pointed

EVOLUTIONARY HOMOLOGS (part 2/4)

Ets target genes

Members of the Ets family of transcription factors mediate transcriptional responses of multiple signaling pathways in diverse cell types and organisms. Targeted deletion of the conserved DNA binding domain of the Ets2 transcription factor results in the retardation and death of homozygous mouse embryos before 8.5 days of embryonic development. Defects in extraembryonic tissue gene expression and function include deficient expression of matrix metalloproteinase-9 (MMP-9, gelatinase B), persistent extracellular matrix, and failure of ectoplacental cone proliferation. Mutant embryos were rescued by aggregation with tetraploid mouse embryos, which complement the developmental defects by providing functional extraembryonic tissues. Rescued Ets2-deficient mice are viable and fertile but have wavy hair, curly whiskers, and abnormal hair follicle shape and arrangement, resembling mice with mutations of the EGF receptor or its ligands. However, these mice are not deficient in the production of TGFalpha or the EGF receptor. Homozygous mutant cell lines respond mitogenically to TGFalpha, EGF, FGF1, and FGF2. However, FGF fails to induce MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in the Ets2-deficient fibroblasts. Ectopic expression of Ets2 in the deficient fibroblasts restores expression of both matrix metalloproteinases. Therefore, Ets2 is essential for placental function and mediating growth factor signaling to key target genes in different cell types, including MMP-3, MMP-9, and MMP-13, and for regulating hair development (Yamamoto, 1998).

Cis-acting DNA sequences in the murine junB promoter are capable of mediating transcriptional activation by the proto-oncogene products c-Ets-1 and c-Ets-2. In vitro DNA binding assays indicate that the elements identified can specifically interact with c-Ets-1 protein. ETS-transactivation of a variety of reporter constructs is dramatically enhanced by introduction of oncogenic Ha-ras. The activation of Ras by extracellular stimuli invokes a phosphorylation cascade that includes the downstream mitogen-activated protein (MAP) kinase p44ERK-1 (Drosophila homolog: Rolled). Addition of activated p44ERK-1 MAP kinase can also enhance ETS-transactivation of junB promoter reporter constructs. Thus ETS-family members play a role in the activation of junB transcription by a Ras-stimulated signal transducing pathway that includes MAP kinase(s) (Coffer, 1994).

Ets-2 is a member of a family of transcription factors implicated in the regulation of gene expression during cell proliferation, cell differentiation, and development. The Ets-2 protein transactivates the promoter of the cdc2 gene which encodes a 34-kDa serine-threonine kinase required for mitotic initiation in mammalian cells. Transactivation occurs via specific interaction with multiple ets binding sites in the 5' flanking region of the gene. Increased cdc2 transcriptional activation correlates with elevated levels of cyclin A but not cyclin B1. Furthermore, ets-2-transfected cells grow under low serum conditions, albeit at a reduced rate. These data demonstrate that Ets-2 plays a direct role in the regulation of cdc2 expression and raise the possibility that Ets-2 participates in the coordinated regulation of cdc2 cyclin A expression (Wen, 1995).

Transforming mutants of p21ras induce the cyclin D1 promoter. Site-directed mutagenesis of AP-1-like sequences at in the cyclin D1 promoter abolishes p21ras-dependent activation of cyclin D1 expression. The AP-1-like sequences are also required for activation of the cyclin D1 promoter by c-Jun. Several AP-1 proteins (c-Jun, JunB, JunD, and c-Fos) bind the cyclin D1 promoter. cyclin D1 promoter activity is stimulated by overexpression of mitogen-activated protein kinase (MAPK) or c-Ets-2 through the proximal 22 base pairs. Expression of plasmids encoding either dominant negative MAPK or dominant negatives of ETS activation, antagonized MAPK-dependent induction of cyclin D1 promoter activity. Epidermal growth factor induction of cyclin D1 transcription, through the proximal promoter region, is antagonized by either dominant negative MAPK or dominant negatives of ETS activation, suggesting that ETS functions downstream of epidermal growth factor and MAPK in the context of the cyclin D1 promoter. The activation of cyclin D1 transcription by p21ras provides evidence for cross-talk between the p21ras and cell cycle regulatory pathways (Albanese, 1995).

Binding sites for the tissue-specific POU-homeodomain transcription factor, Pit-1, are required for basal and hormonally induced prolactin gene transcription. The 3P DNA element or the rat prolactin gene is found to contain a consensus binding site for the Ets family of proteins. Mutation of the Ets binding site greatly decreases the ability of epidermal growth factor, phorbol esters, Ras, or the Raf kinase to induce reporter gene activity. Mutation of the Ets site had little effect on basal enhancer activity. In contrast, mutation of the consensus Pit-1 binding site in the 3P element essentially abolished all basal enhancer activity. Overexpression of Ets-1 in GH3 pituitary cells enhanced both basal and Ras induced activity from the 3P enhancer. These data describe a composite element in the prolactin gene containing binding sites for two different factors and the studies suggest a mechanism by which Ets proteins and Pit-1 functionally cooperate to permit transcriptional regulation by different signaling pathways (Howard, 1995).

