Wang, 1994). This could implicate Jra in formation of adult neural structures.

The Drosophila homolog of c-Jun regulates epithelial cell shape changes during the process of dorsal closure in mid-embryogenesis. A mutation in Fra shows defects in dorsal closure and also interacts with mutations in Jra. Like Jra mutations, Fra null alleles completely block shape changes that normally occur in the leading edge of the lateral epithelium during dorsal closure. In dorsal closure, Fra cooperates with Jra by regulating the expression of dpp; Dpp acts as a relay signal that triggers cell shape changes and Fra expression in neighboring cells (Riesgo-Escovar, 1997b).

In vertebrates, c-Jun and c-Fos activities are regulated at various levels. Whereas c-Jun is widely expressed at low levels and activated primarily by NH2-terminal phosphorylation by JNKs, c-fos expression is dynamic and activated in response to various extracellular stimuli. A similar dichotomy of Jra and Fra regulation occurs in Drosophila: Jra is widely expressed during embryogenesis and is phosphorylated by DJNK (Basket), whereas Fra expression is dynamic. There is strong expression of Fra in leading edge cells and cells of the lateral epithelium. Expression of Fra in the lateral epithelium is reduced in thick veins and punt mutant embryos but is still detectable in the leading edge. Therefore, not only does Fra control expression of dpp in the leading edge, but in a reciprocal manner, Fra expression is dependent on Dpp function in cells of the lateral epithelium. Similarly, in late embryos, Fra expression in the endoderm depends on dpp expression in the overlying visceral mesoderm. Activation of the Dpp signaling pathway is indeed sufficient to activate Fra expression. Ectopic expression of an activated Dpp receptor (Thickveins) in a segmental pattern results in a corresponding pattern for Fra expression. It is concluded that Fra may be required in all ectodermal cells in order to activate target genes required for cell shape changes (Riesgo-Escovar, 1997b).

In addition to the joint requirement of Fra and Jra during dorsal closure, Fra functions independently of Jra during embryogenesis. Early dpp expression on the dorsal side of embryos induces expression of several genes, including race, which encodes a protein with homology to angiotensin-converting enzyme in the amnioserosa. The race cis-acting sequences required for dpp-mediated expression contain AP-1 binding sites. Consistent with Fra-mediated direct activation of race through these AP-1 sites, race expression in the amnioserosa is abolished in Fra mutants. In contrast, race expression is normal in Jra or basket mutant embryos. This early Jra-independent function of Fra may be mediated by a Fra homodimer. During wound healing in vertebrates (a process that exhibits parallels with dorsal closure), TGF-beta induces c-fos expression and AP-1 activity. The reciprocal regulatory relation between Fra and dpp in Drosophila appears to be conserved in mammalian cells. In mammalian myeloid cells, induction of c-jun and c-fos by serum or oncogenic v-src results in expression of TGF-beta1 by direction activation of TGF-beta1 transcription by AP1. TGF-beta induces AP-1 activity in keratinocytes during wound healing. These findings demonstrate common and distinct roles for Fra and Jra during embryogenesis and suggest a conserved link between AP-1 (activating protein-1) and TGF-beta (transforming growth factor-beta) signaling during epithelial cell shape changes (Riesgo-Escovar, 1997b).

Normal dendrite growth in Drosophila motor neurons requires the AP-1 transcription factor

During learning and memory formation, information flow through networks is regulated significantly through structural alterations in neurons. Dendrites, sites of signal integration, are key targets of activity-mediated modifications. Although local mechanisms of dendritic growth ensure synapse-specific changes, global mechanisms linking neural activity to nuclear gene expression may have profound influences on neural function. Fos, being an immediate-early gene, is ideally suited to be an initial transducer of neural activity, but a precise role for the AP-1 transcription factor in dendrite growth remains to be elucidated. This study measured changes in the dendritic fields of identified Drosophila motor neurons in vivo and in primary culture to investigate the role of the immediate-early transcription factor AP-1 in regulating endogenous and activity-induced dendrite growth. The data indicate that (1) increased neural excitability or depolarization stimulates dendrite growth, (2) AP-1 (a Fos, Jun heterodimer) is required for normal motor neuron dendritic growth during development and in response to activity induction, and (3) neuronal Fos protein levels are rapidly but transiently induced in motor neurons following neural activity. Taken together, these results show that AP-1 mediated transcription is important for dendrite growth, and that neural activity influences global dendritic growth through a gene-expression dependent mechanism gated by AP-1 (Hartwig, 2009).

