Other Ets family members

A yeast one-hybrid system has been used to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE. This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities. It therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity. To search for transcriptional regulators, a fragment of the SpHE promoter containing several individual elements has been used instead of the conventional bait that contains a multimerized cis element. This screen yields a number of positive clones that encode a new member of the Ets family, named SpEts4. This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain. Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments. The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts. SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output. As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula (Wei, 1999).

The TEL gene, which is frequently rearranged in human leukemias of both myeloid and lymphoid origin, encodes a member of the Ets family of transcription factors. The TEL gene is widely expressed throughout embryonic development and in the adult. The amino-terminal HLH (or pointed domain) is highly conserved among a subset of Ets proteins including Ets-1, Ets-2, Erg, Fli-1, Yan and Pointed. While this domain facilitates self-association of TEL, such oligomerization properties have not been observed for other members of the family. Nonetheless, it has been established that the corresponding region of Ets-1 and Ets-2 is required for full transactivation and particularly for synergy with the Ras pathway. To determine the requirement for the TEL gene product in development, TEL knockout mice (TEL-/-) were generated by gene targeting in embryonic stem cells. TEL-/- mice are embryonic lethals and die between E10.5-11.5 with defective yolk sac angiogenesis and intra-embryonic apoptosis of mesenchymal and neural cells. Two-thirds of TEL-deficient yolk sacs at E9.5 lack vitelline vessels, yet possess capillaries, indicative of normal vasculogenesis. Vitelline vessels regress by E10.5 in the remaining TEL-/- yolk sacs. However, hematopoiesis at the yolk sac stage appears unaffected in TEL-/- embryos. These findings demonstrate that TEL is required for maintenance of the developing vascular network in the yolk sac and for survival of selected cell types within the embryo proper (Wang, 1997).

Spi-B is a hematopoietic-specific Ets family transcription factor closely related to PU.1. Previous gene targeting experiments have shown that PU.1 is essential for the production of both lymphocytes and monocytes. Mice have been generated with a null mutation at the Spi-B locus. Unlike PU.1 mutant mice, Spi-B-/- mice are viable, fertile and possess mature B and T lymphocytes. However, Spi-B-/- mice exhibit severe abnormalities in B cell function and selective T cell-dependent humoral immune responses:

  1. Although Spi-B-/- splenic B cells respond normally to lipopolysaccharide stimulation in vitro, these B cells proliferate poorly and die in response to B cell receptor (surface IgM) cross-linking.
  2. Spi-B-/- mice display abnormal T-dependent antigenic responses in vivo and produce low levels of antigen-specific IgG1, IgG2a and IgG2b after immunization.
  3. Spi-B-/- mice show a dramatic defect in germinal center formation and maintenance. In contrast to wild-type animals, germinal centers in Spi-B-/- mice are smaller and short-lived with significantly increased numbers of apoptotic B cells.
Taken together, these results demonstrate that Spi-B is essential for antigen-dependent expansion of B cells, T-dependent immune responses and maturation of normal germinal centers in vivo (Su, 1997).

CD18, the beta chain of the leukocyte integrins, plays a crucial role in immune and inflammatory responses. CD18 is expressed exclusively by leukocytes, and it is transcriptionally regulated during the differentiation of myeloid cells. The ets factors, PU.1 and GABP, bind to three ets sites in the CD18 promoter, which are essential for high level myeloid expression of CD18. Two binding sites have been identified for the transcription factor, Sp1; these binding sites flank the ets sites. Sp1 is the only factor from myeloid cells that binds to these sites in a sequence-specific manner. Mutagenesis of these sites abrogates Sp1 binding and significantly reduces the activity of the transfected CD18 promoter in myeloid cells. Transfection of Sp1 into Drosophila Schneider cells, which otherwise lack Sp1, dramatically activates the CD18 promoter. GABP also activates the CD18 promoter in Schneider cells. Co-transfection of Sp1 and GABP activates CD18 more than the sum of their individual effects, indicating that these factors cooperate to transcriptionally activate myeloid expression of CD18. These studies support a model of high level, lineage-restricted gene expression mediated by cooperative interactions between widely expressed transcription factors (Rosmarin, 1998).

Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore, PU.1(-/-) embryonic stem (ES) cells fail to differentiate into Mac-1(+) and F4/80(+) macrophages in vitro. A PU.1 transgene under the control of its own promoter restores the ability of PU. 1(-/-) ES cells to differentiate into macrophages. Advantage was taken of a PU.1(-/-) ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in the assay system, it has been established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, has no effect on the ability of PU.1 to promote macrophage development. Mutagenesis of four independent sites of serine phosphorylation also have no effect on myelopoiesis. Collectively, these results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU.1(-/-) ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages (Fisher, 1998).