Tissue factor (TF) is induced in THP-1 cells stimulated with lipopolysaccharide (LPS). DNase I footprinting identifies six sites of protein-DNA interaction between -383 and the cap site that varies between control and induced extracts. Four footprints show qualitative differences in nuclease sensitivity. Footprints I (-85 to -52) and V (-197 to -175) are induction-specific and localize to regions of the promoter that mediate serum, phorbol ester, partial LPS response (-111 to +14), and the major LPS-inducible element (-231 to -172). Electrophoretic mobility shift assays with the -231 to -172 probe demonstrate JunD and Fos binding in both control and induced nuclear extracts; however, binding of c-Jun is only detected following LPS stimulation. Antibody inhibition studies implicate binding of Ets-1 or Ets-2 to the consensus site between -192 and -177, a region that contains an induction-specific footprint. The proximal region (-85 to -52), containing the second inducible footprint, binds Egr-1 following induction. These data suggest that LPS stimulation of THP-1 cells activate binding of c-Jun, Ets, and Egr-1 to the TF promoter and implicates these factors in the transcriptional activation of TF mRNA synthesis (Groupp, 1997).

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example, in Ewing's sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, two approaches were used. In the first approach, genes were isolated by the RNA differential display technique. Three known genes isolated by differential display are identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, taken to isolate ETS target promoters directly, ETS1 binding was performed with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned. Of the large number of clones isolated, 43 represent unique sequences not previously identified. Three clones turn out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II and Egr1 genes. The ETS binding sites derived from these three regulatory sequences show specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene (Robinson, 1997).

The MafB transcriptional activator, a member of a family of genes encoding bZIP transcription factors, plays a pivotal role in regulating lineage-specific gene expression during hematopoiesis by repressing Ets-1-mediated transcription of key erythroid-specific genes in myeloid cells. To determine the effects of Maf family proteins on the transactivation of myeloid-specific genes in myeloid cells, the ability of c-Maf to influence Ets-1- and c-Myb-dependent CD13/APN transcription was tested (see Drosophila Myb). Expression of c-Maf in human immature myeloblastic cells inhibits CD13/APN-driven reporter gene activity (85 to 95% reduction) and requires the binding of both c-Myb and Ets, but not Maf, to the promoter fragment. c-Maf's inhibition of CD13/APN expression correlates with its ability to physically associate with c-Myb. While c-Maf mRNA and protein levels remain constant during myeloid differentiation, formation of inhibitory Myb-Maf complexes is developmentally regulated, their levels being highest in immature myeloid cell lines and markedly decreased in cell lines representing later developmental stages. This pattern matches that of CD13/APN reporter gene expression, indicating that Maf modulation of c-Myb activity may be an important mechanism for the control of gene transcription during hematopoietic cell development (Hedge, 1998).

ZEB, a vertebrate homolog of Drosophila Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. One target of this positive/negative regulation in vivo is the alpha4 integrin, which plays a key role in normal hematopoiesis and function of mature leukocytes. Stem cell alpha4 integrin, negatively regulated by ZEB, is known to bind VCAM-1 and fibronectin located in the stroma of hematopoetic organs. alpha4 integrin binding to stromal ligands is necessary for lymphoid differentiation (Postigo, 1997b).

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. The nature of Fgf response element (FRE) has been investigated in a posterior neural gene, Xcad3 (Xenopus caudal homolog), which plays a crucial role of posterior neural development. Evidence suggests that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s) (Haremaki, 2003).

The reporter constructs containing the FREs exhibit high dose dependence on Fgf similar to that shown for endogenous Xcad3, when examined in the embryonic cell culture assay. Sequence and mutagenesis analyses reveal that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs (EBMs and TLBMs respectively) that lie in juxtaposition. The EBM is known to serve as the binding site for Ets family transcription factors that are nuclear effectors of the Fgf/Ras/Mapk pathway. Indeed, functional and physical analyses show that Ets proteins are involved in the Fgf response of Xcad3 as transcriptional activators, and that Xcad3 is directly targeted by the Fgf signaling pathway. This conclusion is consistent with the previous observation that Fgf can induce Xcad3 expression in the animal cap assay within 2 hours of its addition and even in the presence of the protein synthesis inhibitor cycloheximide, which indicates that Xcad3 is an immediate early target of Fgf signaling (Haremaki, 2003 and references therein).