Like CREB, AP-1 (Fos and Jun dimer) is likely to be an important activity-dependent regulator of dendritic plasticity. Fos is upregulated rapidly in neuronal populations following activity and can generate a rapid genomic response to incoming stimuli since it is an immediate-early gene not requiring protein synthesis for its own induction. At the Drosophila NMJ AP-1 controls both structural and functional aspects of long-term plasticity (Hartwig, 2009).

To test if AP-1 plays a role in activity-dependent dendritic plasticity, experimental systems were established to study dendrites of the RP2 motor neuron in vivo in the larval CNS, and this was complemented with an in vitro culture system using a strategy to enrich for larval motor neurons. This dual analysis utilizes technical strengths of both systems and permits cross-validation of results. Results reported in this study support three main conclusions: (1) motor neuron dendrites in Drosophila show activity, or depolarization, dependent plasticity, (2) normal dendrite growth requires AP-1, and (3) activity or depolarization driven dendritic growth is gated by AP-1 (Hartwig, 2009).

It was important to initially establish that motor neuron dendrites in Drosophila display robust changes in response to neural activity. Both pharmacological and genetic manipulations were used to alter neural activity in vivo and in vitro. Maintaining cultured motor neurons in high K+ medium enhanced the growth of neurites that have the characteristics of dendrites. Expression of dominant-negative subunits of the potassium channels Eag and Sh, increased excitability and caused increased dendrite growth both in vivo and in vitro. These experiments suggest that a conserved mechanism of plasticity operates in these motor neurons to regulate dendritic growth. Additionally, by developing the first assay for activity-stimulated dendritic growth in Drosophila, a wide range of experiments were enabled to further dissect underlying signaling and cell biological pathways (Hartwig, 2009).

It is worth noting that the cell culture approach allowed the comparison of acute depolarization (high potassium) with the more chronic alteration of excitability caused by the expression of eag and shaker dominant-negative transgenes. Despite the interesting possibility of compensatory changes following long-term transgenic manipulations, the functional properties of larval motor neurons in vivo were stably altered by reducing potassium channel function. By contrast, the effects of elevated potassium reflect an acute manipulation after cells are placed in culture. In spite of these differences the effects of expression of a recombinant including both UAS-Sh(DN) and UAS-eag(DN) termed 'Electrical Knock In (EKI)' on cell growth in vitro were similar to those induced by elevated potassium. The lack of an effect on outgrowth by UAS-Sh(DN) and UAS-Sh[act] (EKO) expression may reflect a cellular compensatory mechanism. Although effects on cell physiology were detected, the difference in excitability measured in vivo was not significant. Two alternatives are that there is a basal level of normal growth that is not activity-sensitive, or that reduced activity had an influence on growth or branching that the analysis did not detect (Hartwig, 2009).

It is suggested that the depolarization by high potassium or the increased excitability caused by EKI expression would lead to an increase in calcium influx through voltage-dependent channels. The voltage-dependent properties of Eag and Shaker potassium channels suggest that EKI expression would not necessarily be expected to alter the resting membrane potential. Even without altering the resting membrane potential, however, the reduction in voltage-dependent potassium currents might allow increased calcium influx in response to spontaneous depolarization or calcium waves. This would be augmented, in vivo, by the higher action potential frequency evoked by depolarization of motor neurons in EKI larvae as compared to wild type. It is also possible that the expression of eag-DN alters a modulatory function of the potassium channel subunit. Consistent with this hypothesis, it was found that inhibiting PLTX-sensitive calcium channels prevents EKI mediated neurite outgrowth. A similar dependence of neuronal growth on calcium channels has been demonstrated convincingly in the Drosophila giant neuron culture system. Ultimately, it will be important to establish the specific parameters of activity and calcium flux that are essential for modulating the intracellular signals that mediate growth plasticity (Hartwig, 2009).

