A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein

TEL is a novel member of the ETS family of transcriptional regulators that is frequently involved in human leukemias as the result of specific chromosomal translocations. Co-immunoprecipitation and GST chromatography analyses has been used to show that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region that is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG (Flybase name: Ets97D) in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators (Jousset, 1997).

Pointed domains are ERK binding sites

The ETS transcription factors perform distinct biological functions despite conserving a highly similar DNA-binding domain. One distinguishing property of a subset of ETS proteins is a conserved region of 80 amino acids termed the Pointed (PNT) domain. Using enzyme kinetics it has been determined that the Ets-1 PNT domain contains an ERK2 docking site. The docking site enhances the efficiency of phosphorylation of a mitogen-activated protein kinase (MAPK) site N-terminal to the PNT domain. The site enhances ERK2 binding rather than catalysis. Three hydrophobic residues are involved in docking, and the previously determined NMR structure indicates that these residues are clustered on the surface of the Ets-1 PNT domain. The docking site function is conserved in the PNT domain of the highly related Ets-2 but not in the ets family member GABPalpha. Ablation of the docking site in Ets-1 and Ets-2 prevents Ras pathway-mediated enhancement of the transactivation function of these proteins. This study provides structural insight into the function of a MAPK docking site and describes a unique activity for the PNT domain among a subset of ets family members. The Drosophila protein Pnt-P2 is encoded by a gene that is an apparent ortholog of the genes encoding Ets-1 and Ets-2. Pnt-P2 conserves the Ras/MAPK-targeted phosphoacceptor found N-terminal to the PNT domain in Ets-1 and Ets-2. Additionally, the PNT domain of Pnt-P2 contains sequence closely resembling the ERK2 docking site of Ets-1 and Ets-2 (L228, I230, and F234). It is likely this sequence functions as a docking site for Rolled, the Drosophila MAPK related to ERK2 (Seidel, 2002).

Invertebrate Pointed homologs

The Ras signaling pathway specifies a variety of cell fates in many organisms. However, little is known about the genes that function downstream of the conserved signaling cassette, or what imparts the specificity necessary to cause Ras activation to trigger different responses in different tissues. In C. elegans, activation of the Ras pathway induces cells in the central body region to generate the vulva. Vulval induction takes place in the domain of the Hox gene lin-39, a homolog of Drosophila Sexcombs reduced. lin-39 is absolutely required for Ras signaling to induce vulval development. During vulval induction, the Ras pathway, together with basal lin-39 activity, up-regulates lin-39 expression in vulval precursor cells. If lin-39 function is absent at this time, no vulval cell divisions occur. If lin-39 is replaced with the posterior Hox gene mab-5, then posterior structures are induced instead of a vulva (Maloof, 1998).

Animal lacking lin-1, which encodes an ETS-like transcription factor, have a multivulva phenotype: in lin-1 mutants, all the vulval precursor cells generate vulval lineages in an anchor-cell independent fashion. Because lin-39 increases in lin-1 mutants, it seems likely that Lin-39 protein acts downstream of lin-1, and thus is required for the multivulva phenotype of lin-1. Lin39 alone cannot trigger vulval development. Thus, the Ras pathway must have other functions in vulval development in addition to inducing lin-39 expression These findings suggest that in addition to permitting vulval cell divisions to occur, lin-39 is also required to specify the outcome of Ras signaling by selectively activating vulva-specific genes (Maloof, 1998).

The C. elegans gene lin-1 appears to act after the Ras-Raf-MEK-MAPK signaling cascade that mediates vulval induction. lin-1 is a negative regulator of vulval cell fates and encodes an ETS-domain putative transcription factor containing potential MAPK phosphorylation sites. In lin-1 null mutants, the vulval precursor cells (VPCs) still respond to signaling from the gonadal anchor cell, indicating that lin-1 defines a branch of the inductive signaling pathway. The inductive and lateral signaling pathways are integrated to control the 1 degree and 2 degrees vulval cell fates after the point at which lin-1 acts in the inductive pathway and that VPCs can assess the relative rather than absolute levels of inductive and lateral signaling in determining whether to express the 1 degree or 2 degrees vulval cell fates (Beitel, 1995).

In Caenorhabditis elegans, the MAP kinase signaling pathway is required for the development of multiple tissues: the hermaphrodite vulva, the male tail, the excretory system, the germline, the sex myoblasts, and possibly the posterior ectoderm as well. Of these tissues, the function of MAP kinase in the vulva has been the best characterized. During the L1 larval stage, six vulval precursor cells are generated along the ventral midline of the worm. LIN-3, a protein similar to epidermal growth factor, is produced by the gonadal anchor cell and initiates vulval development by activating the EGF receptor tyrosine kinase homolog LET-23 in the closest vulval precursor cell. Activation of LET-23 RTK triggers a conserved Grb2/Ras/Raf/MEK/MAP kinase cascade. Thus, the let-23 receptor stimulated mpk-1 MAP kinase signaling pathway functions to induce the vulva in C. elegans. MPK-1 directly regulates both the LIN-31 winged-helix transcription factor (a homolog of Drosophila Forkhead) and the LIN-1 Ets transcription factor (a homolog of Drosophila Pointed) to specify the vulval cell fate. lin-31 and lin-1 act genetically downstream of mpk-1, and both proteins can be directly phosphorylated by MAP kinase. LIN-31 binds to LIN-1, and the LIN-1/LIN-31 complex inhibits vulval induction. Phosphorylation of LIN-31 by MPK-1 disrupts the LIN-1/LIN-31 complex, relieving vulval inhibition. Phosphorylated LIN-31 may also act as a transcriptional activator, promoting vulval cell fates. LIN-31 is a vulval-specific effector of MPK-1, while LIN-1 acts as a general effector. The partnership of tissue-specific and general effectors may confer specificity onto commonly used signaling pathways, creating distinct tissue-specific outcomes (Tan, 1998).

The MAP kinase phosphorylation of general factors is well known, but only recently have tissue-specific effectors been found. In mammals, the bHLH transcription factor Microphthalmia and the PPAR nuclear receptor are tissue-specific effectors of MAP kinase signaling in melanocyte and adipocyte differentiation, respectively. Like LIN-31, these tissue-specific effectors may interact with general effectors (e.g., Elk-1) to initiate new programs of gene expression. In Drosophila, the MAP kinase Rolled may directly regulate at least three transcription factors: Aop/Yan, PointedP2, and dJun. However, none of these transcription factors is tissue specific. Thus, the mechanism of signaling specificity in Drosophila is currenly unknown. Tissue-specific effectors of MAP kinase signaling may form an important class of proteins that confer specificity onto generally used signaling pathways so that diverse cellular outcomes can ultimately be generated (Tan, 1998).

The Caenorhabditis elegans mpk-1 gene encodes a MAP kinase protein that plays an important role in Ras-mediated induction of vulval cell fates. Mutations that eliminate mpk-1 activity result in a highly penetrant, vulvaless phenotype. A double mutant containing a gain-of-function mpk-1 mutation and a gain-of-function mek mutation (MEK phosphorylates and activates MPK-1) exhibits a multivulva phenotype. These results suggest that mpk-1 may transduce most or all of the anchor cell signal. Epistasis analysis suggests that mpk-1 acts downstream of mek-2 (encodes a MEK homolog) and upstream of lin-1 (encodes an Ets transcription factor) in the anchor cell signaling pathway. mpk-1 may act together with let-60 ras in multiple developmental processes, as mpk-1 mutants exhibit nearly the same range of developmental phenotypes as let-60 ras mutants (Lackner, 1998).

