org Interactive Fly, Drosophila pointed: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

Gene name - pointed

Synonyms - Ets-2

Cytological map position - 49E

Function - transcription factor

Keywords - Glial differentiation and agonist of the sevenless/Ras/MAPK pathway in photoreceptor development, oncogene

Symbol - pnt

FlyBase ID:FBgn0003118

Genetic map position - 3-79.0

Classification - ETS family

Cellular location - nuclear ation -



NCBI links: Precomputed BLAST | Entrez Gene |
Recent literature
Peco, E., Davla, S., Camp, D., Stacey, S., Landgraf, M. and van Meyel, D. (2016). Drosophila astrocytes cover specific territories of CNS neuropil and are instructed to differentiate by Prospero, a key effector of Notch. Development [Epub ahead of print]. PubMed ID: 26893340
Summary:
Astrocytes are recognized as critical elements in the formation, fine-tuning, function and plasticity of neural circuits in the central nervous system. However, important questions remain unanswered about the mechanisms instructing astrocyte cell fate. This paper describes a study of astrogenesis in the ventral nerve cord of Drosophila larvae, where astrocytes have remarkable morphological and molecular similarities to astrocytes in mammals. The births of larval astrocytes from a multi-glial lineage are described, their allocation to reproducible positions, and their deployment of ramified arbors to cover specific neuropil territories to form a stereotyped astroglial map. Finally, a molecular pathway was unraveled for astrocyte differentiation in which the Ets protein Pointed and Notch signaling pathway are required for astrogenesis; however, only Notch is sufficient to direct non-astrocytic progenitors toward astrocytic fate. Prospero was found to be a key effector of Notch in this process. These data identify an instructive astrogenic program that acts as a binary switch to distinguish astrocytes from other glial cells.

Li, X., Xie, Y. and Zhu, S. (2016). Notch maintains Drosophila type II neuroblasts by suppressing the expression of the Fez transcription factor Earmuff. Development [Epub ahead of print]. PubMed ID: 27151950
Summary:
Notch signaling is critical for maintaining neural stem cell (NSC) self-renewal and heterogeneity, however the underlying mechanism is not well understood. In Drosophila, loss of Notch prematurely terminates the self-renewal of larval type II neuroblasts (NBs, the Drosophila NSCs) and transforms type II NBs into type I NBs. This study demonstrates that Notch maintains type II NBs by suppressing the activation of earmuff (erm) by Pointed P1 (PntP1). It was shown that loss of Notch or components of its canonical pathway leads to PntP1-dependent ectopic Erm expression in type II NBs. Knockdown of Erm significantly rescues the loss of Notch phenotypes and misexpression of Erm phenocopies the loss of Notch. Ectopically expressed Erm promotes the transformation of type II NBs into type I NBs by inhibiting PntP1's function and expression in type II NBs. These data not only elucidate a critical mechanism of Notch-mediated maintenance of type II NB self-renewal and identity, but also reveals a novel function of Erm.

