ETS1, v-ets avian erythroblastosis virus E26 oncogene homolog 1

ETS1, the founding member of the ETS family, is located on chromosome 11 and is expressed in a variety of cells, including B and T cells, endothelial cells, vascular smooth muscle cells, epithelial cells as well as neoplastic cells.

The major isoform of ETS1 in human is encoded by 8 exons forming the full length p54 (p51 based on predicted molecular weight) composed by 441 amino acids. The ETS1 protein is characterized by a well-conserved winged helix-turn-helix DNA binding domain (DBD) (Fig.8), with which it binds to the specific EBSs on the promoters of its targets genes.

The DNA binding domain is flanked by two autoinhibitory domains composed by two alpha helices, HI-1 and HI-2, and a serin rich region located in the N-terminal part of the ETS domain (exon VII), and by two other alpha helices H4 and H5 in the C-terminal region of the ETS domain. These two regions interact with each other creating an autoinhibitory conformation that prevents the DNA binding. Serines within the exon VII could be phosphorilated by calmodulin kinase (CAM) in a calcium-dependent manner and this phosphorylation cause a decreased in the flexibility that characterized the active DNA binding form of ETS1 leading to a more rigid conformation of the ETS domain and to a progressive reduction in DNA binding activity. Exon VII is absent in the shorter isoform p42 of 356 amino acids long making that isoform more active in binding the DNA than the full length p54 (Fig.8).

ETS domain is also involved in protein-protein interaction allowed by a conserved PNT (or Pointed) domain within ETS1 protein (Fig.8). PNT is structurally related to the larger group of sterile alpha motif (SAM) domains through a common tertiary arrangement of four αlpha helices. SAM domains are typically found in numerous proteins involved in eukaryotic developmental and signal transduction pathways. PNT domain is a docking

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site for mitogen –activated protein (MAP) kinases such as ERK2 that phosphorylate ETS1 at a conserve threonine residue (T38) closed to PNT domain leading to a strong increase in the transcriptional activity of ETS1 mediated by CREB binding protein (CBP) recruitment.

Finally, ETS1 harbors an acidic transactivation domain (TAD) that is essential to activate transcription activity of ETS1. TAD is required for the interaction with CBP and p300 that function as histone acetyl transferases and co-factors.

A third isoform of 225 amino acid long, p27 (Fig.8), arises from splicing out of the threonine-38 residue, the PNT and TAD domains but retains the DNA binding and both the autoinhibitory domains surrounding that domain thus p27 isoform can bind and autoinhibit its binding to the DNA but it cannot transactivates its target genes.

In addition to phosphorylation, ETS1 protein structure can be modify by sumuylation and ubiquitinylation [354,355]. Small ubiquitin-like modifier (or SUMO) proteins are a family of small proteins that modify the function of proteins that bind. Sumoylation is directed by an enzymatic cascade analogous to that involved in ubiquitinylation but in contrast to ubiquitinylation, sumoylation is not use to tag proteins for proteasomal degradation. In fact, SUMO modification appears to play a role in a variety of cellular processes including protein–protein interaction, subcellular localization, protein stabilization and transcriptional regulation [356]. The major sumuoylated sites of ETS1 are lysine 15 and lysine 227, and UBC9 (E2 conjugated enzyme) and PIASy (E3 lygase) are the two proteins involved in sumoylating ETS1 [355, 357]. This post-transcriptional modification does not affect the nuclear localization of ETS1 but inhibits its activity in transactivating genes and could be reverted with the help of the SUMO protease SEMP1.

Moreover, ETS1 is polyubiquitinylated on the lysine 48 residue independently on sumoylation. These modifications appear regulate ETS1 activity in a different manner, with polyubiquitinylation regulating its protein stability and sumoylation reducing its transcriptional activity [354].