Six gain-of-function mutations were identified and characterized that define a new class of lin-1 allele. The new lin-1 mutants displayed protruding vulval tissue and defects in egg laying, possible indications of abnormalities in the vulval passageway. These lin-1 alleles appeared to be constitutively active and unresponsive to negative regulation. Each allele has a single-base change that affects the predicted C terminus of LIN-1, suggesting that this region is required for negative regulation. The C terminus of LIN-1 is a high-affinity substrate for Erk2 in vitro, suggesting that LIN-1 is directly regulated by ERK MAP kinase. Because mpk-1 ERK MAP kinase controls at least one cell-fate decision that does not require lin-1, these results suggest that MPK-1 contributes to the specificity of this receptor tyrosine kinase-Ras-MAP kinase signal transduction pathway by phosphorylating different proteins in different developmental contexts. These lin-1 mutations all affect a four-amino-acid motif, FQFP, which is conserved in vertebrate and Drosophila ETS proteins that are also phosphorylated by ERK MAP kinase. This sequence may be a substrate recognition motif for the ERK subfamily of MAP kinases (Jacobs, 1998).

LIN-1 is an ETS domain protein. A receptor tyrosine kinase/Ras/mitogen-activated protein kinase signaling pathway regulates LIN-1 in the P6.p cell to induce the primary vulval cell fate during Caenorhabditis elegans development. Twenty-three lin-1 loss-of-function mutations were identified by conducting several genetic screens. This study characterizes the molecular lesions in these lin-1 alleles and in several previously identified lin-1 alleles. Nine missense mutations and 10 nonsense mutations were identified. All of these lin-1 missense mutations affect highly conserved residues in the ETS domain. These missense mutations can be arranged in an allelic series; the strongest mutations eliminate most or all lin-1 functions, and the weakest mutation partially reduces lin-1 function. An electrophoretic mobility shift assay was used to demonstrate that purified LIN-1 protein has sequence-specific DNA-binding activity that required the core sequence GGAA. LIN-1 mutant proteins containing the missense substitutions have dramatically reduced DNA binding. These experiments identify eight highly conserved residues of the ETS domain that are necessary for DNA binding. The identification of multiple mutations that reduce the function of lin-1 as an inhibitor of the primary vulval cell fate and also reduce DNA binding suggest that DNA binding is essential for LIN-1 function in an animal (Miley, 2004).

The LIN-1 ETS transcription factor inhibits vulval cell fates during Caenorhabditis elegans development. LIN-1 interacts with UBC-9, a small ubiquitin-related modifier (SUMO; see Drosophila SUMO) conjugating enzyme. This interaction is mediated by two consensus sumoylation motifs in LIN-1. Biochemical studies showed that LIN-1 is covalently modified by SUMO-1. ubc-9 and smo-1, the gene encoding SUMO-1, inhibit vulval cell fates and function at the level of lin-1, indicating that sumoylation promotes LIN-1 inhibition of vulval cell fates. Sumoylation of LIN-1 promotes transcriptional repression and mediates an interaction with MEP-1, a protein shown to associate with the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies show that mep-1 inhibits vulval cell fates and functions at the level of lin-1. It is proposed that sumoylation of LIN-1 mediates an interaction with MEP-1 that contributes to transcriptional repression of genes that promote vulval cell fates. These studies identify a molecular mechanism for SUMO-mediated transcriptional repression (Leight, 2005).

In ascidian embryos, a fibroblast growth factor (FGF) signal induces notochord, mesenchyme, and brain formation. Although a conserved Ras/MAPK pathway is known to be involved in this signaling, the target transcription factor of this signaling cascade has remained unknown. HrEts, an ascidian homolog of vertebrate Ets1 and Ets2, has been isolated to elucidate the transcription factor involved in the FGF signaling pathway in embryos of the ascidian Halocynthia roretzi. Maternal mRNA of HrEts is detected throughout the entire egg cytoplasm and early embryos. Its zygotic expression starts in several tissues, including the notochord and neural plate. Overexpression of HrEts mRNA does not affect the general organization of the tadpoles, but results in formation of excess sensory pigment cells. In contrast, suppression of HrEts function by morpholino antisense oligonucleotide results in severe abnormalities, similar to those of embryos in which the FGF signaling pathway is inhibited. Notochord-specific Brachyury expression at cleavage stage and notochord differentiation at the tailbud stage were abrogated. Formation of mesenchyme cells was also suppressed, and the mesenchyme precursors assumed muscle fate. In addition, expression of Otx in brain-lineage blastomeres was specifically suppressed. These results suggest that an Ets transcription factor, HrEts, is involved in signal transduction of FGF commonly in notochord, mesenchyme, and brain induction in ascidian embryos (Miya, 2003).

Ascidian embryos develop with a fixed cell lineage into simple tadpoles. Their lineage is almost perfectly conserved, even between the evolutionarily distant species Halocynthia roretzi and Ciona intestinalis, which show no detectable sequence conservation in the non-coding regions of studied orthologous genes. To address how a common developmental program can be maintained without detectable cis-regulatory sequence conservation, the regulation of Otx, a gene with a shared complex expression pattern, was studied in both species. It was found that in Halocynthia, the regulatory logic is based on the use of very simple cell line-specific regulatory modules, the activities of which are conserved, in most cases, in the Ciona embryo. The activity of each of these enhancer modules relies on the conservation of a few repeated crucial binding sites for transcriptional activators, without obvious constraints on their precise number, order or orientation, or on the surrounding sequences. It is proposed that a combination of simplicity and degeneracy allows the conservation of the regulatory logic, despite drastic sequence divergence. The regulation of Otx in the anterior endoderm by Lhx and Fox factors may even be conserved with vertebrates (Oda-Ishii, 2005).

In Ciona, it has been shown that Otx is activated in the animal hemisphere (a- and b-line) by the neural inducer Ci-Fgf9/16/20. This signal is mediated by the transcription factors Ci-GATAa and Ci-Ets1/2 via a cluster of GATA- and Ets-binding sites in the Ci-Otx ab-module. In Halocynthia, it has also been shown that Hr-Ets, the ortholog of Ci-Ets1/2, is required for Otx activation in the a- and b-line, and this study shows that the modules driving Hr-Otx expression in this line (#1, #2, #5) also contain clusters of GATA- and Ets-BSs. This suggests that the regulatory logic in this line is conserved between the two species. In addition, #2 and #5 also drive expression in a- and b-line cells when tested in Ciona. However, #1 does not drive expression in Ciona, indicating that the syntax of the module is not entirely degenerate and that it partly differs between the two species. Given the important sequence divergence of at least one of the factors binding to the a-module, Ets1/2 (50% amino acid identity between Halocynthia and Ciona), some co-evolution of the module and its binding factors is not unexpected (Oda-Ishii, 2005).

Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A well-characterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT (Saunders, 2014).