Xie, Y., Li, X., Deng, X., Hou, Y., O'Hara, K., Urso, A., Peng, Y., Chen, L. and Zhu, S. (2016). The Ets protein Pointed prevents both premature differentiation and dedifferentiation of Drosophila intermediate neural progenitors. Development [Epub ahead of print]. PubMed ID: 27510969
Summary:
Intermediate neural progenitor cells (INPs) need to avoid both dedifferentiation and differentiation during neurogenesis, but the mechanisms are not well understood. In Drosophila, the Ets protein Pointed P1 (PntP1) is required to generate INPs from type II neuroblasts. This study investigated how PntP1 promotes INP generation. By generating pntP1-specific mutants and using RNAi knockdown, the loss of PntP1 was shown to lead to both an increase in the type II neuroblast number and the elimination of INPs. The elimination of INPs results from premature differentiation of INPs due to the ectopic Prospero expression in newly generated immature INPs (imINP), whereas the increase in the type II neuroblast number results from the dedifferentiation of imINPs due to a loss of Earmuff at later stages of imINP development. Furthermore, reducing Buttonhead enhances the loss of INPs in pntP1 mutants, suggesting that PntP1 and Buttonhead act cooperatively to prevent premature INP differentiation. These results demonstrate that PntP1 prevents both the premature differentiation and dedifferentiation of INPs by regulating the expression of distinct target genes at different stages of imINP development.
Bartoletti, R., Capozzoli, B., Moore, J., Moran, J., Shrawder, B. and Vivekanand, P. (2017). Short hairpin RNA is more effective than long hairpin RNA in eliciting pointed loss-of-function phenotypes in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 28464429
Summary:
Pointed (Pnt) is a transcriptional activator that functions downstream of the highly conserved Receptor Tyrosine Kinase (RTK) signaling pathway. Pnt is an ETS family transcription factor and encodes for two proteins, PntP1 and PntP2. However, while PntP1 is constitutively active, PntP2 is only active after being phosphorylated by MAPK in the RTK pathway. As mutations in pnt perturb the development of several tissues, the effect and efficacy of using RNAi to target Pnt was examined. pnt RNAi was expressed in the eyes, oocyte, and heart cells using three different RNAi lines: Valium20, Valium10, and VDRC. Valium20 is distinct since it generates a short hairpin RNA (shRNA), while Valium10 and VDRC produce long hairpin dsRNA. It was found that for each tissue examined Valium20 exhibited the strongest phenotype while the Valium10 and VDRC lines produced varying levels of severity; the long hairpin RNA produced by the Valium10 and VDRC lines are unable to effectively knockdown pnt in embryonic tissues.
Wang, G., Gutzwiller, L., Li-Kroeger, D. and Gebelein, B. (2017). A Hox complex activates and potentiates the Epidermal Growth Factor signaling pathway to specify Drosophila oenocytes. PLoS Genet 13(7): e1006910. PubMed ID: 28715417
Summary:
Hox transcription factors specify distinct cell types along the anterior-posterior axis of metazoans by regulating target genes that modulate signaling pathways. A well-established example is the induction of Epidermal Growth Factor (EGF) signaling by an Abdominal-A (Abd-A) Hox complex during the specification of Drosophila hepatocyte-like cells (oenocytes). Previous studies revealed that Abd-A is non-cell autonomously required to promote oenocyte fate by directly activating a gene (rhomboid) that triggers EGF secretion from sensory organ precursor (SOP) cells. Neighboring cells that receive the EGF signal initiate a largely unknown pathway to promote oenocyte fate. This study shows that Abd-A also plays a cell autonomous role in inducing oenocyte fate by activating the expression of the Pointed-P1 (PntP1) ETS transcription factor downstream of EGF signaling. Genetic studies demonstrate that both PntP1 and PntP2 are required for oenocyte specification. Moreover, PntP1 contains a conserved enhancer (PntP1OE) that is activated in oenocyte precursor cells by EGF signaling via direct regulation by the Pnt transcription factors as well as a transcription factor complex consisting of Abd-A, Extradenticle, and Homothorax. These findings demonstrate that the same Abd-A Hox complex required for sending the EGF signal from SOP cells, enhances the competency of receiving cells to select oenocyte cell fate by up-regulating PntP1. Since PntP1 is a downstream effector of EGF signaling, these findings provide insight into how a Hox factor can both trigger and potentiate the EGF signal to promote an essential cell fate along the body plan.
Webber, J. L., Zhang, J., Massey, A., Sanchez-Luege, N. and Rebay, I. (2018). Collaborative repressive action of the antagonistic ETS transcription factors Pointed and Yan fine-tunes gene expression to confer robustness in Drosophila. Development. PubMed ID: 29848501
Summary:
The acquisition of cellular identity during development depends on precise spatiotemporal regulation of gene expression, with combinatorial interactions between transcription factors, accessory proteins and the basal transcription machinery together translating complex signaling inputs into appropriate gene expression outputs. The Drosophila ETS family transcription factors Yan and Pointed, whose opposing repressive and activating inputs orchestrate numerous cell fate transitions downstream of receptor tyrosine kinase signaling, provide one of the premier systems for studying this process. Current models describe the differentiative transition as a switch from Yan-mediated repression to Pointed-mediated activation of common target genes. This paper describes a new layer of regulation whereby Yan and Pointed co-occupy regulatory elements to coordinately repress gene expression, with Pointed unexpectedly required for the genome-wide occupancy of both Yan and the corepressor Groucho. Using even-skipped as a test-case, synergistic genetic interactions between Pointed, Groucho, Yan and components of the RNA polymerase II pausing machinery suggest Pointed integrates multiple scales of repressive regulation to confer robustness. It is speculated that this mechanism may be used broadly to fine-tune the expression of many developmentally critical genes.
Schwarz, B., Hollfelder, D., Scharf, K., Hartmann, L. and Reim, I. (2018). Diversification of heart progenitor cells by EGF signaling and differential modulation of ETS protein activity. Elife 7. PubMed ID: 29869981
Summary:
For coordinated circulation, vertebrate and invertebrate hearts require stereotyped arrangements of diverse cell populations. This study explores the process of cardiac cell diversification in the Drosophila heart, focusing on the two major cardioblast subpopulations: generic working myocardial cells and inflow valve-forming ostial cardioblasts. By screening a large collection of randomly induced mutants several genes involved in cardiac patterning were identified. Further analysis revealed an unexpected, specific requirement of EGF signaling for the specification of generic cardioblasts and a subset of pericardial cells. The Tbx20 ortholog Midline acts as a direct target of the EGFR effector Pointed to repress ostial fates. Furthermore, Edl/Mae, an antagonist of the ETS factor Pointed, was identified as a novel cardiac regulator crucial for ostial cardioblast specification. Combining these findings a regulatory model is proposed in which the balance between activation of Pointed and its inhibition by Edl controls cardioblast subtype-specific gene expression.
BIOLOGICAL OVERVIEW