TLBMs could serve as the binding sites for Tcf/Lef family transcription factors that are nuclear effectors of the Wnt/ß-catenin pathway. It was anticipated that XTcf3 would functioned as a co-activator of Ets proteins, since Wnt signaling has been suggested as being involved in activation of posterior neural genes. Surprisingly, however, functional analysis reveals that XTcf3 acts as a repressor of Xcad3. The data suggest that the endogenous pool of ß-catenin in ectoderm cells is considerably smaller compared with that of XTcf3 co-repressors such as XCtBP and Groucho. This in turn implies that Wnt signaling could activate Xcad3 expression in embryonic cells, when they are provided with a larger pool of ß-catenin. Marginal zone cells of the early gastrula embryo, where Xcad3 is initially expressed, are among such candidate cells, since a relatively large amount of ß-catenin is translocated into the nucleus in these cells. Recently, a mutant function of Tcf3 as a repressor has revealed in the zebrafish headless mutant that carries a mutation in Tcf3. In this mutant, expression of midbrain-hindbrain boundary genes such as En2 and Pax2 is de-repressed in more anterior neural region, leading to severe head defects. It would be interesting to know whether similar anterior expansion is seen in Cdx gene expression in this mutant (Haremaki, 2003 and references therein).

Sox2 is de-repressed by Bmp antagonists in the neurogenic region of ectoderm during neural induction. Sox2, which shares a cognate DNA bindings motif with Tcf/Lef family members, is required as a co-activator for the Fgf response of Xcad3. Sox2 is likely to compete with XTcf3 for TLBMs in the composite FREs to cooperate with Ets proteins that bind to adjacent EBMs. Physical analysis supports this idea. Both Sox and Ets family transcription factors interact with specific partner factors to direct signals to target genes, but direct partnership between them has not been reported. Collectively, these results indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on the FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox proteins and negatively acting Tcf protein (Haremaki, 2003).

Members of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors play key roles in the development and differentiation of numerous cell types during mammalian development, including the vascular endothelium. mef2c is expressed very early in the development of the endothelium, and genetic studies in mice have demonstrated that mef2c is required for vascular development. However, the transcriptional pathways involving MEF2C during endothelial cell development have not been defined. As a first step towards identifying the transcriptional factors upstream of MEF2C in the vascular endothelium, a screened was carried out for transcriptional enhancers from the mouse mef2c gene that regulate vascular expression in vivo. In this study, a transcriptional enhancer was identified from the mouse mef2c gene sufficient to direct expression to the vascular endothelium in transgenic embryos. This enhancer is active in endothelial cells within the developing vascular system from very early stages in vasculogenesis, and the enhancer remains robustly active in the vascular endothelium during embryogenesis and in adulthood. This mef2c endothelial cell enhancer contains four perfect consensus Ets transcription factor binding sites that are efficiently bound by Ets-1 protein in vitro and are required for enhancer function in transgenic embryos. Members of the Ets family are defined by the presence of a conserved DNA binding domain, which folds into a winged helix-turn-helix motif and binds to the consensus core DNA sequence GGAW with variable flanking sequences. Of the nearly 30 mammalian family members so far identified, at least five, Ets-1, Erg, Fli-1, TEL, and NERF-2, are expressed in the vasculature during embryonic development and each has been shown to play an important role in endothelial-restricted gene expression. Thus, these studies identify mef2c as a direct transcriptional target of Ets factors via an evolutionarily conserved transcriptional enhancer and establish a direct link between these two early regulators of vascular gene expression during endothelial cell development in vivo (De Val, 2004).

Ets-2 is a transcriptional activator that can be modulated by ras-dependent phosphorylation. Evidence is presented indicating that ets-2 can also act as a transcriptional repressor. In the breast cancer cell line MCF-7, exogenous ets-2 repressed the activity of a BRCA1 promoter-luciferase reporter dependent on a conserved ets-2-binding site in this promoter. Conditional overproduction of ets-2 in MCF-7 cells resulted in repression of endogenous BRCA1 mRNA expression. To address the mechanism by which ets-2 could act as a repressor, a biochemical approach was used to identify proteins that interacted with the ets-2 pointed domain. From this analysis, components of the mammalian SWI/SNF chromatin remodeling complex were found to interact with ets-2. Brg-1, the ATP-hydrolyzing component of the SWI/SNF complex, along with the BAF57/p50 and Ini1 subunits could be co-immunoprecipitated from cells with ets-2. The pointed domain of ets-2 directly interacts in vitro with the C-terminal region of Brg-1 in a phosphorylation-dependent manner. The combination of Brg-1 and ets-2 could repress the BRCA1 promoter reporter in transfection assays. These results support a role for ets-2 as a repressor and indicate that components of the mammalian SNF/SWI complex are required as co-repressors (Baker, 2003).