It has been shown previously that AP-1 regulates synaptic plasticity at Drosophila motor terminals. The present results uncover a role for AP-1 in regulating dendritic growth. Reduction of AP-1 activity decreases, while enhancement of AP-1 increases, dendritic outgrowth in vivo and in vitro. These conclusions are further strengthened by the fact that known and predicted loss-of-function alleles of kayak (DFos) also reduce dendrite volume significantly, when present in heterozygous combinations with wild-type alleles. It is to be noted that these mutations in Fos are homozygous lethal at early developmental stages, and transgenic strategies for tissue specific genetic manipulations are especially useful in these contexts. Additionally, multiple Fos isoforms have been reported, and using a dominant negative construct offers a good way to inhibit these multiple Fos proteins. In conclusion, the observations that (1) Fos is normally detectable in motor neuron nuclei, (2) Fbz, Jbz and Fos-RNAi inhibit and AP-1 increases dendrite growth, (3) loss-of-function alleles of kayak decrease dendrite volume, and (4) a variety of controls validate the genetic perturbations used in this study, strongly suggest that AP-1 functions physiologically to control dendrite growth (Hartwig, 2009).

To test if neural activity driven growth requires AP-1 dependent transcription, AP-1 function was inhibitied in a background of elevated neural activity. The results indicate that neural activity induced dendrite growth is completely abolished in vivo by the coexpression of Fbz. Further, in vitro experiments with High K+ induced dendrite growth show that expression of either the domainant negative bZip domain of either dFos or dJun (Fbz or Jbz) substantially reduces the extent of depolarization induced growth. The results also indicate that the extent of overgrowth seen through AP-1 induction cannot be increased any further by High K+ induced depolarization. Furthermore, calcium channel inhibition by PLTX toxin does not preclude AP-1 driven neurite growth, suggesting that AP-1 functions downstream of neural activity and calcium entry to enhance neurite growth. These results are consistent with the idea that AP-1 is a major contributor to activity-induced plasticity of dendrites (Hartwig, 2009).

Finally, to ascertain the mechanism by which neural activity might influence AP-1 function, expression of Fos protein was tested following acute induction of activity in cultured motor neurons. Fos protein levels are increased by ~35% as compared to uninduced controls. This induction is rapid, occurring within 4 h, but transient, since Fos levels return to baseline during 20 h of stimulation. Although modest, this change in cellular Fos levels is consistent with previous observations and suggests, though it does not prove, a model where neural activity recruits AP-1 possibly through synthesis of new Fos protein, to promote dendrite growth. Hence, the results confirm that AP-1, positively regulates plasticity in postsynaptic compartments, as has been demonstrated for presynaptic terminals, and establishes AP-1 as a key component in activity-dependent neuronal plasticity (Hartwig, 2009).

A key goal for future studies must be to determine how neural activity is translated into transcription factor activity. Calcium dependent signaling is arguably the most important pathway for the activation of both CREB and Mef2A, two transcription factors that influence plasticity in opposite ways, as well as AP-1. A second question is that of interaction between transcription factors. Transcriptional regulation of Fos by CREB (and vice versa) has been described in some detail, but the temporal sequence in which these proteins function or their relative importance in plasticity have not been assayed rigorously in the same preparation. Finally, further experiments to identify the downstream targets of these transcription factors, will enable the description of a composite transcription factor network that mediates protein synthesis during long-term neural plasticity (Hartwig, 2009).