Pointed is required for the differentiation of glial cells in the ventral nerve cord known as the CNS, and is also required downstream of Ras in the development of the eye. This discussion will deal with the role of pointed in glial differentiation. Glia are companion cells for neurons, providing a substrate for axon growth as well as nourishment, protection and insulation for mature neurons. The central nervous system has two embryonic origins: the mesectoderm, which gives rise to the ventral midline and the neuroectoderm, which gives rise to the CNS proper. Pointed is involved in glial differentiation in both these systems.

Mutations of single-minded, a gene required for the proper formation of the ventral midline, delete the glial cells that express pointed. These cells known as midline glia are absent in pointed mutants. One of two Pointed transcripts, P2, is involved in this functional determination of midline glia. pointed also affects longitudinal glia, another group of glial cells only a few cells away from midline glia. They are not part of the ventral midline, but instead form a part of the CNS proper. pointed mutants result in a misplacement of longitudinal glia, rather their deletion. Glia are required for axon guidance, and a defect in glia results in defective axon guidance. In these cases the pointed P1 transcript is implicated. Proper axon guidance is necessary for the formation of commissures and longitudinal connectives. pointed mutants give rise to fused commissures and thinner than normal longitudinal connectives.

Misplacement of longitudinal glia and improper axon guidance are only part of the disruptive effects caused by pointed mutation. Specific neural cells termed MP2 normally express the antigen Mab22C10. In pointed mutants, MP2 neurons fail to make this antigen. Thus a mutation affecting glial cells results in improper neural functioning. It is possible that the Mab22C10 antigen acts to engender proper axon guidance (Klaes, 1994).

Therefore pointed is involved in glial survival, axon transport dependent on glia, and glial nourishment of neurons. The separate involvement of pointed in the CNS and in the midline, points to the complexity of tissue specific regulation of gene expression.

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral axis and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. The first growth cones navigate toward the anterior most ventral unpaired median (VUM) cell and thus pioneer the prospective posterior commissure. Only when the posterior commissure is established, the anterior commissure forms. In later stages, midline glial cells, migrating toward the posterior, are required to separate anterior and posterior commissures into distinct axon bundles. The VUM neurons reside ventral to the posterior commissure and project in a characteristic axon-bundle to the anterior commissure. Migration of two midline glial cells occurs along these cell processes. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, a saturating ethylmethane sulfonate mutagenesis was conducted, screening for mutations that disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. Two gene functions are required for the initial formation of commissural tracts, in addition to the function of commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures (Hummel, 1999a).

The analysis of mutations reveals two major phenotypic classes: the pointed and the tramtrack groups. pointed and tramtrack mediate different aspects of glial development. In pointed mutants no glial differentiation occurs, whereas ectopic pointed expression results in ectopic glial differentiation. tramtrack, in contrast, does not interfere with actual glial cell differentiation but appears to be required for the repression of neuronal differentiation in these cells. The pointed group consists of pointed itself, rhomboid, kastchen, klotzchen, kette, schmalspur, mochte gern, spitz, Star, cabrio and kubel. Mutations in eight other genes lead to an axon phenotype initially described for tramtrack. In tramtrack-type mutation (tramtrack, shroud, disembodied, spook, shade, shadow, phantom, and rippchen) commissures appear fused, but in contrast to pointed group mutations, connectives are not affected (Hummell, 1999a).

