The ETS family is one of the largest families of transcriptional regulators, which was initially derived from the leukemia virus, E26 (E-twenty-six). This virus carried the v-ets oncogene, which was initially discovered as part of the gag-myb-ets transforming fusion protein of E26 and it can cause mixed erythroid-myeloid and lymphoid leukemia. The founding member of this family is Ets1. Based on their homology with highly conserved DNA-binding domain, the ETS domain, many other ETS-domain proteins have been identified from various organisms (Sharrocks 2001). The number of ETS transcription factors is much more in vertebrates compared to invertebrate metazoans: while in Drosophila and Caenorhabditis elegans, 8 and 10 genes, respectively, are present, in vertebrates 26 or more members of this family have been found (Hart et al. 2000; Hsu and Schulz 2000; Liu and Patient 2008).
The ETS family is defined by a highly conserved DNA-binding domain, the ETS domain, which forms a winged helix-turn-helix structural motif. This domain is composed of three alpha helices and a four-stranded, beta sheet that recognises a core GGAA/T sequence (Ets-binding site, EBS). The second conserved domain found in a subset of ETS genes is the pointed (PNT) domain. This domain has been shown to function in protein–protein interaction and oligomerisation. Based on the sequence similarities in ETS domain and conserved PNT domain, ETS family members are futher classified into several sub-groups (Sharrocks 2001).
Many ETS factors have similar biological abilities involved in a wide variety of developmental processes, including cellular proliferation, differentiation, hematopoiesis, apoptosis, metastasis, tissue remodeling, angiogenesis, and transformation.
Mode of Action
Multiple ETS factors are expressed in spatially and temporally overlapping pattern, suggesting possible functional overlap. Interaction with cofactors might be one general mechanism for ETS transcription factors (Li et al. 2000). The promoters/enhancers of many genes have multiple conserved ETS-binding sites. And more than one ETS family members would bind these promoters to combinatorially regulate the expression of target genes (Yordy and Muise-Helmericks 2000). In loss-of-function experiment, depletion of any of these four ETS genes, Fli1, Fli1b, Ets1, and Etsrp, caused either partial or no defects in endothelial differentiation and hematopoietic development. However, the combined knockdown of these four genes resulted in much more severe vascular and hematopoietic defects (Pham et al. 2007), demonstrating that combinatorial ETS factors are necessary for development. Scl plays a key role in the development of blood and endothelium. Previous experiments identified a bifunctional 5′ enhancer (−3.8 element), which functions in the hematopoietic progenitors and endothelium. This enhancer contains multiple conserved ETS-binding sites and can be bound by ETS factors, including Fli1 and Elf1 (Gottgens et al. 2004).
Several characterized hematopoietic promoters contain both ETS-binding sites and GATA-binding sites, which implies that these two transcription factors may coregulate the expression of these genes. The GATA family is a group of zinc finger transcription factors, which plays an important role in many biological process. The GATA factor Gata1 and the ETS factor Fli1 exbihit combinational DNA binding and synergistically activate a megakaryocyte-specific enhancer GPIX and GPIbα (Eisbacher et al. 2003). Gata2 and Ets1 can upregulate the expression of Ang-1 via cooperative binding of a HIF site (Marie-Pierre Simon, J Cell Physiol. 217:809–18). ETS and GATA sites have also been identified in association with other cis-acting elements to confer the specificity. Tal1, Gata2, and Fli1 combinatorially regulate endothelial and hematopoietic gene expression (Pham et al. 2007).
As noted above, ETS transcription factors play central roles in vascular development and hematopoiesis (Ciau-Uitz et al. 2013). However, there is no single ETS factor that is unique to either blood cells or endothelium. Current evidence supports that ETS factors would bind to lower affinity sites and cooperate with other proteins to achieve tissue-specific activation. The Forkhead (FOX) transcription factors also play critical roles in vascular development, which are helix-turn-helix proteins. It could bind to cis-acting elements, containing the core consensus of RYMAAYA. Recent studies suggest that ETS factors function in cooperation with FoxC transcription factors via a composite element, the FOX:ETS motif, which could be bound and regulated by ETS and FOX transcription factors. FoxC2 and Etv2 can bind this motif simultaneously and activate all of the enhancers that contain this FOX:ETS motif. Coexpression of the FoxC2 and Etv2 causes ectopic expression of endothelial genes in Xenopus embryos, and the combined knockdown of the orthologous genes in zebrafish embryos induces vascular defects. This conserved ETS:FOX motif was identified in numerous endothelial promoters and enhancers throughout the human genome (De Val et al. 2008).