Gene regulatory logic of dopamine neuron differentiation in C. elegans

Dopamine signalling regulates a variety of complex behaviours, and defects in dopamine neuron function or survival result in severe human pathologies, such as Parkinson's disease. The common denominator of all dopamine neurons is the expression of dopamine pathway genes, which code for a set of phylogenetically conserved proteins involved in dopamine synthesis and transport. Gene regulatory mechanisms that result in the direct activation of dopamine pathway genes and thereby ultimately determine the identity of dopamine neurons are poorly understood in all systems studied so far. This study shows that a simple cis-regulatory element, the dopamine (DA) motif, controls the expression of all dopamine pathway genes in all dopaminergic cell types in Caenorhabditis elegans. The DA motif is activated by the ETS transcription factor AST-1. Loss of ast-1 results in the failure of all distinct dopaminergic neuronal subtypes to terminally differentiate. Ectopic expression of ast-1 is sufficient to activate the dopamine pathway in some cellular contexts. Vertebrate dopamine pathway genes also contain phylogenetically conserved DA motifs that can be activated by the mouse ETS transcription factor Etv1 (also known as ER81), and a specific class of dopamine neurons fails to differentiate in mice lacking Etv1. Moreover, ectopic Etv1 expression induces dopaminergic fate marker expression in neuronal primary cultures. Mouse Etv1 can also functionally substitute for ast-1 in C. elegans. These studies reveal a simple and apparently conserved regulatory logic of dopamine neuron terminal differentiation and may provide new entry points into the diagnosis or therapy of conditions in which dopamine neurons are defective (Flames, 2009).

Nervous systems generally harbour distinct populations of dopamine (DA) neurons that derive from different precursor cells. Despite their diverse origin, all DA neurons share the expression of a core set of five genes that code for enzymes and transporters which synthesize, package and re-uptake dopamine ('dopamine pathway genes'). The regulatory logic of the terminal differentiation of DA neurons, manifested by the induction of the DA pathway genes, can, in theory, be described by two distinct models. In model 1, each dopamine pathway gene is independently activated by a distinct set of regulatory factors and, as a reflection of their distinct developmental history, each DA neuron subtype uses a distinct set of regulatory molecules. In model 2, each dopamine pathway gene is regulated by the same regulatory factor(s) and those factor(s) are the same in each DA neuron subtype. These two models make specific predictions about the cis-regulatory architecture of dopamine pathway genes. In model 1, each dopamine pathway gene is controlled by distinct cis-regulatory motifs and different motifs are active in individual DA neuron subtypes. In model 2, there is a single motif for all pathway genes that is used in all different DA neuron subtypes. To test these models, use was made of the DA system of the nematode C. elegans, which contains four distinct, lineally unrelated classes of DA neuron subtypes that express the same set of highly conserved dopamine pathway genes. The cis-regulatory regions of all DA pathways genes was systematically dissected in the context of gfp reporters expressed in transgenic worms. The cis-regulatory analysis of two genes exclusively expressed in the DA neurons, the dopamine transporter gene dat-1 (dopamine transporter) and the tyrosine hydroxylase gene cat-2 (tyrosine hydroxylase), revealed the existence of a small cis-regulatory module (CRM) in each promoter that is required and sufficient to drive expression in all DA neurons. Dopamine pathway genes expressed in both DA and serotonergic (5-HT) neurons (cat-1, vesicular monoamine transporter, cat-4, GTP cyclohydrolase, bas-1, aromatic amino acid decarboxylase) contain separable CRMs for expression in DA and 5-HT neurons (Flames, 2009).

The DA-specific CRM of the dat-1 locus contains a small sequence motif that is conserved in three other Caenorhabditis species; mutational analysis demonstrates that this motif is required for dat-1 expression in all DA neurons. This motif was also sufficient to drive expression in all DA neurons, either when tested in isolation or when appended to the CRM of another neuron-specific gene. Bioinformatics analysis predicted the binding of six different types of transcription factors to this conserved motif. Point mutations that specifically abolished the predicted binding of some factors while keeping others intact revealed that the only predicted motif that can be made responsible for cis-regulatory motif activity in the DA neurons is a predicted ETS transcription factor binding site (EBS) defined by an invariant GGAW core sequence. The DA-expressed CRMs of all other dopamine pathway genes also contain predicted EBSs, and mutational analysis corroborated their requirement for the correct expression in all DA neurons of C. elegans hermaphrodites and in the three additional DA neuron pairs present in the male. All the functionally characterized EBSs are conserved in other Caenorhabditis species; they can occur in either orientation and at different distances from the transcriptional start. The weight matrix generated with all these sequences defines a consensus EBS sequence motif that is termed the 'DA motif' (Flames, 2009).

By analysing the expression of the DA marker dat-1::gfp in mutants that lack each of the ten C. elegans ETS family members, it was found that all ets family mutants showed wild-type dat-1::gfp expression except for animals lacking the axon steering defect-1 (ast-1) gene, previously identified as a gene controlling axon outgrowth in the ventral nerve cord. Moreover, a mutant allele, ot417, which was retrieved from an unbiased forward genetic screen for mutants in which DA fate is inappropriately executed, is an allele of ast-1. The expression of all five dopamine pathway genes was strongly affected if not completely lost in ast-1 mutants. Two other DA terminal differentiation markers, the ion channels asic-1 and trp-4, also failed to be expressed in the DA neurons of ast-1 mutants. Both genes contain phylogenetically conserved DA motifs in their regulatory regions. ast-1 therefore appears to affect DA fate broadly, which is further corroborated by the axon pathfinding defects of DA neurons that were observed in ast-1 mutants. Loss of DA marker gene expression is not a reflection of early lineage specification defects and/or absence of the neurons, as assessed by analysis of additional fate markers (Flames, 2009).

ast-1 is expressed in several neurons, including all DA neurons, and acts cell-autonomously in DA neurons, as the ast-1 mutant phenotype can be rescued by expression of ast-1 specifically in the DA neurons. ast-1 expression persists in DA neurons throughout postembryonic stages, indicating that ast-1 is required not only to initiate DA terminal cell fate but also to maintain DA neuron identity, a notion confirmed through temporally controlled addition and removal of ast-1 gene activity (Flames, 2009).

To address whether ast-1 function is not only necessary for proper DA neuron differentiation but also sufficient, ast-1 expression was ectopically induced throughout all cell and tissue types at different stages of development. Ectopic induction during embryogenesis led to a substantial ectopic expression of both dat-1::gfp and cat-2::gfp. The morphology, location and pan-neuronal fate marker expression of these cells indicates that the effects of ast-1 are confined to the nervous system, in which ~20 cells can be induced to ectopically express both dat-1/DAT and cat-2/TH. Ectopic ast-1 was maximally effective when expressed around the time of neuronal differentiation. Moreover, ectopic ast-1 expression under control of the DA- and 5-HT-specific bas-1 promoter induced dat-1/DAT expression in 5-HT neurons demonstrating that ast-1 acts autonomously to control DA neuron specification and that 5-HT neurons provide the appropriate cellular context to allow ast-1 to induce DA neuron specification. The related ETS domain transcription factor LIN-1 was not able to induce ectopic DA neuron production when expressed under similar condition, demonstrating the specificity of AST-1 function (Flames, 2009).

To assess whether ETS transcription factor(s) have a similar function in vertebrate DA neuron specification, their expression was analyzed in the DA areas of the brain. Distinct ETS factors appeared to be expressed in distinct types of DA neurons and focus was placed on the ETS factor Etv1, which is expressed in the DA neurons of the olfactory bulb. Mice lacking Etv1 displayed a notable reduction in the number of tyrosine hydroxylase (TH)-positive cells in their olfactory bulb compared to wild-type siblings, whereas other periglomerular interneuron subtypes were not affected or were less severely reduced. This phenotype was not paralleled by increased cell death, by a reduction in the overall density of cells in the glomerular layer, by a reduction in overall neuron number or by proliferation defects. Moreover, the identity of DA progenitor cells in the lateral ganglionic eminence, which already express Etv1, appeared unaffected in Etv1 mutants. Therefore, Etv1 may affect a late stage in olfactory DA neuron differentiation (Flames, 2009).