ETS1 is important in various biological processes such as development, differentiation, proliferation, apoptosis, migration and tissue remodelling. ETS1 acts as an oncogene that controls invasive and angiogenic behaviour of malignant cells in multiple human cancers [113, 114]. ETS1 has been implicated in the activation of metastasis-associated molecules and its expression in ovarian carcinoma is associated with poor prognosis [115]. ETS1 has also a role in human breast cancer [116], lung cancer, colorectal and squamous cell carcinoma [117], ETS1 expression is associated with a higher incidence of lymph node metastasis [118-120] while in prostate cancer its expression is associated with a progressive disease [121]. ETS1 expression increases upon treatment with angiogenic

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factors such as tumor necrosis factor 1 (TNF1), phorbol myristate acetate, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). ETS1 binding sites have been identified in numerous promoters of genes that are involved in angiogenesis, including the VEGF receptors, FLT1 and FLK1. Overexpression of ETS1 results in cellular transformation in vitro and fibroblasts expressing high levels of exogenous ETS1 gene product are tumorigenic in vivo [122, 123].

Figure 8: Protein domains representing ETS1 full length and the two splice variants in human. PNT, pointed domain; TAD, transactivation domain; DBD, DNA binding domain; sss, serine-rich domain;

K15/K227, lysines 15 and 227 residues; T38, threonine-38 residue.

ETS1 has an important role in immunity. In the absence of ETS1, T cell numbers are reduced due to impaired survival and ability to respond to proliferative stimuli. A few T cell-specific genes are regulated by ETS1. The enhancer of the gene encoding the α chain of the TCR contains an EBS specific for ETS1 [124]. TCRβ is regulated by ETS1 in a negative manner. Immature T cells without ETS1 coexpress TCRs with different TCRβ chains, indicating that ETS1 is involved in allelic exclusion [125]. The ETS1 levels are high in resting T cells and are reduced after activation suggesting that ETS1 is important for the quiescence of T cells [126]. However, even if the levels of ETS1 are low in activated T cells, ETS1 is involved in the transactivation of the gene encoding granulocyte-macrophage colony-stimulation factor (GM-CSF) following T cell activation, and this activity is repressed when Calmodulin-dependent kinase II (CaMKII) phosphorilates ETS1 protein.

GM-CSF is a cytokine that regulates the growth and differentiation of dendritic cells, myeloid progenitors, and granulocytes and is involved in inflammatory and autoimmune diseases [127]. T cells and macrophages produce the majority of GM-CSF. ETS1 have also demonstrated ability in regulating the activity of IL-5 promoter in activated T and myeloid cells together with the ETS1 closely related factor ETS2 and GATA3 [128]. Depending on the presence of specific cofactors, several ETS factors have this ability to act as either an activator or a repressor of transcription. In contrast to IL-5, also IL-2, important for B and T

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cell proliferation and enhanced B cell Ig secretion, in particular IgM [129], is regulated by ETS1 but in a negative manner [130].

Regarding its role in B cell development, ETS1 is highly expressed in naïve B cells with decreased expression following B cell activation and terminal differentiation. ETS1 has been shown to cooperate with PAX5 in gene target activation. In particular ETS1 forms a ternary complex on the CD79α promoter (mb-1 gene, encoding for the immunoglobulin-associated alpha chain, Igα) with PAX5 and FLI1 [131]. ETS1 also leads to PAX5 up-regulation in B cells [132], and might contribute to the up-regulation of plasmacytic differentiation. Upon TLR9 stimulation, in vitro ETS1^-/- B cells differentiate into IgM-secreting PCs, suggesting that ETS1 functions to limit TLR9 signalling pathway and thereby limiting B cell terminal differentiation [132]. Mice with a deletion of the Pointed domain of ETS1 gene and expressing low levels of an ETS1 protein incapable of being functionally activated exhibit an increased number of IgM-secreting PCs [133]. These mice possess very few marginal zone B cells (CD20^hi CD23^lo) and have an increased expression of activation markers on follicular B cells (CD20^lo CD23^hi). These observations support the idea that ETS1 is a critical negative regulator of B cell terminal differentiation induced by TLR9, and that ETS1-deficient B cells have an intrinsic propensity to undergo differentiation into PCs, confirming the important role that ETS1 plays during B cell developmental process. ETS1 physically interacts with BLIMP1, leading to a block of BLIMP1-mediated repression of its target genes without interfering with BLIMP1 levels [132].