BIOLOGICAL OVERVIEW

anterior open, more often referred to as yan, is a transcription factor that serves to inhibit neural and other types of differentiation. When yan is inactivated, differentiation proceeds. yan is modulated by the effects of Sevenless/Ras/MAPK, a major cytoplasmic signal transduction pathway. This pathway is a lengthy and complicated cascade of events leading to the activation of Pointed, and the inactivation of Yan. A look at Sevenless should prove instructive.

Sevenless is one of at least four Drosophila receptors that utilize the Ras/MAPK pathway to tranduce extracellular signals from outside the cell to the nucleus; the others are EGF-R or Torpedo, the homolog of the vertebrate epidermal growth factor receptor; Torso, the receptor responsible for terminal signaling; Breathless, the Drosophila FGF receptor homolog, and finally, Sevenless, the receptor for BOSS (Bride of Sevenless). Sevenless regulates the determination of cell fate of the R7 photoreceptor in the eye. R7 is the last to develop of the eight photoreceptors in each ommatidium (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation).

To understand how the cascade functions physically, one must look at the proteins implicated in Sevenless signaling, and the signals of other receptor tyrosine kinases, including Ras1 and the mitogen actived protein kinase (MAPK) family of serine/threonine kinases. These include Raf (MAPKKK), DSor (MAPKK) and rolled (MAPK). These three kinases each add phosphate residues to the next protein in the signaling cascade as the activation signal is passed from molecule to molecule. Ultimately, the cascade reaches its targets; the transcription factors Yan and Pointed. While Pointed is a positive regulator of genes, activated by the cascade, Yan is negatively regulated by Sevenless signaling. Yan is an antagonist of the Sevenless/Ras1 proneural signal, meaning when the signal to differentiate is turned on, Yan is turned off.

Yan is a repressor. It is expressed in basally located nuclei of undifferentiated cells in the larval eye imaginal disc. As cells differentiate and their nuclei migrate to an apical position, yan expression is suddenly shut down, or downregulated. Phosphorylation by MAPK regulates the stability and subcellular localization of Yan. A mutant form of Yan, deficient in phosphorylation sites, results in cell death, possibly due to prolonged Yan-mediated inhibition of differentiation (Rebay, 1995).

Although yan is expressed in the embryonic ectoderm and mesoderm, it is absent in the developing central nervous system. Ectopic expression of yan in the CNS inhibits embryonic development. Overexpression of yan in the mesoderm, where it is normally expressed but later downregulated, strongly reduces twist expression. twist is vital to the differentiation of cells that will eventually become mesoderm. Such results indicated that yan is an inhibitor of differentiation, and does not just affect neural cells, but is also relevant in other sites. Yan is not a universal inhibitor of differentiation, since other ectodermal markers such as engrailed show no effects from yan overexpression. Notch function is similar to Yan. It too inhibits differentiation, and provides another example of the importance of negative regulation in the differentiation process (Rebay, 1995).

A key step in development is the establishment of cell type diversity across a cellular field. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for this process. At each parasegment boundary, cells expressing the Wnt family member Wingless confront cells expressing the homeoprotein Engrailed. The Engrailed-expressing cells normally differentiate as one of two alternative cell types. In investigating the generation of this cell type diversity among the 2-cell-wide Engrailed stripe, it has been shown that Wingless, expressed just anterior to the Engrailed cells, is essential for the specification of anterior Engrailed cell fate. In a screen for additional mutations affecting Engrailed cell fate, anterior open (aop) (also known as yan) was identified, a gene encoding an inhibitory ETS-domain transcription factor that is negatively regulated by the Ras-MAP kinase signaling cascade. Anterior open must be inactivated for posterior Engrailed cells to adopt their correct fate. This is achieved by the EGF receptor (Egfr), which is required autonomously in the Engrailed cells to trigger the Ras1-MAP kinase pathway. Localized activation of Egfr is accomplished by restricted processing of the activating ligand, Spitz. Processing is confined to the cell row posterior to the Engrailed domain by the restricted expression of Rhomboid. These cells also express the inhibitory ligand Argos, which attenuates the activation of Egfr in cell rows more distant from the ligand source. Thus, distinct signals flank each border of the Engrailed domain, since Wingless is produced anteriorly and Spitz posteriorly. Since En cells have the capacity to respond to either Wingless or Spitz, these cells must choose their fate depending on the relative level of activation of the two pathways (O'Keefe, 1997).

The larval cuticle comprises a repeated array of precisely patterned denticle belts interspersed with smooth cuticle. In abdominal segments, each of these belts is made up of 6 rows of denticles, where each row is of a characteristic size and orientation reflecting fate decisions made by the underlying cells. Using a lacZ reporter gene expressed in the En cells, it has been demonstrated that the anterior En cells normally produce smooth cuticle, while the posterior En cells produce denticles and, thereby, form the first row of each belt. Thus, cells in the En domain adopt either a smooth or denticle fate depending on their position. To identify genes involved in specifying En cell fates, existing collections of mutants were screened for those in which anterior En cells inappropriately produce denticles. Ectopic denticles are observed immediately anterior to the denticle belts in aop mutants. The extra denticles are located at the lateral edges of denticle belts, and are more commonly observed in the posterior segments. To determine whether the En cell fates were altered in these mutants, the En cells were visualized with a lacZ reporter construct. Anterior En cells produce denticles instead of the normal smooth cuticle. Thus, aop function is required for some anterior En cells to adopt the smooth cell fate (O'Keefe, 1997).