Like ast-1, Etv1 appears not only required for DA neuron differentiation but also sufficient, because ectopic expression of Etv1 in olfactory bulb primary cell culture increases the number of cells expressing the DA marker TH. Etv1 is also able to activate directly the cis-regulatory region of the mouse TH locus in a heterologous context. This activation depends on the presence of two phylogenetically conserved DA motifs. Like in C. elegans, phylogenetically conserved DA motifs can also be found in the 5' upstream regulatory region of all four other mouse dopamine pathway genes. Another indicator for a conserved function of mouse Etv1 and worm AST-1 is that mouse Etv1 is able to rescue the ast-1 mutant phenotype when expressed in transgenic worms (Flames, 2009).

In conclusion, this study reports a surprisingly simple regulatory logic for DA specification. cis-regulatory analysis in worms reveals that all dopamine pathway genes are co-regulated through a similar cis-regulatory motif and trans-acting factor, and this regulatory logic applies to DA neurons of distinct lineage origin. This analysis demonstrates that the ETS factor ast-1 is a terminal selector gene for DA cell fate, akin to other terminal selector genes that control the terminal identity of other neuron. Terminal selector genes are transcription factors that directly regulate the 'nut-and-bolts' differentiation gene batteries that determine the specific properties of a neuron by binding to simple cis-regulatory motifs shared by members of the terminal gene batteries, termed 'terminal selector motifs' (or, in the case of the DA neurons, the DA motif). As exemplified by AST-1, terminal selector genes are continuously expressed throughout the life of a neuron to ensure that the terminal differentiation state is properly maintained (Flames, 2009).

The regulatory logic of DA neuron specification seems to be phylogenetically conserved. Vertebrate dopamine pathway genes also contain DA motifs that are required for the activation by a trans-acting factor that is homologous to the C. elegans trans-acting factor. Loss of the trans-acting factor either in worms or in mice leads to a loss of the DA phenotype. Both Etv1 and ast-1 are continuously expressed throughout the postmitotic life of DA neurons, and this analysis in worms indicates that these factors also maintain the terminal identity of DA neurons. The function of vertebrate ETS proteins in DA specification may have been distributed over several different ETS domain transcription factors, as Etv1 is not expressed in other DA neuron populations in the brain and as it does not affect the generation of these other types of DA neurons. Those other areas express a related ETS factor, Etv5, which may fulfil a role similar to that of Etv1 in olfactory DA neurons; in support of this notion, Etv5 can also transactivate the TH promoter in a heterologous assay system. The logic of distributing an ancestral gene function, observed in an invertebrate species, over several vertebrate paralogues of the ancestral invertebrate orthologue has been noted for other transcription factors as well and seems an important component of driving neuronal diversification processes in more complex brains (Flames, 2009).

AST-1 and Etv1 both act as terminal selector genes for DA terminal differentiation, but their presence is not strictly sufficient to activate DA genes because both AST-1 and Etv1 are also expressed in neurons other than DA neurons. Ectopic expression experiments also show that ability of AST-1 to induce ectopic DA fate is restricted to some cellular and temporal contexts. Classic 'master regulators', such as fly Eyeless or mouse MyoD (also known as Myod1), also show similar context-dependencies in their mode of action. AST-1 and Etv1 function may be actively inhibited in cells 'refractory' to AST-1/Etv1 activity. Alternatively, AST-1/Etv1 function may require additional, cell-type-specific factors for appropriate function in DA neurons. Such 'combinatorial coding' mechanisms are a common theme in neuron type specification, and identification of a conserved role of ETS factors as a central component of such a code is the first important step in decoding the regulatory logic of DA neuron specification. It will be interesting to see whether the additional specificity determinants of ETS factors are also conserved from worms to vertebrates (Flames, 2009).

Anti-intron antisense RNA blocks embryonic ets1 gene expression

Many genes, and particularly regulatory genes, are utilized multiple times in unrelated phases of development. For studies of gene function during embryogenesis, there is often need of a method for interfering with expression only at a specific developmental time or place. In sea urchin embryos cis-regulatory control systems which operate only at specific times and places can be used to drive expression of short designed sequences targeting given primary transcripts, thereby effectively taking out the function of the target genes. The active sequences are designed to be complementary to intronic sequences of the primary transcript of the target genes. In this work, the target genes were the transcription factors alx1 and ets1, both required for skeletogenesis, and the regulatory drivers were from the sm30 and tbr genes. The sm30 gene is expressed only after skeletogenic cell ingression. When its regulatory apparatus was used as driver, the alx1 and ets1 repression constructs had the effect of preventing postgastrular skeletogenesis, while not interfering with earlier alx1 and ets1 function in promoting skeletogenic mesenchyme ingression. In contrast, repression constructs using the tbr driver, which is active in blastula stage, block ingression. This method thus provides the opportunity to study regulatory requirements of skeletogenesis after ingression, and may be similarly useful in many other developmental contexts (Smith, 2008).

The following experiment shows the potential effectiveness and specificity of anti-intron antisense RNA (aiRNA), transcribed from a cis-regulatory expression vector, for blocking target gene expression. Use was made of the fact that sea urchin eggs concatenate injected linear DNA, and whatever constructs are injected, stably incorporate these together into a blastomere chromosome, whereafter the exogenous concatenate replicates together with the host DNA. Thus a mixture of a marker construct, an aiRNA generating construct, and a target construct was injected. The marker consisted of tbr cis-regulatory DNA driving an RFP gene as a reporter (tbr > RFP). It will express only in skeletogenic cells and will identify those cells which contain the exogenous mix of constructs. The target construct consisted of an alx1 BAC, containing its own endogenous cis-regulatory information as well as the complete gene, into the 5' UTR of which a GFP coding sequence had been inserted by homologous recombination. The aiRNA generating construct (tbr > aiRNA) consisted of the same tbr cis-regulatory sequence as in the marker construct, but here used to drive expression of the antisense transcripts. In one version, the construct produced an antisense transcript targeting the intron1/exon1 splice junction of the alx1 gene [as would be targeted by a splice-blocking MASO (morpholino-substituted antisense oligonucleotides)]; and in a second version, it produced an antisense transcript targeting an internal region of intron1. These aiRNA transcripts were generated off 24 bp antisense oligonucleotides terminated with three tandem p(A) addition sites, cloned into the tbr expression vector. The results were monitored by QPCR measurement of GFP mRNA, normalized to the RFP mRNA in the same embryos. Both aiRNA constructs almost eliminated GFP mRNA production. Constructs generating sense rather than antisense transcripts of the same intronic sequences had no effect. Nor was expression of the alx1 BAC-GFP reporter affected by an aiRNA construct targeted against the ets1 gene, which is active in the same cells, excluding a nonspecific interference with expression. The effect of aiRNA constructs on endogenous alx1 transcripts could not be directly measurede in the same experiment by QPCR, due to the mosaic incorporation of the targeting vector, since about 3/4 of the skeletogenic cells lack the exogenous DNA, and produce normal levels of alx1 in the same embryos. In contrast, since the constructs are co-incorporated, as noted above, in the alx1 BAC-GFP experiment, the three- to four-fold reduction in GFP transcript is the actual gene knockdown effect in those cells carrying the aiRNA construct. It is concluded (1) that intranuclear stoichiometry does indeed appear to favor efficient target acquisition by endogenously produced antisense transcripts; (2) that the interference with GFP production was not a general effect of interference with splicing machinery; (3) that this interference operates on internal as well as junctional intronic sequences; and (4) that it causes destruction or inactivation of the whole target transcript since the target sequences are all downstream of the intact GFP sequence. In other words, it is likely that the primary transcript is targeted for degradation. Though it is expected that the p(A) sites would ultimately result in short aiRNA transcripts, it is not known whether the active inhibitory form is a longer readthrough pre-poly(A) RNA, or the terminated polyadenylated product (Smith, 2008).