Physiological Functions in Hematopoiesis and Vasculogenesis
In endothelial and hematopoietic cells, a number of ETS transcription factors are expressed and play essential roles (Ciau-Uitz et al. 2013; Craig and Sumanas 2016). Fli1 is one of the earliest transcription factors which can regulate the endothelial and hematopoietic cell development. Loss- and gain-of-function experiments suggest that Fli1 plays a predominant role in regulation of hemangioblast development. Fli1 knockdown reduced the expression of Gata2 and other genes, including Scl, Lmo2, and Flk1, which suggests that Fli1 may act upstream of Gata2 in hemangioblast formation (Liu et al. 2008). Tel1 is one of the ETS transcription factors with repressor activity, which plays a critical role in vascular development. Knockdown of Tel1 in mouse induced death of embryos at E10.5 and E11.5 because of the defective yolk sac angiogenesis. Expression analysis of Tel1 in Xenopus embryos showed that Tel1 expression appears in the ventral wall of the dorsal aorta from stage 41. And in loss-of-function experiment, HSC emergence was inhibited in the embryos. Therefore, Tel1 is required for the formation of HSCs (Aldo Ciau-Uitz 2010). Vegfa can rescue the phenotype of Tel1 morphants, and knockdown of Vegfa or its receptors, Flk1 showed the similar phenotype to the Tel1 morphants (Aldo Ciau-Uitz 2010). These observations suggested that Tel1 plays a key role in HSC development by regulating Vegf production in LPM and somite. Etsrp has been confirmed to be a key regulator in the initiation of vasculogenesis in zebrafish. Overexpression of the Etsrp orthologue in mice, ER71, enhanced hematopoietic cell generation, while in ER71−/− mouse, hematopoietic and endothelial cells were completely absent. Zebrafish studies showed that Etsrp plays a critical role in definitive hematopoiesis. In Etsrp morphants, Scl-α overexpression can restore the expression of HSC and angioblast markers, whereas Scl-β is only sufficient to partially rescue the Runx1 expression (Ren et al. 2010). When the VEGF signaling pathway is inhibited, both Scl isoforms partially rescue the Runx1 expression. This study suggests that Etsrp acts upstream of Scl isoforms in angioblast specification and the emergence of definitive hematopoiesis (Ren et al. 2010). PU.1 is one of the ETS transcription factors and is named by a PU-box binding sites on C-terminus. PU.1 is an important regulator of hematopoiesis, especially in the development of myeloid cell. PU.1−/− embryos die at E18.5 because of the complete absence of B cells, mature T cells, and macrophages (Scott et al. 1994). The ETS transcription factor, Fev (also known as Pet1 in mammals), is also expressed in blood/endothelial cells in zebrafish. Fev deficiency results in a reduction of Runx1 expression in the dorsal aorta and fewer T cells in the thymus, and that Fev regulates HSC specification by transcriptionally activating its target, Erk2. Thus, Fev is a new ETS transcription factor identified to be involved in the generation of HSCs (Wang et al. 2013).
ETS in Cancer
Ets1, as the founding member of ETS family, was firstly identified as an oncogenic fusion gene with c-Myb proto-oncogene in E26 leukemia. Since then, the relationship of ETS transcription factors and cancer was gradually recognized. Many ETS proteins have been linked with cancer. Their downregulation or multiple point mutations have been shown in cancers. In contrast, elevated expression of ETS genes has also been identified in leukemia and solid tumors. For example, increased level of Ets1, Pea3, Erm, Ets2, Fli1, and Elf1 has been observed in breast, colon, lung, and prostate cancers (Sharrocks 2001; Seth and Watson 2005).
The ETS genes were identified to be translocated to new chromosomal position close to breakpoints in childhood-associated leukemia. Several chromosomal breakpoints result in a fusion protein which may lead to carcinogenic transformation. In this instance, the EWS-FLI1 fusion protein results from the translocation of Fli1 to chromosome 22, which fuses the carboxyl terminal DNA-binding domain of the Fli1 to the amino terminal region of the Ews (Arvand and Denny 2001). It has been shown that this fusion protein is a more potent transcriptional activator than Fli1 protein alone and controls various oncogenic pathways.
In other cancers, translocations generate the fusion of the EWS gene with other members of the ETS family, including Erg, Etv1, Etv4, and Fev. Tel1 is rearranged in CMML, acute myelogenous leukemia (AML), acute myeloblastic leukemia (AML-M2), MDS, and acute lymphoblastic leukemia. Either the PNT domain or the ETS domain as well as both domains of Tel have been identified in over 20 different translocations observed in human leukemia and more rarely-occurred solid tumors (Mavrothalassitis and Ghysdael 2000).
ETS transcription factors are involved in a variety of biological processes including cell proliferation, differentiation, hematopoiesis, angiogenesis, and tumorigenesis, acting as either transcriptional activators or repressors. By interacting with their cofactors, ETS-domain proteins can bind to various downstream targets in a context-dependent manner. In addition to their roles in embryogenesis and adulthood, many ETS factors have been demonstrated to involve in leukemia and solid tumors because of their altered expression patterns, copy numbers and forming oncogenic fusion proteins after translocation.
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