Since aop activity is required for anterior En cells to adopt the smooth cell fate, Aop activity was tested to see if it was sufficient to force posterior En cells to produce smooth cuticle instead of first row denticles. A constitutively active form of Aop was examined, where all eight MAP kinase consensus phosphorylation sites were mutated, and its expression was driven in the En cells using the UAS/GAL4 system. While En-GAL4 embryos carrying UAS-Aop WT exhibit normal denticle pattern, such embryos carrying UAS-Aop Act are missing the normal first denticle row of each belt. Thus, if posterior En cells express a form of Aop that can not be inhibited by MAP kinase, then these cells adopt the smooth fate. This suggests that, normally, Aop must be inactivated in the posterior En cells for them to adopt denticle fates. Given that the Ras1-MAP kinase cascade is responsible for inhibiting Aop function in other tissues, it became a good candidate for inactivating Aop in the posterior En cells. If this pathway is indeed involved, then inappropriate activation of the pathway should mimic the aop mutant phenotype and allow anterior En cells to incorrectly produce denticles. To test this, embryos expressing a constitutively active form of Ras (UAS-Ras1 val-12 ) in the En cells were examined. These embryos have an ectopic row of denticles anterior to the normal first row, corresponding to the location of the anterior En cells. Thus, the anterior En cells are mis-specified by ectopic Ras1-MAP kinase activity, similar to the effects of loss of aop function. This suggests that Ras1-MAP kinase activity may normally be responsible for inactivating Aop in the posterior En cells, allowing them to adopt the denticle fate (O'Keefe, 1997).

Since the Ras1-MAP kinase cascade is activated by receptor tyrosine kinases, a test was performed to see whether such a receptor could be involved in specifying En cell fate. For several reasons, the best candidate was the Drosophila EGF Receptor (Egfr). (1) In the eye, an allele of aop was isolated as an enhancer of mutations in Ellipse, a gain-of-function allele of Egfr. (2) Egfr is ubiquitously expressed epidermis throughout embryogenesis and is required early for ventral-to-lateral patterning, as is Aop. Finally, at later stages, Egfr is required for cells to adopt denticle fates (O'Keefe, 1997 and references).

To address whether Egfr function is required for posterior En cells to adopt their correct fate, a dominant negative form of Egfr was expressed specifically in En cells. These embryos lack the first denticle row, corresponding to the position of the posterior En cells. Therefore, Egfr is autonomously required for the posterior En cells to adopt a denticle fate. It was next determined whether there is a source of Egfr ligand positioned appropriately to signal to the En cells. The Spitz source is posteriorly adjacent to En cells. It seemed likely that Egfr would be activated by Spi, its ligand in many other contexts. Spi is ubiquitously expressed as an inactive membrane-bound molecule with homology to TGF-alpha. A processing event, which requires Rhomboid (Rho) activity, releases active ligand. Thus, the spatially regulated expression of Rho marks cells that are the source for active, secreted Spi. These cells can trigger activation of Egfr in adjacent cells. The expression of Rhomboid suggests that there is a novel source of active Spi ligand at the appropriate time and place to influence En cell fate (O'Keefe, 1997).

To test directly whether the Egfr pathway is activated in these transverse stripes, the spatial distribution of activated MAP kinase was examined, using an antibody that is specific to the di-phosphorylated (active) form of MAP kinase (dp-ERK). In late stage embryos (9.5 hours AEL), a stripe of activated MAP kinase is detected just posterior to the En cells. This stripe is dependent on Egfr, since it is selectively removed in flb mutant embryos. In wild type, active MAP kinase is detectable within the En cells themselves, although at low levels. Thus, it appears that Egfr activation indeed spreads into the En cells. It could not be determined whether there is a difference between the anterior and posterior En cells. Activation of the Egfr pathway was confirmed by testing for the induction of a Egfr target gene, argos, the expression of which is closely correlated with regions of maximal Egfr activation. For instance, during earlier ventral-to-lateral patterning, argos is expressed in the ventralmost 1- to 2-cell rows, the point of highest Egfr activation. However, at later stages Argos mRNA is expressed in a stripe of cells posterior to the En cells, coincident with the expression of Rho and the highest levels of activated MAP kinase. Taken together, these data demonstrate that a secreted Egfr ligand (probably secreted Spitz), produced by cells just posterior to En cells, activates Egfr. Furthermore, it appears that the activation of Egfr is graded; highest posterior to En cells and at lower levels within the En cells. This signaling corresponds to the time when fates of the En cells are being determined, which is consistent with a role for Egfr in determining the fates of En cells. Experiments were carried out that revealed that anterior En cells can, in fact respond to Spitz (O'Keefe, 1997).