If the effect of an aiRNA construct is indeed the functional inactivation of the target transcript so that it cannot be expressed, then introduction of tbr > aiRNA against alx1 should produce the same morphological effect on the skeletogenic cells carrying it as does injection into the egg of MASO against alx1, since the tbr cis-regulatory control system initiates expression very early in development. The alx1 MASO effect is the total prevention of ingression: alx1 regulates downstream differentiation genes required for this distinct function. The result of introducing tbr > aiRNA against alx1 is indeed that in 80%-90% of embryos bearing tbr > aiRNA targeted to alx1, no skeletogenic cells bearing the construct whatsoever emerge from the vegetal wall of the embryo, and in the remainder only a few do. The skeletogenic cells bearing the construct are marked by expression of tbr > GFP (i.e., the 'marker' in these experiments is a tbr > GFP construct as opposed to the tbr > RFP construct. Expression of ets1 is also required for ingression, as shown by its MASO phenotype, and again, this phenotype is seen as well with tbr > aiRNA directed against an ets1 intron. Quantitatively, both aiRNA constructs are extremely effective in arresting ingression (Smith, 2008).

The problem outlined above can now be approached: how to study late alx1 and ets1 function by knocking out expression in skeletogenic cells only after allowing these genes to function long enough to permit complete ingression. To this end, the cis-regulatory system of the sm30 gene was used; it is turned on only after ingression. Sm30 > aiRNA constructs targeted against the same intronic sequences of either alx1 or ets1 as in the tbr > aiRNA constructs were introduced, together with the tbr > GFP marker construct. Assuming that sm30 cis-regulatory control is sufficiently tight, the expectation is that there will be no expression of the aiRNAs prior to ingression and thus that neither construct will interfere with ingression. In fact, both sets of embryos displayed control levels of ingression. However, subsequent skeletogenesis was dramatically affected, though in a very specific way. In normal postgastrular embryos, the skeletogenic cells migrate about the inner walls of the blastocoel, and then read signals displayed by the ectoderm cells, which specify the bilateral, branched form of the skeletal spicules. In response, they arrange themselves in highly reproducible, ordered, linear arrays. The cells then fuse laterally and secrete the skeleton into extracellular cables by which they are connected to one another. But the cells bearing sm30 > aiRNA targeted to either alx1 or ets1 fail entirely to form these arrays, or to participate in secretion of organized spicule rods. The cells instead assume random positions on the inner wall of the blastocoel: thus they retain their motility, but it would appear that they have failed to respond to the spatial information presented on the blastocoel wall. That this information is being normally expressed in the same embryos can be seen by the presence of morphologically normal skeletal elements formed by cells not bearing the aiRNA constructs, i.e., not expressing GFP. Secondary skeletogenic cells were not observed up to 72 h post-fertilization. The basic biomineralization functions are also severely affected. Thus instead of all tbr > GFP cells producing biomineral as in controls, only about 3% of green cells in the aiRNA embryos are associated with rudimentary accumulations of biomineral, which can be detected in polarized light. In summary, the experiment shows that expression of alx1 and ets1 after ingression is required for alignment of the cells in response to ectodermal patterning information; whether these genes are needed for syncytial cable formation is moot since they never get in position to form linear cables. Both alx1 and ets1 are clearly required for completion of the skeletogenic program. These functions are consistent with the character of the gene regulatory network linkages set up by the time of ingression, which include, for both genes, inputs into signal receptors and into biomineralization differentiation genes. It is now clear that these regulatory linkages are set up to be utilized only after ingression, and that they and no doubt many others of similar nature are requisite for mature skeletogenic function (Smith, 2008).

This study has shown that, in sea urchin embryos, expression of RNA complementary to intronic sequence, under control of selected cis-regulatory modules, can be used to effect spatially and temporally targeted gene expression knockdown. The results of the ets1 and alx1 aiRNA experiments are exactly consistent with expectation from the model experiment that demonstrated aiRNA efficacy against the alx1 BAC GFP construct (Smith, 2008).

The mechanism by which these interference constructs work is not known. It is clearly distinct from that of classical RNAi since the latter causes destruction of target mRNAs in the cytoplasm, a process nucleated on the RNAi:3' trailer complex. Messenger RNA destruction mediated by RNAi is effected by cytoplasmic proteins, while in the current case the sequence targeted, i.e., the intron, exists only in the nucleus. Nor does the mechanism of interference with expression seem the same as that of splice-blocking morpholinos, even though this project began with the thought that because of favorable stoichiometry the function of splice blocking morpholinos could be duplicated by use of endogenously synthesized antisense RNAs. Splice blocking morpholinos leave unspliced primary transcript fragments to accumulate in the nucleus where they are easily detected. But, as pointed out above, the aiRNA vectors apparently cause the destruction of the whole the transcript since even portions upstream of the targeted intron disappear. The actual mechanism by which formation of a 24 bp intron-antisense duplex effects primary transcript destruction will be most interesting to determine (Smith, 2008).

In the meantime, at the very least, this advance opens the way to exploration of a plethora of interesting problems in the regulatory control of postgastrular development and morphogenesis in the sea urchin embryo. The result will be to extend gene regulatory network analysis to the later development of this model embryonic system. The effectiveness of the method may depend on the intra-nuclear stoichiometric ratios of transcripts made on exogenous constructs to endogenous pre-mRNAs. Thus, the generality of its application in other model organisms would need to be determined (Smith, 2008).

An ETS domain protein involved in gastrulation

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. Xegr-1, a zinc finger protein-encoding gene, has been identified as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. The Ets-SRF complexes are known to act downstream of the MAP kinase pathway, and in the case of Xegr-1 the complex is shown to function downstream of FGF signaling. The finding that Xegr-1 activation requires Ets-SRF complexes provides the first indication for Ets-SRF complexes binds to serum response elements that are activated during gastrulation. MAP kinase signaling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra is found to induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the activation of FGF signaling and the subsequent signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis (Panitz, 1998).

In chordates, formation of neural tissue from ectodermal cells requires an induction. The molecular nature of the inducer remains controversial in vertebrates. Using the early neural marker Otx as an entry point, the neural induction pathway in the simple embryos of Ciona intestinalis was dissected. The regulatory element driving Otx expression in the prospective neural tissue was isolated; this element directly responds to FGF signaling and FGF9/16/20 acts as an endogenous neural inducer. Binding site analysis and gene loss of function established that FGF9/16/20 induces neural tissue in the ectoderm via a synergy between two maternal response factors. Ets1/2 mediates general FGF responsiveness, while the restricted activity of GATAa targets the neural program to the ectoderm. Thus, this study identifies an endogenous FGF neural inducer and its early downstream gene cascade. It also reveals a role for GATA factors in FGF signaling (Bertrand, 2003).

Otx expression starts in the animal a6.5 pair of blastomeres as they become restricted to anterior neural fate, at the onset of the neural induction process. At this stage, Otx is also activated in the animal b6.5 pair of blastomeres (precursors of the posterior dorsal neural tube and of the dorsal midline which constitutes a neurogenic region and in some vegetal B-line blastomeres (precursors of the posterior mesendoderm). Interestingly, Otx activation in b6.5, as in a6.5, requires an induction from vegetal blastomeres (Bertrand, 2003 and references therein).