The VEGF receptor, FLK1, is essential for differentiation of the endothelial lineage and for embryonic vascular development. Using comparative genomics, conserved ETS and Krüppel-like factor (KLF) binding sites have been identified within the Flk1 enhancer. In transgenic studies, mutation of either site results in dramatic reduction of Flk1 reporter expression. Overexpression of KLF2 or the ETS transcription factor ERG is sufficient to induce ectopic Flk1 expression in the Xenopus embryo. Inhibition of KLF2 function in the Xenopus embryo results in a dramatic reduction in Flk1 transcript levels. Furthermore, KLF2 and ERG are shown to associate in a physical complex, and the two proteins synergistically activate transcription of Flk1. Since the ETS and KLF protein families have independently been recognized as important regulators of endothelial gene expression, cooperation between the two families has broad implications for gene regulation during development, normal physiology and vascular disease (Meadows, 2009).

The otic placode, a specialized region of ectoderm, gives rise to components of the inner ear and shares many characteristics with the neural crest, including expression of the key transcription factor Sox10. This study shows that in avian embryos, a highly conserved cranial neural crest enhancer, Sox10E2, also controls the onset of Sox10 expression in the otic placode. Interestingly, this study showed that different combinations of paralogous transcription factors (Sox8, Pea3 and cMyb versus Sox9, Ets1 and cMyb) are required to mediate Sox10E2 activity in the ear and neural crest, respectively. Mutating their binding motifs within Sox10E2 greatly reduces enhancer activity in the ear. Moreover, simultaneous knockdown of Sox8, Pea3 and cMyb eliminates not only the enhancer-driven reporter expression, but also the onset of endogenous Sox10 expression in the ear. Rescue experiments confirm that the specific combination of Myb together with Sox8 and Pea3 is responsible for the onset of Sox10 expression in the otic placode, as opposed to Myb plus Sox9 and Ets1 for neural crest Sox10 expression. Whereas SUMOylation of Sox8 is not required for the initial onset of Sox10 expression, it is necessary for later otic vesicle formation. This new role of Sox8, Pea3 and cMyb in controlling Sox10 expression via a common otic/neural crest enhancer suggests an evolutionarily conserved function for the combination of paralogous transcription factors in these tissues of distinct embryological origin (Betancur, 2011).

Ets interaction with CBF, a Runt homolog, on the T-cell receptor promoter

An examination was made of the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple proteins was examined. Reconstitution of TCR alpha enhancer function in nonlymphoid cells requires expression of the lymphoid-specific proteins LEF-1, Ets-1 and PEBP2 alpha (CBF alpha: Drosophila homolog Runt), and a specific arrangement of their binding sites in the enhancer. Ets-1 cooperates with PEBP2 alpha to bind adjacent sites at one end of the enhancer, forming a ternary complex that is unstable by itself. Stable occupancy of the Ets-1- and PEBP2 alpha-binding sites in a DNase I protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 bends the DNA helix in the center of the TCR alpha enhancer. The HMG domain of the distantly related SRY protein, which also bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins bound at nonadjacent sites (Giese, 1995).

Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bind individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. CBF and Ets-1 bind together to beta E2 and beta E3: Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increases the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes are not observed when either the Ets or core site is mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appears to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription (Wotton, 1994).

The transcription factors Ets-1 and AML1 (the alphaBl subunit of PEBP2/CBF) play critical roles in hematopoiesis and leukemogenesis, and cooperate in the transactivation of the T cell receptor (TCR) beta chain enhancer. The DNA binding capacity of both factors is blocked intramolecularly but can be activated by the removal of negative regulatory domains. These include the exon VII domain for Ets-1 and the negative regulatory domain for DNA binding (NRDB) for alphaB1. The direct interaction between the two factors leads to a reciprocal stimulation of their DNA binding activity and activation of their transactivation function. Detailed mapping reveals two independent contact points involving the exon VII and NRDB regions as well as the two DNA binding domains. Using deletion variants and dominant interfering mutants, it has been demonstrated that the interaction between exon VII and NRDB is necessary and sufficient for cooperative DNA binding. The exon VII and NRDB motifs are highly conserved in evolution yet deleted in natural variants, suggesting that the mechanism described is of biological relevance. The mutual activation of DNA binding of Ets and AML1 through the intermolecular interaction of autoinhibitory domains may represent a novel principle for the regulation of transcription factor function (Kim, 1999).

Ets regulation of the Fos promoter

Continued Pointed Evolutionary homologs part 3/4 | part 4/4 | back to part 1/4

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