Spitz and Wingless signaling have been shown to have competing affects on En cell fate. Anterior En cells assume a denticle fate when wg function is eliminated at 8 hours AEL. Wg is expressed just anterior to the En domain, in a region of smooth cuticle. Thus, while Wg input instructs cells to adopt the smooth fate, activation of Egfr instructs cells to adopt denticle fates. The opposite response of En cells to these two signals raises the question of what fate these cells would adopt in the absence of both signals. To determine this, Egfr signaling was blocked by expressing Aop Act in En cells while concomitantly removing wg function using a conditional allele. When wg ts embryos carrying both En-GAL4 and UAS-Aop Act are shifted to non-permissive temperature at 8 hours AEL, the En cells adopt smooth fates. This suggests that smooth cuticle is the default cell fate. Wg signaling in this context is required primarily for antagonizing the effect of DER signaling in anterior En cells (O'Keefe, 1997).

The posterior En cells, which adopt a denticle fate, either cannot respond to Wg due to the absence of key signal transducers, or they do not see effective concentrations of Wg. In fact, it appears that the posterior En cell does not receive Wg input. The presence of downstream signal transducers was tested in posterior En cells. Cells expressing either an activated form of Armadillo or higher levels of wild-type Disheveled respond as if they have received the Wg signal. In embryos carrying both En-GAL4 and UAS-Arm S10, the expression of activated Armadillo causes the posterior En cells to inappropriately adopt the smooth cell fate. Identical results were obtained expressing Disheveled. Thus, Wg signal transducers downstream of Disheveled are present in posterior En cells. During normal patterning, these cells are probably not exposed to sufficient Wg levels to antagonize the effects of Egfr in these cells (O'Keefe, 1997).

A model is presented for the cooperation between Wingless and Spitz in specifying cell fate in Engrailed expressing cells. The En-expressing cells are flanked anteriorly by a cell row producing Wg and posteriorly by a cell row expressing Rhomboid, which produces secreted Spitz. The En cell nearest the Spi source receives a higher concentration of Spi, and thus activates the Egfr pathway sufficiently to specify a denticle fate. Reciprocally, the En cell nearest the Wg source receives a higher concentration of Wg and adopts a smooth fate. Spi also activates the Egfr pathway in the Rho-expressing cell, which therefore produces and secretes Argos. Argos can inhibit Spi activation of the Egfr pathway at a distance. As a consequence, the Egfr pathway is not sufficiently activated in the anterior En cell to out compete Wg signaling in this cell, and it adopts a smooth fate. In fact, the specific targets of Egfr signaling responsible for conferring the denticle fate are unknown (O'Keefe, 1997).

Combinatorial signaling by the Frizzled/PCP and Egfr pathways during planar cell polarity establishment in the Drosophila eye

Frizzled (Fz)/PCP signaling regulates planar, vectorial orientation of cells or groups of cells within whole tissues. Although Fz/PCP signaling has been analyzed in several contexts, little is known about nuclear events acting downstream of Fz/PCP signaling in the R3/R4 cell fate decision in the Drosophila eye or in other contexts. This study demonstrates a specific requirement for Egfr-signaling and the transcription factors Fos (AP-1), Yan and Pnt in PCP dependent R3/R4 specification. Loss and gain-of-function assays suggest that the transcription factors integrate input from Fz/PCP and Egfr-signaling and that the ETS factors Pnt and Yan cooperate with Fos (and Jun) in the PCP-specific R3/R4 determination. The data indicate that Fos (either downstream of Fz/PCP signaling or parallel to it) and Yan are required in R3 to specify its fate (Fos) or inhibit R4 fate (Yan) and that Egfr-signaling is required in R4 via Pnt for its fate specification. Taken together with previous work establishing a Notch-dependent Su(H) function in R4, it is concluded that Fos, Yan, Pnt, and Su(H) integrate Egfr, Fz, and Notch signaling input in R3 or R4 to establish cell fate and ommatidial polarity (Weber, 2008).

Previous studies established that Fz is required cell-autonomously for R3 fate induction. The current analyses of kay/fos LOF alleles indicate that Fos is also required cell-autonomously in R3 for its fate determination. When overexpressed, Fos also acts like Fz in R3/R4 photoreceptors at the time of PCP establishment, with the cell of the pair that has higher Fos levels adopting the R3 fate. Based on its requirement in R3 and genetic interactions, Fos could act as a nuclear effector of Fz/PCP signaling. This is supported by the observation that it is able to suppress sev-dsh induced PCP defects; the genetic data can however not rule out that Fos could act in parallel to Fz/Dsh-PCP signaling). The subtle differences observed between fz and kay/fos LOF requirements (in fz⁻ R3/R4 mosaics the wild-type cell adopts the R3 fate often causing chirality inversions, while in kay/fos mosaic pairs with a mutant R3 the pair often adopts symmetrical R4/R4 appearance) is likely due either to the hypomorphic nature of the kay/fos alleles that had to be used in the analysis or potential redundancy with jun (Weber, 2008).

In addition to the positive Fos signaling input, R3 specification also requires the repressor function of Yan, with Yan inhibiting R4 fate in the R3 precursor. This is evident by the cellular requirement of Yan and highlighted by the increased defects in a kay/fos and yan double mutant scenario, where both aspects are partially impaired causing frequent R3/R4 fate decision defects. The dominant enhancement of kay² by yan LOF suggests that keeping the R4 fate off in R3 precursors is as important as inducing the R3 fate (Weber, 2008).