Little is known about the transcription factors that mediate lineage commitment of multipotent hematopoietic precursors. One candidate is the Ets family transcription factor PU.1, which is expressed in myeloid and B cells and is required for the development of both these lineages. The factor specifically instructs transformed multipotent hematopoietic progenitors to differentiate along the myeloid lineage. This involves not only the up-regulation of myeloid-specific cell surface antigens and the acquisition of myeloid growth-factor dependence but also the down-regulation of progenitor/thrombocyte-specific cell-surface markers and GATA-1. Both effects require an intact PU.1 transactivation domain. Whereas sustained activation of an inducible form of the factor leads to myeloid lineage commitment, short-term activation leads to the formation of immature eosinophils, indicating the existence of a bilineage intermediate. These results suggest that PU.1 induces myeloid lineage commitment by the suppression of a master regulator of nonmyeloid genes (such as GATA-1) and the concomitant activation of multiple myeloid genes (Nerlov, 1998).

Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which synergistic, tissue-specific activation can be studied. Myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors that are crucial for normal hematopoiesis: PU.1 (an ETS domain transcription factor), AML1 (the mammalian homolog of Drosophila Runt), and C/EBPalpha (see Drosophila Slbo. These proteins interact in such a way as to demonstrate at least two examples of synergistic activation. AML1 and C/EBPalpha are shown to activate the M-CSF receptor promoter in a synergistic manner. AML1 also synergizes, and interacts physically, with PU.1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPalpha has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPalpha, but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPalpha. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPalpha, and PU.1 (Petrovick, 1998).

PU.1 is a unique regulatory protein required for the generation of both the innate and the adaptive immune system. It functions exclusively in a cell-intrinsic manner to control the development of granulocytes, macrophages, and B and T lymphocytes. Mutation of the PU.1 gene causes a severe reduction in myeloid (granulocyte/macrophage) progenitors. PU.1 -/- myeloid progenitors can proliferate in vitro in response to the multilineage cytokines interleukin-3 (IL-3), IL-6 and stem cell factor but are unresponsive to the myeloid-specific cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and M-CSF. The failure of PU.1 -/- progenitors to respond to G-CSF is bypassed by transient signaling with IL-3. In the presence of IL-3 and G-CSF, PU.1 -/- progenitors can differentiate into granulocytic precursors containing myeloperoxidase-positive granules. Thus PU.1 is not essential for specification of granulocytic precursors, but is required for their further differentiation. The failure of PU.1 -/- progenitors to respond to M-CSF is due to lack of c-fms gene (coding for the CSF receptor) transcription. Transduction of c-fms into PU.1 -/- myeloid progenitors bypasses the block to M-CSF-dependent proliferation but does not induce detectable macrophage differentiation. Therefore, PU.1 appears to be essential for specification of monocytic precursors. Importantly, retroviral transduction of PU.1 into mutant progenitors restores responsiveness to myeloid-specific cytokines and development of mature granulocytes and macrophages. Thus PU.1 controls myelopoiesis by regulating both proliferation and differentiation pathways (DeKoter, 1998).

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated by multiple pathways. Agrin, which is the signal for one pathway, stimulates a redistribution of previously unlocalized AChRs to synaptic sites. The signal for a second pathway is not known, but this signal stimulates selective transcription of AChR genes in myofiber nuclei located near the synaptic site. Neuregulin (NRG) is a good candidate for the extracellular signal that induces synapse-specific gene expression, since NRG is concentrated at synaptic sites and activates AChR gene expression in cultured muscle cells. Previous studies have demonstrated that 181 bp of 5' flanking DNA from the AChR delta-subunit gene are sufficient to confer synapse-specific transcription in transgenic mice and NRG responsiveness in cultured muscle cells, but the critical sequences within this cis-acting regulatory region have not been identified. AChR delta-subunit-hGH gene fusions were transfected into a muscle cell line, and it has been shown that a potential binding site for Ets proteins is required for NRG-induced gene expression. Furthermore, transgenic mice were produced carrying AChR delta-subunit-hGH gene fusions with a mutation in this NRG-response element (NRE), and this NRE was shown to be necessary for synapse-specific transcription in mice. The NRE binds proteins in myotube nuclear extracts; nucleotides that are important for NRG responsiveness are likewise critical for formation of the protein-DNA complex. This complex contains GABPalpha, an Ets protein, and GABPbeta, a protein that lacks an Ets domain but dimerizes with GABPalpha, because formation of the protein-DNA complex is inhibited by antibodies to either GABPalpha or GABPbeta. These results demonstrate that synapse-specific and NRG-induced gene expression require an Ets-binding site and suggest that GABPalpha/GABPbeta mediates the transcriptional response of the AChR delta-subunit gene to synaptic signals, including NRG (Fromm, 1998).