HP1a/KDM4A is involved in the autoregulatory loop of the oncogene gene c-Jun

The proto-oncogene c-Jun plays crucial roles in tumorigenesis, and its aberrant expression has been implicated in many cancers. Previous studies have shown that the c-Jun gene is positively autoregulated by its product. Notably, it has also been reported that c-Jun proteins are enriched in its gene body region. However, the role of c-Jun proteins in its gene body region has yet to be uncovered. HP1a is an evolutionarily conserved heterochromatin-associated protein, which plays an essential role in heterochromatin-mediated gene silencing. Interestingly, accumulating evidence shows that HP1a is also localized to euchromatic regions to positively regulate gene transcription. However, the underlying mechanism has not been defined. This study demonstrates that HP1a is involved in the positive autoregulatory loop of the Jra gene, the c-Jun homologue in Drosophila. Jra recruited the HP1a/KDM4A complex to its gene body region upon osmotic stress to reduce H3K36 methylation levels and disrupt H3K36 methylation-dependent histone deacetylation, resulting in high levels of histone acetylation in the Jra gene body region, thus promoting gene transcription. These results not only expand knowledge towards the mechanism of c-Jun regulation, but also reveal the mechanism by which HP1a exerts its positive regulatory function in gene expression (Liu, 2015).

In order to identify Jra interacting partners in Drosophila, the Jra complex was purified from S2 cells expressing FLAG-tagged Jra. Cells were first treated with or without 500 mM sorbitol for 30 minutes, which induces osmotic stress and activates the MAPK signaling pathway and Jra. The nuclear extracts prepared from these cells were subjected to complex purification using anti-FLAG antibody. The mass spectrometry data showed that, among other Jra interacting partners, heterochromatin protein HP1a co-purifies with Jra-FLAG under osmotic stress. The interaction between Jra-FLAG and HP1a was further confirmed by Western blot analysis. Interestingly, Jra only co-immunoprecipitates with HP1a under osmotic stress, but not under unstressed conditions (Liu, 2015).

To further determine whether HP1a interacts with endogenous Jra, nuclear extracts from S2 cells treated with or without osmotic stress were subjected to co-immunoprecipitation assay using anti-Jra antibody. The results confirmed that endogenous Jra co-immunoprecipitates with HP1a under osmotic stress. Taken together, these data demonstrate that HP1a interacts with Jra under osmotic stress (Liu, 2015).

Jra has been previously shown to bind to its gene body region. Having found that Jra interacts with HP1a, it was reasoned that Jra might recruit HP1a to its gene body region. To answer this question, a chromatin immunoprecipitation (ChIP) assay was performed to determine whether HP1a binds to the Jra gene locus. HP1a did not localized to either the promoter or the gene body region of Jra under unstressed conditions. However, under osmotic stress, HP1a is enriched in the gene body region of Jra, but not in the promoter region. Western blot assay confirmed that Jra is phosphorylated under osmotic stress. Phosphorylation is an important post-translational modification for c-Jun regulation, which has previously been shown to regulate its transcriptional activity and its protein stability. The ChIP result combined with co-immunoprecipitation data indicate that phosphorylated Jra, but not unphosphorylated Jra, interacts with HP1a (Liu, 2015).

Next, attempts were made to determine if HP1a binding to the Jra gene body region is dependent on Jra phosphorylation. To address this concern, S2 cells were treated with JNK dsRNA to knock down JNK expression. JNK is the protein kinase in MAPK signaling pathway, which is responsible for c-Jun phosphorylation. S2 cells were also treated with GFP dsRNA as a control. Western blot result confirmed that the expression levels of JNK were dramatically reduced in in JNK dsRNA treated cells, and that the phosphorylation of Jra was greatly reduced in JNK-depleted cells under osmotic stress. These cells were then analyzed by ChIP assay using anti-HP1a antibody. The data showed that upon the depletion of JNK, HP1a lost its binding to the Jra gene body under osmotic stress, suggesting that phosphorylated Jra recruits HP1a to its gene body region (Liu, 2015).