Previous work has demonstrated that Fz/PCP signaling leads to Dl and neur upregulation in R3, activating Notch signaling in the neighboring R4 precursor. This study shows that Egfr-signaling is also specifically required for R4 fate determination. The ETS factors Yan and Pnt are nuclear effectors of Egfr-signaling in many contexts including photoreceptor induction, and the data indicate that they act also in R3/R4 determination. Egfr-signaling leads to an inactivation of Yan and an activation of Pnt through their phosphorylation by the Rl/Erk MAPK. As Yan represses the R4 fate it needs to get inactivated in the R4 precursor by Egfr-signaling and conversely Pnt is activated in R4. Together with the Notch-Su(H) activity this leads to R4 fate induction. Thus, for R3 determination Fz/PCP signaling and its nuclear effectors Fos (and Jun) are sufficient, along with Yan mediated repression of the R4 fate in R3 precursors. R4 fate determination, on the other hand, requires the joint activity of two pathways, Notch and Egfr-signaling and their nuclear effectors. A similar Egfr-Notch cooperation is observed in R7 induction and in cone cells (Weber, 2008).

These data support a complex interaction scenario between Fz/PCP, Notch, and Egfr-signaling in R3/R4 fate determination. Whereas the Notch-Su(H) activation in R4 depends on Fz/PCP signaling in the R3 precursor, the Fz/PCP and Egfr-signaling pathways require a fine balance. This is reflected by their genetic interactions, both at the level of the receptors fz and Egfr and their nuclear effectors Fos/Jun and the ETS factors Pnt and Yan, suggesting a cooperative involvement between the Fz/PCP and Egfr pathways (Weber, 2008).

The nuclear Egfr-signaling response is very likely mediated by Pnt in R4. Although this could not be addressed in pnt LOF clones due to the non-autonomous defects, which are caused by feedback loop requirements in which Pnt participates. The sufficiency experiments fully support a cell-autonomous requirement of Pnt in R4 to specify R4 fate, consistent with the Egfr requirement (Weber, 2008).

In summary, the behavior of the nuclear effectors of the respective signaling pathways involved in R3/R4 specification reflects the combinatorial nature of the signaling pathway input into the R3 and R4 fates (Weber, 2008).

Although in the embryo Fos and Jun need to act as heterodimeric partners in a non-redundant manner, in imaginal discs the scenario is more complicated. Whereas jun mutant clones display only mild phenotypes and do not affect proliferation/survival, strong kay/fos LOF alleles show severe defects, suggesting that kay/fos is the main AP-1 component acting in imaginal discs. This is supported by recent studies on the role of Fos in cell cycle regulation and proliferation (Hyun, 2006). Nevertheless, the double mutant combination of kay and jun revealed a requirement of both as no kay/fos, jun double mutant cells are recovered, suggesting a partially redundant function of kay/fos and jun in imaginal discs (Weber, 2008).

The specific role of the possible distinct heterodimers between the different Fos isoforms and Jun, or the different Fos isoforms themselves, could be very complex. This complexity is also evident in the fact that overexpression of a dominant-negative Fos protein form or a single wild-type isoform (transcript RA, according to Flybase) causes similar phenotypic defects (e.g. in the eye or in thorax closure). Future experiments will have to address which of the Fos isoforms is required in which context and if and how they interact with Jun (Weber, 2008).

Cooperative recruitment of Yan via a high-affinity ETS supersite organizes repression to confer specificity and robustness to cardiac cell fate specification

Cis-regulatory modules (CRMs) are defined by unique combinations of transcription factor-binding sites. Emerging evidence suggests that the number, affinity, and organization of sites play important roles in regulating enhancer output and, ultimately, gene expression. This study investigated how the cis-regulatory logic of a tissue-specific CRM responsible for even-skipped (eve) induction during cardiogenesis organizes the competing inputs of two E-twenty-six (ETS) members: the activator Pointed (Pnt) and the repressor Yan. Using a combination of reporter gene assays and CRISPR-Cas9 gene editing, it is suggested that Yan and Pnt have distinct syntax preferences. Not only does Yan prefer high-affinity sites, but an overlapping pair of such sites is necessary and sufficient for Yan to tune Eve expression levels in newly specified cardioblasts and block ectopic Eve induction and cell fate specification in surrounding progenitors. Mechanistically, the efficient Yan recruitment promoted by this high-affinity ETS supersite not only biases Yan-Pnt competition at the specific CRM but also organizes Yan-repressive complexes in three dimensions across the eve locus. Taken together, these results uncover a novel mechanism by which differential interpretation of CRM syntax by a competing repressor-activator pair can confer both specificity and robustness to developmental transitions (Boisclair Lachance, 2018).

Development of a multicellular organism relies on tissue-specific gene expression programs to establish distinct cell fates and morphologies. The requisite patterns of gene expression must be both spatiotemporally precise and robust in the face of genetic and environmental variation; this is achieved through the action of transcription factors (TFs), whose activating and repressive inputs are integrated at the cis-regulatory modules (CRMs) or enhancers of their target genes. Consequently, the sequence of each CRM provides a physical blueprint for a combinatorial regulatory code that translates upstream signaling information into downstream gene expression. While significant advances have been made in the ability to distinguish regulatory elements from background noncoding genomic DNA and identify consensus TF-binding motifs within them, understanding of how the intrinsic logic of the cis-regulatory syntax (namely, the number, affinity, position, spacing, and orientation of binding sites) organizes the necessary set of protein-protein and protein-DNA interactions remains poor. Because single-nucleotide polymorphisms in TF-binding sites are being increasingly correlated with altered gene expression and disease susceptibility, the ability to deduce the regulatory logic of an enhancer based on its sequence is important (Boisclair Lachance, 2018).