How do functionally related motor neurons and sensory neurons form selectively connected circuits? In other words, what is the basis of recognition between these two types of neurons that exhibit an independent developmental origin? Motor function depends on the formation of selective connections between sensory and motor neurons and their muscle targets. The molecular basis of the specificity inherent in this sensory-motor circuit remains unclear. Insight into the origins of specificity in motor circuitry has emerged from an analysis of the innervation of limb muscles. Two levels of organization are evident in the arrangement of motor neuron subtypes that project to the limb: (1) motor neurons exhibit a columnar organization in which the cell bodies of motor neurons that innervate limb muscles are confined to the lateral motor column (LMC). There is a further subdivision of LMC neurons that reflects the position of their target muscles. Motor neurons that innervate ventrally and dorsally derived limb muscles are located, respectively, within the medial and lateral divisions of the LMC. (2) At a second level of organization, motor neurons that project to individual limb muscles are segregated into discrete clusters, termed motor pools. The pool identity of LMC neurons is reflected in their pattern of innervation by the afferent neurons that provide sensory feedback from limb muscles. Muscle sensory afferents form selective monosynaptic connections with functionally related motor pools. The selectivity of these connections is evident at the time that muscle afferents first contact motor neurons and endures both in the absence of neural activity and under conditions of inappropriate activity. Selective recognition between subsets of muscle sensory afferents and motor neurons may therefore underlie the formation of functionally appropriate connections in this neural circuit. Nevertheless, the central connections of muscle sensory afferents appear to be influenced by signals from the periphery. The molecular basis of the subtype distinctions that permit motor neurons to establish specific connections in the periphery and to receive selective sensory inputs remains unclear. Columnar subclasses of motor neurons can be distinguished by the expression of members of a family of LIM homeodomain (LIM-HD) proteins, which may control the ability of motor neurons to select distinct axon pathways in the periphery. The expression of LIM-HD proteins is insufficient, however, to account for the organization and diversity of motor neuron pools within the LMC. The delineation of columnar subclasses of motor neurons by LIM-HD protein expression, however, raises the possibility that motor pool identity is defined by a distinct class of transcription factors (Lin, 1998 and references).

Motor neuron pools and subsets of muscle sensory afferents can be defined by the expression of ETS genes, notably the closely releated PEA3 and ER81 ETS genes. Functionally interconnecting motor and sensory motor neurons exhibit a match in their expression of PEA3 and ER81 expression. ETS gene expression by motor and sensory neurons fails to occur after limb ablation, suggesting that their expression is coordinated by signals from the periphery. ETS genes may therefore participate in the development of selective sensory-motor circuits in the spinal cord. The matching of ETS protein expression by sensory and motor neurons raises the possibility of a corresponding match in the cell surface properties of these neurons. One strategy for selective recognition between cells involves homophilic molecular interactions. The cadherins are prevalent homophilic recognition molecules, and analysis of their patterns of expression in the vertebrate CNS has led to the proposal that matching cadherin expression by pre- and post-synaptic neurons participates in the establishment of regional-specific neuronal connections. At least one member of this family, T-cadherin, is expressed in a pool-specific manner within the LMC, and the transcription of certain cadherin genes is controlled by ETS proteins. Analysis of the relationship between ETS protein and cadherin expression might therefore provide additional insight into the process of selective recognition between sensory afferents and motor neurons (Lin, 1998).

Previous studies have suggested that embryonic vascular endothelial, endocardial and myocardial lineages originate from multipotential cardiovascular progenitors. However, their existence in vivo has been debated and molecular mechanisms that regulate specification of different cardiovascular lineages are poorly understood. An ETS domain transcription factor Etv2/Etsrp/ER71 has been recently established as a crucial regulator of vascular endothelial differentiation in zebrafish and mouse embryos. This study shows that etsrp-expressing vascular endothelial/endocardial progenitors differentiate as cardiomyocytes in the absence of Etsrp function during zebrafish embryonic development. Expression of multiple endocardial specific markers is absent or greatly reduced in Etsrp knockdown or mutant embryos. Etsrp regulates endocardial differentiation by directly inducing endocardial nfatc1 expression. In addition, Etsrp function is required to inhibit myocardial differentiation. In the absence of Etsrp function, etsrp-expressing endothelial and endocardial progenitors initiate myocardial marker hand2 and cmlc2 expression. Furthermore, Foxc1a function and interaction between Foxc1a and Etsrp is required to initiate endocardial development, but is dispensable for the inhibition of myocardial differentiation. These results argue that Etsrp initiates endothelial and endocardial, and inhibits myocardial, differentiation by two distinct mechanisms. These findings are important for the understanding of genetic pathways that control cardiovascular differentiation during normal vertebrate development and will also greatly contribute to the stem cell research aimed at regenerating heart tissues (Palencia-Desai, 2011).

Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network

The transcriptional pathways activated downstream of Vascular Endothelial Growth Factor (VEGF; see Drosophila Pvf1) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand, Delta-Like 4 (DLL4; see Drosophila Delta) in endothelial cells this study found that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG (see Drosophila Pointed), a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300 (see Drosophila Nejire). Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG ChIP-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG, and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (a transcription factor implicated in sprouting angiogenesis; see Drosophila Homeodomain protein 2.0) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis (Fish, 2017).

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

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