Most of the neurons of the ventral nerve cord send out long projecting axons that cross the midline. In the Drosophila CNS, cells of the midline give rise to neuronal and glial lineages with different functions during the establishment of the commissural pattern. The development of midline cells is fairly well understood. In the developing ventral neural cord, 7-8 midline progenitor cells per abdominal segment generate about 26 glial and neuronal cells, i.e. 3-4 midline glial cells, 2 MP1 neurons, 6 VUM neurons, 2 UMI neurons, as well as the median neuroblast and its support cells. The VUM neurons comprise motoneurons as well as interneurons, which project through the anterior and posterior commissures. Genetic studies indicate that the VUM neurons are involved in the initial attraction of commissural growth cones. The MP1 neurons are ipsilateral projecting interneurons, which participate in the formation of specific longitudinal axon pathways. The median neuroblast divides during larval and pupal stages. Contrary to what occurs in the grasshopper CNS, the Drosophila median neuroblast does not generate midline glial cells. In Drosophila, the midline glial cells develop from a set of 2-3 progenitors located in the anterior part of each segment. A function of the midline glial cells during the maturation of the segmental commissures has been found, such that two midline glial cells migrate along cell processes of the VUM-midline neurons to separate anterior and posterior axon commissures. If this migration is blocked, a typical fused commissure phenotype develops. Toward the end of embryogenesis, midline glial cells are required for the formation of individual fascicles within the commissures (Hummel, 1999b and references).

The formation of distinct anterior and posterior commissures requires intercalating migration of midline glial cells. This migration depends on a neuron-glia interaction at the midline. Thus perturbation of midline glial migration could be either due to cell autonomous defects in the midline glia itself or could be due to defects in the interacting midline neurons. Many genes have been identified that appear to be required for midline glia migration and show fused commissure phenotypes upon mutation. Based on additional phenotypic similarities, 12 of these genes have been placed in the so called pointed group. In order to define further functional relationships of the pointed group genes, a number of double mutant combinations were examined. pointed is expressed and required in the midline glial cells. It is expected that embryos homozygous mutant for both pointed and other members of the pointed group would show a pointed-like CNS phenotype if both gene functions are required only in the glial cells. pointed kästchen or pointed schmalspur or kästchen klötzchen double mutant embryos show a slightly enhanced fused commissure phenotype, as compared to the phenotypes of the single mutants. In pointed klötzchen double mutant embryos a more severe fused commissure phenotype is observed. In order to correlate these axonal phenotypes with the number and location of midline glial cells, single and double mutant embryos were analyzed with different enhancer trap markers. In pointed embryos the midline glial cells fail to migrate at all. In contrast, partial midline glial cell migration combined with a slight reduction of the glial cell number, is observed in stage 16 kästchen, schmalspur and klötzchen embryos. In embryos double mutant for kästchen klötzchen or pointed schmalspur or pointed klötzchen reduced numbers of midline glial cells are detected. This reduction corresponds well to the strength of the fusion of commissures. In the most extreme case only a few glial cells are present per embryo. In addition these cells express the midline glial enhancer trap marker at low levels. Thus, it is concluded that klötzchen, kästchen and schmalspur mainly function in the midline glial cells but compared to pointed have weaker effects on glial differentiation (Hummel, 1999a). In contrast a qualitatively different phenotype is observed in pointed kette double mutant embryos. Here commissures fail to develop in most neuromeres, suggesting that kette does not function in the midline glial cells but rather might act in the midline neurons. To date the only other gene known to be required for the development of midline neurons is orthodenticle (otd), which encodes a transcription factor expressed in these cells. In otd mutant embryos the posterior commissures fail to develop. Interestingly, double mutant embryos for otd and pointed show a phenotype comparable to the pointed kette mutant. Furthermore, otd;kette double mutant embryos display an otd-like phenotype. This supports the notion that kette acts in the midline neurons as does orthodenticle. The failure of midline glial cell migration in kette mutant embryos would then be a non cell autonomous consequence of a defect in the midline neurons (Hummel, 1999b).

In summary, the genetic analyses show that pointed, kästchen, schmalspur and klötzchen act in the midline glia, whereas kette acts in the midline neurons. Only when the development of neurons and glial cells are disrupted in the midline as in otd pointed or pointed;kette double mutant embryos do no commissures develop. However, in klötzchen;kette or kästchen;kette double mutant embryos, a fused commissure phenotype is found. This supports the previous notion that mutations in klötzchen or kästchen have weaker effects on midline glia development than does pointed (Hummel, 1999b).