The tendencies for TFs to cluster into superfamilies and for cells to coexpress multiple nonredundant members of the same superfamily imply that enhancer syntax must enable TFs with very similar DNA-binding preferences to compete, cooperate, and discriminate between binding sites to achieve appropriate gene expression output. Recent insight into these behaviors has come from studies of Hox family TFs. The emerging model suggests a specificity-affinity trade-off such that low-affinity sites are best discriminated, while high-affinity sites can be bound by many different Hox factors. Clustering multiple low-affinity Hox sites permits the cooperative and additive interactions needed for robust gene activation responses without compromising specificity. Whether analogous syntax rules apply to other TF superfamilies is not known, and how transcriptional repressors solve the specificity-affinity problem remains to be tested (Boisclair Lachance, 2018).

The ETS superfamily includes both activators and repressors, all of which recognize the same core DNA sequence, 5'-GGAA/T-3'. ETS TFs are found across metazoan phyla and play key roles in regulating the gene expression programs that direct many aspects of normal development and patterning. Exemplifying this, the Drosophila transcriptional activator Pointed (Pnt) and the repressor Yan operate downstream from receptor tyrosine kinase (RTK) signaling pathways to orchestrate numerous cell fate transitions. Much of the current understanding of Yan and Pnt stems from studying their regulation of even-skipped (eve) expression during cardiac muscle precursor specification at stage 11 of embryogenesis and prospero (pros) expression during R7 photoreceptor specification in the developing eye. Abrogating Pnt-mediated activation or Yan-mediated repression of eve or pros leads to respective loss or ectopic induction of the associated cell fate. Gel shift assays using probes from eve or pros CRMs revealed that most Yan-bound ETS sites are also bound by Pnt, and subsequent high-throughput assays confirm their preferences for very similar sequences. Because none of the in vitro biochemistry has been done with full-length proteins, how accurately the results will predict the outcome of Yan-Pnt competition for ETS sites in CRMs in vivo is uncertain (Boisclair Lachance, 2018).

Hints that binding site syntax might influence Yan recruitment come from in vitro binding studies with TEL1, the human counterpart of Drosophila Yan, and from mathematical modeling of Yan's ETS site occupancy. TEL1 and Yan, unlike Pnt or its mammalian counterpart, ETS1, self-associate via their sterile α motifs (SAMs), and this homotypic interaction is essential for transcriptional repression in both flies and humans. Using gel shift assays, SAM-SAM interactions were shown to mediate cooperative binding of TEL1 at paired ETS sites. A recent theoretical analysis of TEL1/Yan occupancy at equilibrium explains how such cooperative SAM-SAM interactions might promote preferential recruitment to tandem ETS-binding sites. Because neither study examined repressive output, the question of whether preferential or cooperative binding of Yan to closely apposed ETS sites might bias Yan-Pnt competition to permit more complex discrimination of CRM syntax than current models assume remains pressing (Boisclair Lachance, 2018).

To evaluate how CRM syntax organizes the opposing repressive and activating inputs from Yan and Pnt to dictate precise transcriptional output, this study assessed the impact of mutating the eight putative ETS-binding sites identified in the eve muscle heart enhancer (MHE) that drives eve expression in 10 segmentally arrayed clusters of pericardial and muscle cells. This study found that sites with strong affinity best discriminate between Yan and Pnt, with paired sites showing the strongest bias. Thus, mutating a pair of overlapping, conserved, high-affinity ETS sites significantly elevated or expanded MHE reporter expression, consistent with compromised repression. Using CRISPR/Cas9 gene editing of the endogenous MHE, it was shown that mutation of this high-affinity ETS supersite reduced Yan recruitment to not only the MHE but also two other CRMs across the eve locus. Mesodermal Eve expression was elevated, consistent with compromised Yan recruitment, resulting in inadequate repression. In this compromised background, environmental and genetic stresses that would normally be buffered against were now sufficient to induce specification of ectopic Eve-positive (Eve+) cells and reduce survival. It is concluded that the conserved high-affinity ETS pair within the MHE plays a unique and pivotal role in not just recruiting Yan-repressive complexes to the isolated enhancer but also longer-range coordination of transcriptional complex organization and function across the locus (Boisclair Lachance, 2018).

Focusing on ETS-binding motifs within a conserved regulatory module, the eve MHE, this study identified a simple syntax that allows for robust qualitative and quantitative control of enhancer output. Based on extensive mutagenesis of this enhancer, it is proposed that Yan's and Pnt's respective preferences for high- and low-affinity ETS sites provide a mechanism for integrating their competing repressive and activating inputs at individual CRMs. In particular, it was found that the use of paired strong-affinity sites appears critical to the assembly of repressive complexes that dampen eve expression in newly specified cardiac precursors where Yan levels are low and prevent ectopic eve induction in the surrounding mesoderm where levels of activating TFs such as Twi are high. CRISPR/Cas9-mediated mutation of the endogenous MHE confirmed the importance of such optimized syntax for precise Eve expression levels and uncovered an unexpected role of the ETS2,3 dual Yan-binding supersite in longer-range organization of Yan complexes across the locus. It is speculated that efficient Yan recruitment to high-affinity supersites not only influences short-range interactions at the specific enhancer but also fosters longer-range communication across multiple CRMs (Boisclair Lachance, 2018).