The region in Otx located between -1541 and -1417 is required for expression in the a6.5 lineage, and is referred to as the a-element. Consistent with the simultaneous induction of Otx in a6.5 and b6.5 by vegetal cells, deletion of the a-element also reduces the activity in the b6.5 lineage. Finally, regions located between positions -1417 to -1133, and -706 to -271 are required for expression in A-line, and B/b-lines respectively (Bertrand, 2003).

Otx activation in the a6.5 neural precursors requires an interaction with the anterior vegetal blastomeres (A-line). Thus, the inducing FGF should be expressed in A-line blastomeres, before the onset of Otx expression at the 32-cell stage. The Ciona intestinalis genome contains 6 members of the FGF family. By in situ hybridization, only detect one FGF, FGF9/16/20, could be detected that was expressed at the right time and place to be the inducer. Its expression starts at the 16 cell-stage in the A-line and some B-line cells. Expression is stronger in the A-line than in the B-line, and this difference is further enhanced at the early 32-cell stage. This expression pattern is similar to that of the Ciona savignyi ortholog and is consistent with a role for FGF9/16/20 as endogenous neural inducer (Bertrand, 2003).

By both gene loss of function and binding sites analysis it has been determined that cooperation between the maternal transcription factors, Ets1/2 and GATAa, mediates the initial transcriptional response to FGF. Ets transcription factors have already been shown to act in the FGF pathway in vertebrates, and the members of the Ets1/2 subfamily can be directly phosphorylated and activated by Erk. A role for GATAa in this process was more unexpected, since GATA factors have so far not been implicated in the FGF pathway. However, the fact that multimerized GATA binding sites mediate FGF responsiveness indicate that, in this system, GATA does not act solely to modify or enhance Ets activity but functions as an FGF-activated transcription factor. Consistent with the proposal of a direct involvement of GATA factors in the FGF pathway in vivo, it has recently been shown, in vitro, that vertebrate GATA4 can be directly phosphorylated and activated by Erk (Bertrand, 2003).

Could members of the Ets1/2 and GATA families also play a role in neural induction in vertebrates? Ets2 messenger is present maternally in Xenopus eggs and has recently been shown to be required for the induction of Brachyury by FGF in mesodermal cells. It will be interesting to test whether it also acts in the neural induction pathway. Vertebrate GATA factors are thought to antagonize rather than promote neural tissue formation; GATA1/2/3 family members are expressed during gastrulation in the nonneural ectoderm in zebrafish, Xenopus, and chick and GATA1 has an antineuralizing activity when overexpressed in Xenopus. However, GATA2 has no antineuralizing activity, showing that this is not a general property of GATA factors. GATA2 and GATA5 are present in Xenopus eggs but the early role of these maternal GATA factors has not been studied, leaving open the possibility of an involvement in neural induction. Finally, it is proposed that, in ascidians, the use of different response factors accounts for the activation of different target genes in neuroectoderm and mesoderm. It will be interesting to test whether the same logic is used in vertebrates or whether the increase in gene number has led to the recruitment of different FGF inducers or receptors in these two lineages (Bertrand, 2003 and references therein).

Physical interaction of Pointed homologs with other transcription factors

Interactions between Ets family members and a variety of other transcription factors serve important functions during development and differentiation processes, e.g. in the hematopoietic system. The endothelial basic helix-loop-helix PAS domain transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha) (but not its close relative HIF-1alpha), cooperates with Ets-1 in activating transcription of the vascular endothelial growth factor receptor-2 (VEGF-2) gene (Flk-1). The receptor tyrosine kinase Flk-1 is indispensable for angiogenesis, and its expression is closely regulated during development. Consistent with the hypothesis that HIF-2alpha controls the expression of Flk-1 in vivo, HIF-2alpha and Flk-1 are co-regulated in postnatal mouse brain capillaries. A tandem HIF-2alpha/Ets binding site was identified within the Flk-1 promoter that acted as a strong enhancer element. Based on the analysis of transgenic mouse embryos, these motifs are essential for endothelial cell-specific reporter gene expression. A single HIF-2alpha/Ets element confers strong cooperative induction by HIF-2alpha and Ets-1 when fused to a heterologous promoter and is most active in endothelial cells. The physical interaction of HIF-2alpha with Ets-1 was demonstrated and localized to the HIF-2alpha carboxyl terminus and the autoinhibitory exon VII domain of Ets-1, respectively. The deletion of the DNA binding and carboxyl-terminal transactivation domains of HIF-2alpha, respectively, created dominant negative mutants that suppressed transactivation by the wild type protein and failed to synergize with Ets-1. These results suggest that the interaction between HIF-2alpha and endothelial Ets factors is required for the full transcriptional activation of Flk-1 in endothelial cells and may therefore represent a future target for the manipulation of angiogenesis (Elvert, 2003).

The distal enhancer region of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) is known to be essential for HIV replication and to contain immediately adjacent E-box and Ets binding sites. Based on a yeast one-hybrid screen the E-box binding protein, USF-1 was identified as a direct interaction partner of Ets-1 and it was found that the complex acts on this distal enhancer element. The binding surfaces of USF-1 and Ets-1 map to their DNA-binding domains and although these domains are highly conserved, the interaction is very selective within the respective protein family. USF-1 and Ets-1 synergize in specific DNA binding as well as in the transactivation of reporter constructs containing the enhancer element, and mutations of the individual binding sites dramatically reduce reporter activity in T cells. In addition, a dominant negative Ets-1 mutant inhibits both USF-1-mediated transactivation and the activity of the HIV-1 LTR in T cells. The inhibition is independent of Ets DNA-binding sites but requires the Ets binding surface on USF-1, highlighting the importance of the direct protein-protein interaction. Together these results indicate that the interaction between Ets-1 and USF-1 is required for full transcriptional activity of the HIV-1 LTR in T cells (Sieweke, 1998).

Different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b, have been isolated. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specific blk promoter. NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 binds to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 leads to cooperative transactivation of the blk promoter, whereas the interaction between AML1 and NERF-1a leads to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, it was determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia (Cho, 2004).

Adipocyte differentiation is orchestrated by multiple signaling pathways and a temporally regulated transcriptional cascade. However, the mechanisms by which insulin signaling is linked to this cascade remain unclear. This study shows that the Med23 subunit of the Mediator Complex and its interacting transcription factor ETS transcription factor Elk1 are critical regulators of adipogenesis. Med23(-/-) embryonic fibroblast cells were refractory to hormone-induced adipogenesis. Knockdown of either Med23 or Elk1, or overexpression of dominant-negative Elk1, inhibited adipogenesis. In the absence of either Elk1 or Med23, Krox20, an immediate early gene stimulated by insulin during adipogenesis, was uninducible. Moreover, the adipogenic defect in Med23-deficient cells was rescued by ectopic expression of Krox20 or one of its downstream factors, C/EBPbeta or PPARgamma. Mechanistically, the insulin-stimulated, Med23-deficient preinitiation complex failed to initiate robust transcription of Krox20. Collectively, these results suggest that Med23 serves as a critical link transducing insulin signaling to the transcriptional cascade during adipocyte differentiation (Wang, 2009).

Signaling upstream of Pointed homologs

In transient transfection assay, reporters containing Ras-responsive enhancers (RREs) composed of Ets-AP-1 binding sites could be activated 30-fold by the combination of Ets1 or Ets2 and Ras (See Drosophila Ras) but not by several other Ets factors that were tested in the assay. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors (Yang, 1996).