How are anterior and posterior commissures separated? Beside guiding commissural axons, the midline glial cells are required for the shaping of the two segmental commissures. The formation of two distinct segmental commissures in each segment requires an intercalating migration of two of the 4-6 midline glial cells. This migration is preceded by a neuron-glia interaction at the midline. A surprisingly high number of genes appear to be required for this process. To be routed into the appropriate commissures, axons that cross the midline have to differentiate between the dorsal and ventral side of the combined signals represented by the migrating midline glial cells and by the extending VUM neurons. Thus, some mutations which lead to a fused commissure phenotype could stem from glial cells defects where the dorsal and ventral sides cannot be distinguished any more. Subsequently anterior commissure neurons would grow on the dorsal side of the CNS next to the posterior commissure. However, following migration, the midline glial cells would then be located below the segmental commissures. Some of the mutations will not affect glial migration in the first place but will rather control the general differentiation of midline glial cells, as for example pointed does. Since neuronal migration along glial processes in the vertebrate system depends on several receptor systems, it appears possible that some components have been identified that are required specifically for glial migration along neuronal processes; this also occurs in the visual system of Drosophila. Based on the double mutant analysis, at least one of the genes, kette, appears to be required in the midline neurons. Indeed, a P-element induced kette mutation has been isolated that shows a specific beta-galactosidase expression in the VUM-neurons, suggesting that kette is expressed in neuronal midline cells. Here kette could either control neuronal development or could be involved in the actual neuron-glia interaction at the midline. But why do commissures fail to develop in pointed;kette double mutant embryos? If attraction of first commissural growth cones is mediated by signals emanating from the midline neurons, this would imply that neuronal differentiation at the midline is more defective in the double mutant, when compared to either of the single mutants. As a consequence, neuronal differentiation as well as glial differentiation in the midline must depends on pointed function. In the CNS, pointed is expressed only in glial cells and in pointed mutant embryos the VUM neurons are present and appear to project in their normal pattern. However, they fail to properly differentiate and do not express orthodenticle at high levels. It is presently unknown whether this disruption of VUM glia differentiation is due to a lack of pointed in the midline glia or depends on pointed function in the VUM support glial cells. Based on this evidence it can also be deduced that klötzchen and kästchen do not influence the development of the midline neurons but encode new components regulating glial development (Hummel, 1999b).

The following model is proposed for commissure formation. The initial growth of commissural growth cones towards the midline in stage 12 embryos is guided by an attractive signal expressed by the midline neurons. Presumably, this attraction is mediated by early Netrin expression in the midline neurons or alternatively by the action of a Schizo/Weniger attractive system. At this early developmental stage the midline glial cells are elongated in shape, contacting the epidermis with their basal side and are assumed to send out cellular processes contacting the VUM-midline neurons at the dorsal side of the nervous system. The midline glial cells express a repulsive signal that is conveyed to lateral axons via the Robo receptor and/or the karussell gene product. This repulsive function restricts the first axons to cross the midline just anterior of the VUM neurons. The midline glial cells also express a contact dependent permissive guidance cue helping the axons to cross the midline. Subsequently, neuron-glia interaction at the midline results in the migration of the midline glial cells along processes of the VUM neurons (Hummel, 1999b).

Ets transcription factor Pointed promotes the generation of intermediate neural progenitors in Drosophila larval brains

Intermediate neural progenitor (INP) cells are transient amplifying neurogenic precursor cells generated from neural stem cells. Amplification of INPs significantly increases the number of neurons and glia produced from neural stem cells. In Drosophila larval brains, INPs are produced from type II neuroblasts (NBs, Drosophila neural stem cells), which lack the proneural protein Asense (Ase) but not from Ase-expressing type I NBs. To date, little is known about how Ase is suppressed in type II NBs and how the generation of INPs is controlled. This study shows that one isoform of the Ets transcription factor Pointed (Pnt), PntP1, is specifically expressed in type II NBs, immature INPs, and newly mature INPs in type II NB lineages. Partial loss of PntP1 in genetic mosaic clones or ectopic expression of the Pnt antagonist Yan, an Ets family transcriptional repressor, results in a reduction or elimination of INPs and ectopic expression of Ase in type II NBs. Conversely, ectopic expression of PntP1 in type I NBs suppresses Ase expression the NB and induces ectopic INP-like cells in a process that depends on the activity of the tumor suppressor Brain tumor. These findings suggest that PntP1 is both necessary and sufficient for the suppression of Ase in type II NBs and the generation of INPs in Drosophila larval brains (Zhu, 2011).