The unique repressive contribution of the ETS2,3 pair may reflect an unconventional form of cooperative recruitment that provides novel regulatory capabilities to Yan-repressive complexes. Specifically, even though the two sites in the ETS2,3 pair are probably too close to permit simultaneous occupancy, their immediate juxtaposition may significantly increase the probability of stable Yan binding. For example, although nothing is known about the kinetics and dynamics of Yan-DNA interactions, the presence of two overlapping high-affinity binding sites could promote stable occupancy by increasing the chance that a newly dissociated Yan molecule would immediately rebind. The syntax could also support a more organized dynamic in which the two molecules of a Yan dimer toggle back and forth rapidly between bound and unbound states at the two sites. Even more speculatively, because SAM-mediated dimerization is required for Yan-mediated repression, if the configuration of the ETS2,3 pair ensured that one molecule of the dimer was always bound, this could leave the second free to interact with either an adjacent nonspecific sequence, as was modeled previously (Hope, 2017); another high-affinity ETS site elsewhere in the MHE (for example, site 8); or, even more speculatively, an ETS motif in the D1 or D2 CRM. It is also possible that the in vivo mechanism by which full-length Yan contacts DNA is different from that suggested by the in vitro assays. For example, interactions with other TFs and cofactors might somehow mitigate the steric clash to allow simultaneous occupancy by a Yan dimer. In this case, higher-order Yan complexes (for example, trimers or tetramers) could mediate the requisite longer-range contacts. Regardless of specific mechanism, the idea that high-affinity 'supersites' might be used to anchor longer-range TF-TF and TF-DNA interactions will be an interesting direction for future investigations (Boisclair Lachance, 2018).

Previous work exploring the in vivo functionality of a Yan protein in which the SAM-SAM interface has been mutated to prevent self-association further supports the importance of SAM-mediated repressor cooperativity. Specifically, it was found that although Yan monomers are recruited to enhancers genome-wide in a pattern close to that of wild-type Yan, adequate repression does not occur, and phenotypes consistent with yan loss of function ensue (Webber, 2013a). This work also noted the prevalence of clustered high-affinity ETS sites across a number of Yan ChIP targets, suggesting that the mechanisms uncovered in the dissection of MHE ETS site syntax might be broadly applicable. Focusing on eve, it is suspected that at the resolution of individual ETS sites, in the absence of SAM-mediated cooperativity, Yan occupancy of the ETS2,3 tandem would be insufficiently stable to either compete appropriately with Pnt at the MHE or organize the necessary 3D communication across the locus (Boisclair Lachance, 2018).

A parallel is noted between the consequences of mutating the high-affinity ETS2,3 supersite in the endogenous eve locus and the findings of an earlier analysis in which three different Yan-bound CRMs were deleted within a genomic Eve-YFP BAC transgene (Webber, 2013b). In this earlier study, while deleting the pattern-driving MHE almost completely ablated mesodermal Eve-YFP induction, deleting a 'repressive' Yan-bound element (referred to as the D1) increased Eve-YFP expression ∼1.5-fold and led to the specification of extra Eve+cells. Additionally, deletion of either the MHE or the D1 in the BAC transgene reduced Yan occupancy at not only the deleted element but also the remaining intact CRMs. This study reports a comparable loss of Yan occupancy across the eve locus upon mutation of the MHE ETS2,3 supersite but only a modest increase in Eve levels and no cell fate specification defects. The discrepancy between reduced Yan occupancy and increased Eve levels in the eveMHEmut2,3 mutant relative to the D1 deletion mutant suggests that deleting an entire CRM not only compromises Yan occupancy across the locus but also disrupts additional repressive inputs. Consistent with this interpretation, the eveMHEmut2,3 background appeared highly sensitized, with the increase in Eve levels and Eve+ cell fate specification associated with a twofold increase in pnt dose almost exactly matching the effects of deleting an entire 'repressive' CRM. Further exploration of how high-affinity ETS pairs organize Yan repression at and between CRMs and how this coordinates the competing and collaborating inputs from other TFs will be needed to test these ideas at eve and, more broadly, other target genes (Boisclair Lachance, 2018).