In cell culture systems, the TCF Elk-1 represents a convergence point for extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) subclasses of mitogen-activated protein kinase (MAPK) cascades. Its phosphorylation strongly potentiates its ability to activate transcription of the c-fos promoter through a ternary complex assembled on the c-fos serum response element. In rat brain postmitotic neurons, Elk-1 is strongly expressed. However, its physiological role in these postmitotic neurons remains to be established. To investigate biochemically the signaling pathways targeting Elk-1 and c-fos in mature neurons, a semi-in vivo system was used, composed of brain slices stimulated with the excitatory neurotransmitter glutamate. Glutamate treatment leads to a robust, progressive activation of the ERK and JNK/SAPK MAPK cascades. This corresponds kinetically to a significant increase in Ser383-phosphorylated Elk-1 and the appearance of c-fos mRNA. Glutamate also causes increased levels of Ser133-phosphorylated cyclic AMP-responsive element-binding protein (CREB) but only transiently relative to Elk-1 and c-fos. ERK and Elk-1 phosphorylation are blocked by the MAPK kinase inhibitor PD98059, indicating the primary role of the ERK cascade in mediating glutamate signaling to Elk-1 in the rat striatum in vivo. Glutamate-mediated CREB phosphorylation is also inhibited by PD98059 treatment. Interestingly, KN62, which interferes with calcium-calmodulin kinase (CaM-K) activity, leads to a reduction of glutamate-induced ERK activation and of CREB phosphorylation. These data indicate that ERK functions as a common component in two signaling pathways (ERK/Elk-1 and ERK/?/CREB) converging on the c-fos promoter in postmitotic neuronal cells and that CaM-Ks act as positive regulators of these pathways (Vanhoutte, 1999)

Target innervation by specific neuronal populations involves still incompletely understood interactions between central and peripheral factors. Glial cell line-derived neurotrophic factor (GDNF), initially characterized for its role as a survival factor, is present early in the plexus of the developing forelimb and later in two muscles: the cutaneus maximus and latissimus dorsi. In the absence of GDNF signaling, motor neurons that normally innervate these muscles are mispositioned within the spinal cord and muscle invasion by their axons is dramatically reduced. The ETS transcription factor PEA3 is normally expressed by these motor neurons and fails to be induced in most of them in GDNF signaling mutants. Thus, GDNF acts as a peripheral signal to induce PEA3 expression in specific motor neuron pools, thereby regulating both cell body position and muscle innervation (Haase, 2002).

During somite development, a fibroblast growth factor (FGF) signal secreted from the myotome induces formation of a scleraxis (Scx)-expressing tendon progenitor population in the sclerotome, at the juncture between the future lineages of muscle and cartilage. Scx is a tendon-specific bHLH transcription factor. While overexpression studies show that the entire sclerotome is competent to express Scx in response to FGF signaling, the normal Scx expression domain includes only the anterior and posterior dorsal sclerotome. To understand the molecular basis for this restriction, the expression of a set of genes involved in FGF signaling was examined and it was found that several members of the Fgf8 synexpression group are co-expressed with Scx in the dorsal sclerotome. Of particular interest were the Ets transcription factors Pea3 and Erm, which function as transcriptional effectors of FGF signaling. Transcriptional activation by Pea3 and Erm in response to FGF signaling is both necessary and sufficient for Scx expression in the somite, and it is proposed that the domain of the somitic tendon progenitors is regulated both by the restricted expression of Pea3 and Erm, and by the precise spatial relationship between these Ets transcription factors and the FGF signal originating in the myotome (Brent, 2004).

Phosphorylation of Pointed homologs activation and inhibition

The phosphorylation of transcription factors by mitogen-activated protein kinases (MAP) is a pivotal event in the cellular response to the activation of MAP kinase signal transduction pathways. Mitogenic and stress stimuli activate different pathways and lead to the activation of distinct groups of target proteins. Elk-1 is targeted by three distinct MAP kinase pathways. This study demonstrates that the MAP kinase ERK2 is targeted to Elk-1 by a domain that is distinct from, and located N-terminal to, its phosphoacceptor motifs. Targeting via this domain is essential for the efficient and rapid phosphorylation of Elk-1 in vitro and full and rapid activation in vivo. Specific residues involved in ERK targeting have been identified. These data indicate that the targeting of different classes of MAP kinases to their nuclear substrates may be a common mechanism to increase the specificity and efficiency of this signal transduction pathway (S.-H. Yang, 1998a).

The activation of MAP kinase (MAPK) signal transduction pathways results in the phosphorylation of transcription factors by the terminal kinases in these cascades. Different pathways are activated by mitogenic and stress stimuli, which lead to the activation of distinct groups of target proteins. The ETS-domain transcription factor Elk-1 is a substrate for three distinct classes of MAPKs. Elk-1 contains a targeting domain, the D-domain, which is distinct from the phosphoacceptor motifs and is required for efficient phosphorylation and activation by the ERK MAPKs. Members of the JNK subfamily of MAPKs are also targeted to Elk-1 by this domain. Targeting via this domain is essential for the efficient and rapid phosphorylation and activation of Elk-1 both in vitro and in vivo. The ERK and JNK MAPKs use overlapping yet distinct determinants in the D-domain for targeting to Elk-1. In contrast, members of the p38 subfamily of MAPKs are not targeted to Elk-1 via this domain. These data therefore demonstrate that different classes of MAPKs exhibit differential requirements for targeting to Elk-1 (S.-H. Yang, 1998b).

Protein phosphorylation represents one of the major mechanisms for transcription factor activation. A molecular mechanism by which phosphorylation by mitogen-activated protein (MAP) kinases leads to changes in transcription factor activity is demonstrated. MAP kinases stimulate DNA binding and transcriptional activation mediated by the mammalian ETS-domain transcription factor Elk-1. Phosphorylation of the C-terminal transcriptional activation domain induces a conformational change in Elk-1, which accompanies the stimulation of DNA binding. C-terminal phosphorylation is coupled to activation of DNA binding by the N-terminal DNA-binding domain via an additional intermediary domain. Activation of DNA binding is mediated by an allosteric mechanism involving the key phosphoacceptor residues. Together, these results provide a molecular model for how phosphorylation induces changes in Elk-1 activity (Yang, 1999).

Based on the results of this study, a model is proposed for the mechanism of phosphorylation-inducible Elk-1 activation. In the unphosphorylated state, DNA binding by Elk-1 is inhibited by a combination of the B-box and C-terminal TAD. A regulatory region is located in the TAD, which encompasses the key phosphoacceptor motifs (Ser383/Ser389) and acts as a phosphorylation-inducible switch that controls DNA binding. Phosphorylation initiates both a local change in the intramolecular interactions and a complex conformational change in the whole protein that relieves the inhibitory interactions. The addition of inhibitory peptides can then reverse this activation process by binding to phosphorylated Elk-1 and acting as a phosphorylation-regulatable allosteric switch. These peptides are, however, unable to gain access to the unphosphorylated protein due to its 'closed' conformation. Although phosphorylation of Ser383/Ser389 is a key regulatory event in DNA binding, it is clear that the overall stoichiometry of phosphorylation is important in inducing both DNA binding and conformational changes in Elk-1. Other phosphoacceptor motifs are, therefore, likely to play a role in this process (Yang, 1999).