In the heterochromatin region, HP1a binding is dependent on H3K9 methylation. Therefore, whether H3K9 methylation also plays a role in HP1a recruitment to the Jra gene body was examined. Cells treated with or without osmotic stress were subjected to ChIP assay using anti-H3K9me2 antibody. The data showed that there was no significant change in H3K9me2 levels in the Jra gene body region, eliminating the regulatory role of H3K9 methylation in the recruitment of HP1a to the Jra gene body region. Taken together, these results showed that Jra recruits HP1a to its gene body region under osmotic stress, which is independent of H3K9 methylation (Liu, 2015).

Since an increased enrichment of HP1a was observed in the Jra gene body region under osmotic stress, attempts were made to examine the effect of HP1a on Jra gene expression. S2 cells were treated with HP1a dsRNA to knock down HP1a expression. S2 cells treated with GFP dsRNA were used as a control. The depletion of HP1a was confirmed by Western blot assay. The total RNA extracted from these cells was then analyzed by qRT-PCR. The results showed that HP1a knockdown significantly reduced Jra mRNA levels, indicating HP1a is positively involved in Jra transcription (Liu, 2015).

Having defined a role for HP1a in positively regulating Jra transcription, the mechanism by which this occurs was explored. A previous study reported that HP1a interacts with Drosophila KDM4A demethylase and stimulates its H3K36 demethylation activity (Lin, 2008). Since the enrichment of HP1a in the Jra gene body region was observed, and H3K36me3 modification has been reported to be enriched in the gene body region, it was asked whether HP1a collaborates with KDM4A to reduce H3K36me3 levels in the Jra gene body region. To test this idea, the H3K36me3 levels were measured of the Jra gene body region upon HP1a depletion. The results showed that HP1a depletion significantly elevated H3K36me3 levels in the Jra gene body region, indicating a potential involvement of KDM4A in Jra transcription. To verify the recruitment of KDM4A to the Jra gene body, an S2 cell line was established stably expressing KDM4A-FLAG. These cells were treated with or without osmotic stress and subjected to ChIP analysis. The results demonstrate that KDM4A is enriched in Jra gene body region upon osmotic stress, and the depletion of HP1a abolishes its binding to the Jra gene body region (Liu, 2015).

H3K36me3 has been shown to be essential in preventing cryptic transcription from the gene body region. Following RNAP II elongation, histone acetylation marks are removed by the Rpd3S complex, which is directed by H3K36me3 in the gene body region. Because elevated levels of H3K36me3 upon HP1a depletion were observed, the histone acetylation levels were examined in this region. As expected, the overall histone acetylation levels were significantly reduced upon HP1a depletion (Liu, 2015).

It should be noted that a previous study has demonstrated that HP1a positively regulates euchromatic gene expression through its involvement in RNA packaging and stability. To determine if the reduced mRNA levels of Jra was due the decreased mRNA stability in HP1a-depleted cells, cells were first treated with 500mM sorbitol and then with actinomycin D for 30 minutes, 60 minutes, and 90 minutes. Total RNA was subsequently isolated for quantitative real-time PCR analysis. The result showed that in both wild-type S2 cells and HP1a-depleted S2 cells, Jra mRNA levels were slightly reduced. However, HP1a depletion did not significantly accelerate Jra mRNA turnover after actinomycin D treatment. Therefore, it is proposed that the reduced Jra expression that was observed is primarily due to the disruption of the positive role of HP1a in Jra transcription (Liu, 2015).

Although many studies reported that HP1a is not only localized to heterochromatin, but also localized to euchromatic regions to regulate gene transcription, the mechanism by which HP1a facilitates gene transcription is still unclear. It has also been established that c-Jun gene is under the control of a positive autoregulatory loop. In addition, c-Jun proteins have also been shown to bind to its own gene body region. However, what c-Jun does in its own gene body region has yet to be uncovered. This study has presented evidence showing that HP1a is involved in the positive autoregulatory loop of the Jra gene. Based on the data, a model is proposed in which Jra recruits the HP1a/KDM4A complex to its gene body region upon osmotic stress to reduce H3K36 methylation levels and disrupt H3K36 methylation-dependent histone deacetylation, resulting in high levels of histone acetylation in the Jra gene body region, and thus promotes gene transcription. These results not only expand knowledge of the mechanism by which the c-Jun oncogene is regulated, but also reveal the mechanism by which HP1a exerts its positive regulatory function in gene expression (Liu, 2015).