To conclude, a working model is proposed in which Yan's and Pnt's differential interpretation of ETS syntax adds a 'dimmer' capability to the classic on/off switch, thereby refining its sensitivity and tunability. Focusing on eve as an example, prior to the onset of RTK-induced cardiac cell fate specification or in cells subject to submaximal signaling, it is suggested that Yan's bias for high-affinity sites ensures an effectively 100% probability of occupancy at the ETS2,3 supersite and hence stable repression. In this regime, Yan could also outcompete Pnt at the lower-affinity sites to occupy fully the CRM, or, if Yan levels are limiting, as the data suggest, its preference for high-affinity sites and relative 'distaste' for lower-affinity sites could offer Pnt an opportunity to occupy the latter and perhaps influence Yan repression. In contrast, if Yan and Pnt had identical ETS-binding preferences, a less tuned response to RTK signaling would be expected; indeed, when the high-affinity 2,3 pair was removed and hence the strong bias toward Yan occupancy and repression at the MHE, stochastic ectopic expression was induced in the surrounding mesoderm where RTK levels are submaximal. Thus, their distinct preferences ensure that only maximal RTK activation will trigger the necessary shift in Yan-Pnt occupancy and activity to activate eve expression. Furthermore, while previous models assumed a complete switch from total Yan occupancy to total Pnt occupancy as Eve+ cell fates are specified, this work suggests that the ETS2,3 supersite still recruits Yan-repressive input even in Eve+ cells with very low Yan concentration. It is speculated that the ability to apply continued Yan-repressive input after cell fate induction may contribute to the robustness of certain developmental transitions by stabilizing the newly acquired cell fate. In agreement with this, in the context of the endogenous eve locus, disruption of the ETS2,3 pair sensitized eve to both fluctuations in upstream signaling and environmental conditions (Boisclair Lachance, 2018).

More broadly, it is speculated that the interplay between the cis-regulatory logic of a CRM and the unique biophysical parameters of different TFs permits evolution to fine-tune gene expression output to a specific threshold depending on each cell's developmental requirement. In the case of Yan-Pnt-regulated genes, the interplay between the degree of Yan SAM-mediated self-association and ETS syntax enables this repressor-activator pair to discriminate between ETS sites with unexpected precision. Furthermore, instead of RTK activation inducing a complete switch from Yan occupancy to Pnt occupancy as cell fates are induced, the cooperative recruitment of Yan to supersites may enable newly differentiating cells with lower Yan:Pnt ratios to sustain the Yan-repressive influence needed to ensure precision and robustness of the gene expression patterns. It is suggested that these ideas provide an interesting new vantage point for considering how single-nucleotide polymorphisms in TF-binding sites may heighten susceptibility to disease by compromising the robustness of gene regulatory networks (Boisclair Lachance, 2018).

Longevity is determined by ETS transcription factors in multiple tissues and diverse species

Ageing populations pose a major public health crises. Reprogramming gene expression by altering the activities of sequence-specific transcription factors (TFs) can ameliorate deleterious effects of age. This explore how a circuit of TFs coordinates pro-longevity transcriptional outcomes, which reveals a multi-tissue and multi-species role for an entire protein family: the E-twenty-six (ETS) TFs. In Drosophila, reduced insulin/IGF signalling (IIS) extends lifespan by coordinating activation of Aop, an ETS transcriptional repressor, and Foxo, a Forkhead transcriptional activator. Aop and Foxo bind the same genomic loci, and this study shows that, individually, they effect similar transcriptional programmes in vivo. In combination, Aop can both moderate or synergise with Foxo, dependent on promoter context. Moreover, Foxo and Aop oppose the gene-regulatory activity of Pnt, an ETS transcriptional activator. Directly knocking down Pnt recapitulates aspects of the Aop/Foxo transcriptional programme and is sufficient to extend lifespan. The lifespan-limiting role of Pnt appears to be balanced by a requirement for metabolic regulation in young flies, in which the Aop-Pnt-Foxo circuit determines expression of metabolic genes, and Pnt regulates lipolysis and responses to nutrient stress. Molecular functions are often conserved amongst ETS TFs, prompting examination of whether other Drosophila ETS-coding genes may also affect ageing. This study shows that five out of eight Drosophila ETS TFs play a role in fly ageing, acting from a range of organs and cells including the intestine, adipose and neurons. This study expands the repertoire of lifespan-limiting ETS TFs in C. elegans, confirming their conserved function in ageing and revealing that the roles of ETS TFs in physiology and lifespan are conserved throughout the family, both within and between species (Dodson, 2019).

Ageing is characterised by a steady systematic decline in biological function, and increased likelihood of disease. Understanding the basic biology of ageing therefore promises to help improve the overall health of older people, who constitute an ever-increasing proportion of populations. In experimental systems, healthy lifespan can be extended by altered transcriptional regulation, coordinated by sequence-specific TFs. Thus, understanding TFs' functions can reveal how to promote health in late life. Forkhead family TFs, especially Forkhead Box O (Foxo) orthologues, have been studied extensively in this context. This effort has been driven by the association of Foxo3a alleles with human longevity; and the findings that the activation of Foxos is necessary and sufficient to explain the extension of lifespan observed following reduced insulin/IGF signalling (IIS) in model organisms. Foxos interact with additional TFs in regulatory circuits, and it is in this context that their function must be understood. For example, in Caenorhabditis elegans, the pro-longevity activity of Daf-16 is orchestrated with further TFs including Hsf, Elt-2, Skn-1, Pqm-1 and Hlh-30/Tfeb. Examining regions bound by Foxos across animals has highlighted the conserved presence of sites to bind ETS family TFs. In Drosophila, two members of this family, namely Aop (a.k.a. Yan) and Pnt, have been linked to ageing via genetic interactions with Foxo and IIS, and similar interactions are evident in C. elegans. These findings raise questions of the overall roles of ETS factors in ageing, and their relationship to the activities of Foxos (Dodson, 2019).