This study has demonstrated that Drosophila PntP1 is a key molecule that suppresses Ase expression in type II NBs and promotes the generation of INPs. PntP1 is specifically expressed in type II NB lineages. Ectopic PntP1 expression suppresses Ase expression in type I NBs and induces ectopic INP-like cells. The generation of ectopic INP-like cells requires prior suppression of Ase in the NBs and Brat activity. Conversely, partial loss of PntP1 or inhibiting PntP1 activity results in the reduction or elimination of INPs and activation of Ase expression in type II NBs (Zhu, 2011).

How does PntP1 suppress Ase expression? Given that Pnt was known to function mainly as a transcriptional activator, it is likely PntP1 suppresses Ase expression indirectly by activating the expression of yet to be identified target genes. However, Ets family proteins with transcriptional activation activity, such as Pnt homolog Ets1, could also function as transcriptional repressors in a cell type- and promoter-dependent manner. It cannot be entirely ruled out that PntP1 acts as a transcriptional repressor to suppress Ase expression in type II NBs (Zhu, 2011).

Although results from this study as well as others suggest that suppressing Ase expression in NBs is necessary for the generation of INPs, loss of Ase expression alone in type I NB lineages does not lead to the generation of ectopic INPs. Therefore, in addition to suppressing Ase expression, PntP1 must activate other target genes to promote the generation of INPs. One of the major features that distinguish INPs from GMCs is that INPs undergo several rounds of self-renewing divisions, whereas GMCs divide terminally. Thus, among PntP1 target genes could be cell-cycle regulators that promote INPs to undergo self-renewing divisions. This finding is consistent with the well-established function of Ets transcription factors in cell-cycle control and tumorigenesis. Ets proteins stimulate cell proliferation by inducing the expression of cell-cycle regulators, such as Cyclin D1, Cdc2 kinase, and Myc. In the developing Drosophila eye disk, Pnt up-regulates the expression of String, the Drosophila homolog of Cdc25, to promote the G2-M transition. Therefore, PntP1 likely activates the expression of cell-cycle regulators to promote the self-renewing division of INPs. A partial loss of PntP1 in pntδ33 mutant clones or inhibition of PntP1 activity by Yan may lead to reduced expression of cell-cycle regulators and subsequent precocious termination of self-renewing divisions of INPs, resulting in a reduction or elimination of INPs in type II NB lineages (Zhu, 2011).

Like in type II NB lineages, the results show that the induction of INP-like cells by ectopic expression of PntP1 involves a Brat-mediated maturation process. Brat functions as a translational repressor, but exact targets of Brat in immature INPs remain unknown. The results show that when Brat is knocked down via RNAi, ectopic PntP1 expression leads to overproliferation of type II NB-like cells in type I NB lineages, similar to the phenotype observed in type II NB lineages when Brat is lost (Zhu, 2011).

However, neither ectopic PntP1 expression nor Brat knockdown alone causes similar overproliferation phenotypes in type I NB lineages. Therefore, it is possible that Brat promotes the maturation of INPs in part by translationally suppressing the expression of PntP1 target genes, particularly cell cycle-related genes, in immature INPs, thus preventing PntP1 from activating cell cycle progression in immature INPs before they fully mature (Zhu, 2011).

Although this study shows that PntP1 is able to induce the generation of ectopic INP-like cells, not every type I NB lineage ectopically expressing PntP1 produces INP-like cells. One explanation could be that additional factors may be required for the generation of INPs. These factors could be specifically expressed in type II NB lineages and function either independently, or together with PntP1 as cofactors, to promote the generation of INPs. In the absence of these factors, PntP1 promotes the generation of INPs at a much reduced efficiency. Ets transcription factors often regulate target-gene expression by recruiting other proteins. For example, Pnt interacts with Jun to induce R7 cell-fate specification in the Drosophila retina. It is possible that PntP1 needs cofactors to efficiently activate the expression of target genes that are involved in the generation of INPs. However, it is also possible that Ase and Pros in GMCs in type I NB lineages might counteract the role of PntP1 in cell-cycle progression by activating the expression of the cell-cycle inhibitor Dacapo, thus preventing the generation of self-renewing INP-like cells (Zhu, 2011).