Phosphorylation of transcription factors is a key link between cell signaling and the control of gene expression. An autoinhibitory mechanism regulates Ets-1 DNA binding. Three alpha-helices (HI-1, HI-2, and H4) interact with one another and H1 of the ETS domain to produce a metastable inhibitory module. A conformational change of the inhibitory module accompanies DNA binding. This structural transition includes the unfolding of helix HI-1 that is detected as DNA-induced protease sensitivity. The coupling of helix unfolding to DNA binding is thought to be the basis of Ets-1 autoinhibition. It is proposed that this DNA-induced conformational change provides a structural switch to modulate Ets-1 DNA binding. To determine the mechanism by which calcium-dependent signaling pathways regulate Ets-1 DNA binding, an in vitro system suitable for quantitative and structural analysis was developed. T-cell nuclear extract and calmodulin-dependent kinase II (CaMKII) phosphorylates bacterially expressed, purified Ets-1 on approximately six and four sites, respectively. Approximately one to two sites are modified by nuclear extract without calcium. Together, these results indicate that four to five sites are targets of calcium-dependent phosphorylation by either nuclear extract or CaMKII in vitro. In vivo studies with transiently overexpressed Ets-1 suggest that at least four sites are phosphorylated following calcium-ionophore treatment of cells, demonstrating that the in vitro system is suitable for mechanistic studies. Phosphorylation regulates DNA binding of the Ets-1 transcription factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show that calcium-dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four serines that mediate this inhibitory effect are distant from the DNA-binding domain but near structural elements required for autoinhibition. Mutational analyses demonstrate that an intact inhibitory module is required for phosphorylation-dependent regulation. Partial proteolysis studies indicate that phosphorylation stabilizes an inhibitory conformation. These findings provide a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as structural mediators of negative signaling events (Cowley, 2000).

Contributions of various phosphorylation sites to inhibition were explored. Four serines are known to be targets of calcium-dependent phosphorylation in vivo. All four serines were mutated together or in pairs to alanine and the phosphorylation and inhibition of these mutants was tested. Mutation of all four serines to alanine reduces phosphorylation by either nuclear extract or CaMKII to a level of approximately one mole of phosphate per mole of Ets-1. The residual phosphorylation of this mutant has very little effect on its DNA-binding affinity, indicating that at least one of these serines is required to mediate dramatic effects on Ets-1 DNA binding. Mutation of the serines in pairs results in intermediate phosphorylation levels of two to three phosphates per Ets-1 molecule by nuclear extract (S251A;S257A, S282A;S285A). However, phosphorylation of these double mutants decreases their DNA-binding affinities only four- and six-fold, respectively. This result suggests that the 50-fold inhibition of wild-type Ets-1 is due to a synergistic effect of the two pairs of phosphorylated residues. The cooperative requirement for multiple phosphoserines in inhibition suggests that structural elements might coordinate the interdependent activity of each phosphate. The phosphorylation sites are near the amino-terminal inhibitory helices, suggesting that phosphorylation might inhibit DNA binding by regulating the function of the inhibitory module. Specifically, the model of autoinhibition predicts that the conformational equilibrium of the inhibitory module determines the affinity of Ets-1 for DNA. Phosphorylation could inhibit Ets-1 DNA binding by affecting this equilibrium. This model makes two principal predictions: (1) the structure and function of the inhibitory module should be critical for phosphorylation-dependent inhibition; (2) an increase in the stability of the inhibitory module should be detectable following phosphorylation (Cowley, 2000).

Three lines of evidence in this report suggest that phosphorylation inhibits Ets-1 DNA binding by shifting this equilibrium toward the folded state. (1) Phosphorylation of serines 251, 257, 282, and 285 synergistically inhibits Ets-1 DNA binding, suggesting the involvement of structural elements. (2) Ets-1 mutants that display constitutively disrupted inhibitory modules are resistant to inhibition by phosphorylation, indicating that the folding of the inhibitory module is critical for this inhibition. (3) Protease experiments indicate that phosphorylation reduces cleavage within the inhibitory module, consistent with a shift of the conformational equilibrium towards the folded state. Together, these three observations support the model that phosphorylation inhibits Ets-1 DNA binding by stabilizing the inhibitory module. How might phosphorylation stabilize the inhibitory module? It is hypothesized that negatively charged phosphate groups promote electrostatic interactions between the phosphorylated region and the inhibitory module. The calcium-dependent phosphorylation sites are found in primary sequence environments punctuated by regularly spaced acidic residues. Phosphorylation of the four serines would increase the local negative charge substantially, facilitating ion-pairing interactions with positively charged residues of the inhibitory module. Inhibitory helix HI-1 is within a 21-residue segment of Ets-1 that contains eight basic amino acids. Interactions between these basic residues and the phosphorylated serines could reduce local unfolding, thus stabilizing the entire inhibitory module (Cowley, 2000).

The ETS domain transcription factor Elk-1 is a direct target of the MAP kinase pathways. Phosphorylation of the Elk-1 transcriptional activation domain by MAP kinases triggers its activation. However, Elk-1 also contains two domains with repressive activities. One of these, the R motif, appears to function by suppressing the activity of the activation domain. SUMO modification of the R motif is required for this repressive activity. A dynamic interplay exists between the activating ERK MAP kinase pathway and the repressive SUMO pathway. ERK pathway activation leads to both phosphorylation of Elk-1 and loss of SUMO conjugation and, hence, to the loss of the repressive activity of the R motif. Thus, the reciprocal regulation of the activation and repressive activities are coupled by MAP kinase modification of Elk-1 (Yang, 2003).

Cell signaling affects gene expression by regulating the activity of transcription factors. Mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2 occurs at a conserved site N terminal to their Pointed (PNT) domains. This results in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. This phosphorylation-augmented interaction was discovered in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins has demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain are required for the interaction. Minimal regions that are competent for induced CBP/p300 binding in vitro also support MAPK-enhanced transcription in vivo. CBP coexpression potentiates MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interact in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, these findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis (Foulds, 2004).

Sumoylation of ETS transcription factors

Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide a molecular framework for understanding this regulation, the conjugation of Ets-1 with SUMO-1 was characterized. Ets-1 is modified in vivo predominantly at a consensus sumoylation motif containing Lys-15. This lysine is located within the unstructured N-terminal segment of Ets-1 preceding its PNT domain. Using NMR spectroscopy, it has been demonstrated that the Ets-1 sumoylation motif associates with the substrate binding site on the SUMO-conjugating enzyme UBC9 [K(d) approximately 400 microm] and that the PNT domain is not involved in this interaction. Ets-1 with Lys-15 mutated to an arginine still binds UBC9 with an affinity similar to the wild type protein, but is no longer sumoylated. NMR chemical shift and relaxation measurements reveal that the covalent attachment of mature SUMO-1, via its flexible C-terminal Gly-97, to Lys-15 of Ets-1 does not perturb the structure or dynamic properties of either protein. Therefore sumoylated Ets-1 behaves as 'beads-on-a-string' with the two proteins tethered by flexible polypeptide segments containing the isopeptide linkage. Accordingly, SUMO-1 may mediate interactions of Ets-1 with signaling or transcriptional regulatory macromolecules by acting as a structurally independent docking module, rather than through the induction of a conformational change in either protein upon their covalent linkage. It is also hypothesized that the flexibility of the linking polypeptide sequence may be a general feature contributing to the recognition of SUMO-modified proteins by their downstream effectors (Macauley, 2006).

Ets target genes

Continued Pointed Evolutionary homologs part 2/4 | part 3/4 | part 4/4

pointed : Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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