The ETS TFs are conserved across animals, including 28 representatives in humans. Their shared, defining feature is a core helix-turn-helix DNA-binding domain, which binds DNA on 5'-GGA(A/T)-3' ETS-binding motifs (EBMs). They are differentiated by tissue-specific expression, and variation in peripheral amino acid residues which, along with variation in nucleotides flanking the core EBM, confers DNA-binding specificity. ETS TFs generally function as transcriptional activators, but a few repress transcription. Aop is one such repressor in Drosophila. Aop and its human orthologue Tel are thought to repress transcription by competing with activators for binding sites, recruiting co-repressors, and forming homo-oligomers that limit activator access to euchromatin. Consequently, Aop's role in physiology must be explored in the context of its interactions with additional TFs, especially activators. Foxo is one such activator. Both Foxo and Aop are required for longevity by IIS inhibition, each is individually sufficient to extend lifespan, and both are recruited to the same genomic loci in vivo. Whilst activating either in the gut and fat body extends lifespan, the effect of activating both is not additive. Furthermore, if Aop is knocked down, activating Foxo not only ceases to extend lifespan, but even becomes deleterious for lifespan. Overall, these findings suggest that gene expression downstream of IIS is orchestrated by the coordinated activity of Aop and Foxo, and that there is a redundancy in the function of the two TFs, even though Foxo is a transcriptional activator and Aop a transcriptional repressor. This study started by characterising Aop and its relationship with relevant transcriptional activators, including Foxo. This led revealing that roles in ageing are widespread throughout the ETS TF family, extending across multiple fly tissues and diverse animal taxa (Dodson, 2019).

Promoting healthy ageing by transcriptional control is an attractive prospect, because targeting one specific protein can restructure global gene expression to provide broad-scale benefits. This study suggests key roles for ETS TFs in such optimisation. The results show dual roles for Aop: balancing Foxo's outputs, and opposing Pnt's outputs. These functions coordinate transcriptional changes that correspond to lifespan. Repressing transcription from the ETS site appears to be the key longevity-promoting step, and indeed lifespan was extended by limiting multiple ETS TFs, in multiple fly tissues, and in multiple taxa. Altogether, these results show that inhibiting lifespan is a general feature of ETS transcriptional activators. Presumably the expression of these TFs is maintained, despite costs in late life, because of benefits in other contexts. For example, Pnt is important during development, and expression may simply run-on into adulthood. This study now shows that Pnt is also important for adults facing nutritional variation or stress, and genomic evidence suggests equivalent functions for Ets-4 in C. elegans. In addition, Ets21C is required to mount an effective immune response, and both Ets21C and Pnt control gut homeostasis. Tissue environment appears to be another important contextual factor that determines the lifespan effects of specific ETS TFs. Differences between tissues in chromatin architecture are likely to alter the capacity of a given TF to bind a given site, and the current results show that a given TF, and also upstream RTKs, do not necessarily lead to the same lifespan effect across all tissues. The tissue-specific functions that are shown for ETS TFs, Foxo and RTKs, suggests that transcription is locally coordinated by distinct receptors and TFs in distinct tissues, but that lifespan-regulatory signalling nevertheless converges on the ETS site. This differentiation makes it all the more remarkable that roles in lifespan appear to be conserved amongst ETS family TFs, even in diverse tissue contexts (Dodson, 2019).

The structure of molecular networks and their integration amongst tissues underpins phenotype, including into old age. Unravelling the basics of these networks is a critical step in identifying precise anti-ageing molecular targets. Identifying the least disruptive perturbation of these networks, by targeting the 'correct' effector, is a key goal in order to achieve desirable outcomes without undesirable trade-offs that may ensue from broader-scale perturbation. This targeting can be at the level of specific proteins, cell types, points in the life-course, or a combination of all three. The tissue-specific expression pattern of ETS TFs, and the apparent conservation of their roles in longevity, highlights them as important regulators of tissue-specific programs that may be useful in precise medical targeting of specific senescent pathologies (Dodson, 2019).

GENE STRUCTURE

Exons - two, separated by 16 kb

Bases in 3' UTR - 1683

PROTEIN STRUCTURE

Amino Acids - 732

Structural Domains

Yan has an ETS domain, bracketed by glutamate rich regions, two preceding it, and one following (Lai, 1992).

The members of the ets gene family of transcription factors are characterized by a conserved 85-residue DNA-binding region, termed the ETS domain, that lacks sequence homology to structurally characterized DNA-binding motifs. The ETS domain is composed of three alpha-helices (H) and four beta-strands (S) arranged in the order H1-S1-S2-H2-H3-S3-S4. The four-stranded antiparallel beta-sheet is the scaffold for a putative helix-turn-helix DNA recognition motif formed by helices 2 and 3. The 25 residues extending beyond the ETS domain to the native C-terminus of the truncated Ets-1 also contain a helical segment. On the basis of the similarity of this topology with that of catabolite activator protein (CAP), heat shock factor (HSF), and hepatocyte nuclear factor (HNF-3 gamma), it is proposed that ets proteins are members of the superfamily of winged helix-turn-helix DNA-binding proteins (Donaldson, 1994).

There are eight MAPK phosphorylation consensus repeats on Yan as well as six PEST sequences conferring rapid turnover (Rebay, 1995).

date revised: 5 August 2023

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