The transcription factor Ets21C drives tumor growth by cooperating with AP-1

Tumorigenesis is driven by genetic alterations that perturb the signaling networks regulating proliferation or cell death. In order to block tumor growth, one has to precisely know how these signaling pathways function and interplay. This study has identified the transcription factor Ets21C as a pivotal regulator of tumor growth, and a new model is proposed of how Ets21C could affect this process. A depletion of Ets21C strongly suppressed tumor growth while ectopic expression of Ets21C further increased tumor size. It was confirmed that Ets21C expression is regulated by the JNK pathway, and Ets21C was shown to acts via a positive feed-forward mechanism to induce a specific set of target genes that is critical for tumor growth. These genes are known downstream targets of the JNK pathway, and their expression was demonstrated to not only depend on the transcription factor AP-1, but also on Ets21C, suggesting a cooperative transcriptional activation mechanism. Taken together this study shows that Ets21C is a crucial player in regulating the transcriptional program of the JNK pathway and enhances understanding of the mechanisms that govern neoplastic growth (Toggweiler, 2016).

This study has identified the transcription factor Ets21C as a crucial regulator of tumor growth and demonstrates that its expression is activated by the JNK pathway. Moreover, Ets21C was shown to regulate the expression of specific target genes that induce and sustain growth and invasiveness of Ras^V12 dlg^RNAi tumors, possibly via a cooperation with AP-1 (Toggweiler, 2016).

The closest human orthologs of Ets21C ETS-related gene (ERG) and Friend leukemia virus-induced erythroleukemia 1 (FLI-1), have also been linked to tumorigenesis. ERG is overexpressed in acute myeloid leukemia (AML) and is associated with a poor prognosis, whereas higher FLI-1 expression has been detected in triple negative breast cancer or in metastatic melanoma. This study shows that Ets21C is not only sufficient to induce tumorigenesis, but required for tumor growth as a depletion of Ets21C strongly reduced tumor size. The smaller tumor size was accompanied by decreased levels of target genes known to drive tumor growth and malignancy, further underscoring the relevance of Ets21C. The finding that an Ets21C loss of function is blocking tumor growth so efficiently, suggests a more fundamental role for Ets21C in tumor growth than previously assumed. Kulshammer (2015) also tested an Ets21C^RNAi in Ras^V12, scrib^-/- tumors, but was not able to observe any remarkable effects besides a partial rescue of the pupariation delay that is commonly associated with neoplastic tumor growth in flies. An explanation for this discrepancy might simply be different strengths of the RNAi lines used (Toggweiler, 2016).

Loss of epithelial polarity caused by a depletion of dlg activates the JNK pathway that critically affects tumor growth. In agreement with previous studies, this study found elevated Ets21C transcript levels in Ras^V12 dlg^RNAi tumors and showed that JNK signaling is required for the upregulation. The JNK pathway activates the AP-1 transcription factor, which in its prototypical form is a dimer consisting of Jun and Fos proteins. In the Ras^V12, scrib^-/- context, Fos has been described as the main effector of the JNK pathway, as a depletion diminishes tumor growth and abrogates induction of target genes, while no such effect have been observed for Jun. However, the current data point towards at least a partial role for Jun activating target genes, since a knockdown of Jun reduced expression of Ets21C and Mmp1 (Toggweiler, 2016).