In conclusion, this study demonstrates that PntP1 is responsible for the suppression of Ase in type II NBs and the generation of INPs. Suppression of Ase is likely a prerequisite for PntP1 to induce the generation of INPs. Furthermore, PntP1 possibly activates yet-to-be identified target genes, including cellcycle regulators, to induce the generation of INPs and to promote their self-renewing divisions. In addition, the data suggest that, at least in part, Brat promotes the maturation of INPs likely by suppressing the expression PntP1-regulated cell cycle-related genes in immature INPs, thus preventing immature INPs from entering self-renewing divisions before they fully mature (Zhu, 2011).

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).


GENE STRUCTURE

TRANSCRIPT #1 (P1)

Amino Acids - 623

Bases in 5' UTR - 1046

Exons - four: the first is not shared with transcript #2 and encodes 229 amino acids.

Bases in 3' UTR - 233


TRANSCRIPT #2 (P2)

Bases in 5' UTR - 775

Exons - eight exons, spanning over 50 kb. The first five are not shared with transcript #1 and encode 324 amino acids.

Bases in 3' UTR - 233


PROTEIN STRUCTURE

Amino Acids 718

Structural Domains

Pointed has an ETS oncogene domain and a second evolutionally conserved domain, the pointed domain, present in the N-terminal region of P2 but not P1. The pointed domain is shared with murine ETS 1, GA binding protein alpha, and other ETS homologs (Klambt, 1993).

Homology of Pointed ETS domain to mouse or human ELK 1 is 95% in the central ETS domain (Klambt, 1993).

Genetic analysis of lin-1 loss-of-function mutations suggests that lin-1 controls multiple cell-fate decisions during Caenorhabditis elegans development and is negatively regulated by a conserved receptor tyrosine kinase-Ras-ERK mitogen-activated protein (MAP) kinase signal transduction pathway. LIN-1 protein contains an ETS domain and presumably regulates transcription. The vertebrate proteins Elk-1, SAP-1a, and Net/ERP/SAP-2 are classified as members of the Elk subfamily of ETS proteins because they share three regions of significant sequence conservation: an N-terminal ETS domain, a centrally positioned B box, and a C-terminal C box. Based on the positions and sequences of their ETS domains and the positions and sequences of regions similar to the C box, it is proposed that LIN-1 and Drosophila Aop are both members of the Elk subfamily. The ETS domain of LIN-1 shares more sequence identity with the ETS domain of human Elk-1 (67% identity) and human SAP-1a (61% identity) than with any other ETS domain. Likewise, the ETS domain of Aop is most similar to the ETS domain of Elk-1 (51% identity). The ETS domains of LIN-1 and Aop are somewhat less similar (41% identity). LIN-1 (441 residues), Elk-1 (428 residues), SAP-1a (453 residues) and Net (409 residues) are similarly sized and have ETS domains similarly positioned in the N-terminal region. By comparison, Aop (688 residues) is larger and has more residues N-terminal to the ETS domain, which is located near the center of the protein. However, the number of residues C-terminal to the ETS domain is similar among all five proteins analyzed. Sequence similarity outside the ETS domain provides further evidence that ETS proteins are members of a subfamily. By studying the C termini of these proteins, it was found that LIN-1, Elk-1, SAP-1a, and Net each have the sequence FQFP, while Aop has the sequence FQFHP. In Elk-1, SAP-1a, and Net, the FQFP sequence is at the end of the C box. The C boxes of ELK-1, SAP-1a, and Net are characterized by five or six S/TP sequences, which are potential MAP kinase phosphorylation sites. In the corresponding regions, LIN-1 has five S/TP sequences and Aop has three. Elk-1, SAP-1a, and Net have additional identities in the C box that are not conserved in LIN-1 and Aop. These observations suggest that LIN-1 and Aop contain divergent C boxes. Thus, lin-1, aop, elk-1, sap-1, and net appear to be derived from an ancestral gene that encoded a protein with an N-terminal ETS domain and a C-terminal C box (Jacobs, 1998 and references).

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies: protein interactions and posttranslational modifications, respectively. By using NMR, the structure of a 110-residue fragment of murine Ets-1 has been determined that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site (Slupsky, 1998).


pointed : Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

date revised:  12 December 99

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