Given the strong effects that Ets21C exerts on tumor growth, attempts were made to identify Ets21C dependent target genes that could explain this phenomenon. Transcriptional profiles revealed an upregulation of Mmp1, Pvf1 and upd1 in tumors that overexpressed Ets21C and downregulated in tumors depleted of Ets21C. Mmp1, Pvf1 and upd1 have previously been shown to be induced by the JNK pathway and to be essential for tumor growth and invasion. Besides upd1, also upd2 and upd3 have been reported to be induced in neoplastic tumors in a JNK pathway dependent manner. In agreement with these reports, an induction was observed of upd2 and upd3 in Ras^V12 dlg^RNAi Ets21C tumors, but to a lesser extent than upd1. This might on the one hand depend on the use of dlg instead of scrib a stronger activation of Upd cytokines has been found in scrib mutant wing discs compared to dlg mutants. On the other hand, additional factors might influence transcriptional activation such as the presence of Ras^V12, the exact timing of sample collection or the genetic system used. Furthermore, the strength of induction might not necessarily correlate with the effect on tumor growth. Although upd3 exhibits the strongest upregulation in neoplastic tumors, it has been shown that co-expression of Upd1 and Upd2 together with Ras^V12 leads to much larger tumors than the combination of Ras^V12 and Upd3 ^{(Toggweiler, 2016).}

While Ets21C is able to stimulate the expression of the JNK downstream targets Mmp1, Pvf1 and upd1, no obvious regulation was observed of canonical JNK pathway targets such as the phosphatase puc or the apoptosis inducers hid or rpr, suggesting that Ets21C only regulates a specific set of JNK pathway targets. In contrast, a previous study has described a slight upregulation of puc in Ras^V12 Ets21C tumors. Trivial differences such as when the samples were collected, transgene strength, or genetic background could account for this difference. For example, an elevation of puc expression could also originate from an indirect increase in JNK activity due to stresses in an older, larger tumor. These results are entirely consistent with the model that Ets21C can activate certain JNK targets in a context dependent manner (Toggweiler, 2016).

Finally, it was asked if and how Ets21C regulates transcriptional outputs of the JNK pathway. (1) Ets21C could interact with Jun and/or Fos. (2) Ets21C could activate or interact with an unknown factor that feeds back on the JNK pathway. (3) Ets21C could use a combination of both (1) and (2). This study showed genetically that in Ras^V12 dlg^RNAi tumors the effects of Ets21C, both phenotypically and on a target genes level, fully depend on an active JNK pathway. If the latter is blocked, for example, by co-expressing Bsk^DN with Ras^V12 dlg^RNAi and Ets21C^HA, tumors remain small and there is no induction of Mmp1, Pvf1 or upd1. These results support all possibilities (1)-(3), but exclude an autonomous function of Ets21C. A physical interaction between Ets21C and Jun and Fos has previously been proposed based on large scale mass-spectroscopy data. This study shows that HA-tagged Ets21C does indeed bind FLAG-tagged Jun or Fos, showing that the proteins could interact physically to regulate target gene expression. The binding is consistent with studies in mammalian systems that have shown a physical interaction between AP-1 and ETS proteins including ERG, the mammalian homolog of Ets21C. In agreement with a cooperative transcriptional activation, Ets21C and AP-1 binding sites were found co-occurring in the putative regulatory regions of the analyzed target genes. It is therefore thought that Ets21C likely activates target genes via a cooperation with AP-1. However, additional factors that are activated by the JNK pathway might contribute to target gene activation as well and could explain why Ets21C activates certain JNK downstream target genes and others not (Toggweiler, 2016).

In summary, this study shows that Ets21C fulfills a crucial role in the regulation of neoplastic tumor growth as a loss of function critically reduced tumor growth, whereas an excess of Ets21C further increased tumor size. While Ets21C was previously accredited only with a role in fine-tuning the transcriptional program of neoplastic tumors, the current results point towards a more fundamental role as activator of a specific set of target genes that drive tumor growth and invasion (Toggweiler, 2016).

GENE STRUCTURE

Exons - one

PROTEIN STRUCTURE

Amino Acids - 289

Structural Domains

The JRA protein is 58% homologous to human proto-oncogene cJUN and has high homology in both the basic DNA binding and leucine zipper regions (Perkins, 1988, Zhang, 1990 and Wang, 1994).

date revised:   5 August 2021

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