In contrast to many other transcription factors, Ets proteins bind to DNA as monomers. Most eukaryotic cells express a variety of Ets proteins at the same time. To achieve functional specificity, Ets proteins display differences in preference for certain nucleotides flanking the core motif in the Ets-responsive DNA element and, more important, for certain cooperating partners. A strong interaction with a cooperating partner may even force Ets proteins to bind to an unfavorable DNA binding site, such as GGAG (Pax5/Ets1 partnership). In many cases, interactions with other proteins depend upon particular protein domains. For example, for the cooperation with SRF, the so-called B-domain is required, which is found in the proteins of the TCF subfamily and the Fli-1 protein. The Pointed domain, named after the Drosophila Ets Protein Pointed and shared by many Ets proteins of different subfamilies, shows similarities to the sterile alpha motif (SAM) domain and is an interface for homotypic and heterotypic protein–protein interactions. In Ets1 and Ets2 proteins, the Pointed domain is the docking site for ERK1/2, allowing these kinases to phosphorylate Ets1 and Ets2 at an N-terminal threonine. In contrast, the Pointed domain of the TEL protein mediates homo-oligomerization. Most Ets proteins are transcriptional activators; others (ERF, NET, Tel, Drosophila YAN, Caenorhabditis lin-1) act as repressors. Some, such as Elk-1 and Net, can undergo activator–repressor switching (Fig. 2).
Ets proteins play an important role in transcriptional regulation. Many eukaryotic genes contain Ets DNA binding sites and are responsive to Ets proteins. Ets-responsive genes are found among critical genes that regulate fundamental cellular processes such as proliferation, differentiation, invasion, and adhesion.
Ets Factors and Development
Some Ets factors, including Ets2, Esx, Ese-2, Fli-1, Pu.1, GABPα, and Tel, are essential for embryonic development. Disruption of the ets2, ese-2, fli-1, pu.1, gabpα, or tel genes in mice results in early death of the embryo. Lack of Ets2 or Ese-2 leads to defects in trophoblast development and to the absence of extraembryonic ectoderm markers. Ese-2 is also involved in mammary alveolar morphogenesis. Tel null mutant embryos fail to develop a vascular network in the yolk sac. Pu.1 is necessary for B- and T-cell development, erythropoiesis, terminal myeloid cell differentiation, and maintenance of hematopoietic stem cells. Fli-1 null embryos die of aberrant hematopoiesis and hemorrhaging. Deficiency of GABPα which is expressed in embryonal stem cells leads to embryonic death prior to implantation. GABPα is also required for the function of neuromuscular junctions. Mice lacking Esx die early after birth. Their intestinal epithelial cells fail to differentiate and polarize as a result of reduced levels of the TGFβII receptor (Transforming Growth Factor-β). Ets1 deficiency leads to defects in B- and T-cell development. In ER81 null mice, two types of mechanoreceptors, muscle spindles and the Pacinian corpuscles, are either absent or degenerated. MEF is involved in osteogenic differentiation.
Regulation of Ets Protein Activities
The activities of Ets proteins are controlled transcriptionally and posttranslationally. The expression of many Ets genes is restricted to certain cell types and/or can be induced by specific extracellular stimuli. For example, the transcription from the Ets1 gene can be activated by a variety of factors including phorbol ester, AP-1, TP53, retinoic acid, ERK1/2 (MAP kinase), and HIF-1 (Hypoxia-Inducible Factor-1). Many Ets proteins undergo posttranslational modifications, which have an impact on their activities. The most common posttranslational modification of Ets proteins is phosphorylation by MAP kinases, such as ERK1/2. Phosphorylation by MAPK leads to activation of activating Ets proteins, such as Ets1, Ets2, Er81, Erm, Sap1, Elk1, Pea3, or GABPα, and loss of activity of repressing Ets proteins, such as Tel or Erf. When phosphorylated by ERK1/2, Net even switches from a repressor to an activator phenotype. It seems that MAPK-dependent phosphorylation (phosphorylation of proteins) shifts the balance between Ets-dependent activation and repression toward activation. In the case of Ets1, MAPK-dependent phosphorylation enhances the transcriptional activity by recruitment of the coactivator CBP/p300 (P300/CBP Co-Activators). Some Ets proteins are also targets of PKA, PKC (Protein Kinase C Family), CaMKII (Calcium Binding Proteins and Cancer), CKII, and cyclin A-dependent cdk2 (Cyclin-Dependent Kinase). CKII increases the activity of Pu.1 and Spi-B, and PKCα activates Ets1. In contrast, PKA inhibits the DNA binding activities of Er81 and Erm, whereas CaMKII and cdk2 inhibit Ets1 and GABPα, respectively. CaMKII phosphorylates Ets1 on serines of a serine-rich region flanking an autoinhibitory module that regulates Ets1 DNA binding activity. The inhibitory effect of CaMKII on the Ets1 protein increases with each serine that is phosphorylated within the serine-rich region allowing fine-tuning of Ets1-dependent transcription.
A few Ets proteins, Ets1, Elk-1, and Tel, have been shown to undergo sumoylation. This posttranslational modification inhibits transcriptional activity of Ets1 and Elk-1 and abrogates the repressing activity of Tel. When sumoylated, the activating Elk-1 protein even transforms to a repressor. Acetylation is another means nature uses to modify the activities of Ets proteins, such as Ets1 and Er81. Er81 becomes acetylated and phosphorylated in response to Her2/neu-stimulated signaling. Acetylation takes place on two lysines within the transactivation domain of Er81, increasing its DNA binding affinity and protein stability. Elf-1 is an example of an Ets protein that becomes glycosylated. Glycosylation affects the subcellular localization and DNA binding activity of Elf-1.
Ets Proteins and Cancer
The Ets proteins Ets1, Ets2, Fli-1, and Erg are able to transform murine cells. These and other Ets proteins are also involved in human carcinogenesis and/or tumor progression. This is in line with the fact that many of these Ets proteins are targets of the Ras/Raf/MEK/ERK signaling pathway which is often deregulated in human tumors. The Ras-responsive Ets1 protein is found in different types of solid tumors, including carcinomas and sarcomas. Its overexpression often correlates with increased invasion, higher tumor microvessel density, higher grading, and unfavorable prognosis. Ets1 has been linked to the regulation of key proteases, such as matrix metalloproteases, involved in the degradation of the extracellular matrix. In tumors, Ets1 is expressed by tumor cells as well as by stromal cells. Due to its ability to convert endothelial cells to an angiogenic phenotype, Ets1 is involved in tumor-dependent angiogenesis. A number of other Ets proteins, such as ERG, PEA3, and E1AF, are capable of upregulating proteases and supposed to be involved in tumor progression. PEA3 has particularly been linked to mammary gland development and oncogenesis. Fli-1 and Ets1 has been shown to regulate tenascin C (tenascin and cancer), an extracellular matrix protein, associated with tumor progression. In some tumors, Ets genes are subject to mutations and recombinations. The inhibitory Ets protein Tel2 has been shown to induce myeloproliferative diseases in mice by cooperating with the Myc oncogene and stimulating proliferation. Elf-1 has been implicated in tumor-associated angiogenesis. A target of Elf-1 is Tie2 (Receptor Tyrosine Kinases), a receptor tyrosine kinase involved in the activation of endothelial cells. Chromosomal translocations leading to fusion proteins containing Ets proteins are observed in Ewing tumors and certain types of leukemias. EWS–Ets fusion proteins (EWS-FLI (ets) Fusion Transcripts), most often containing Fli-1 or Erg and rarely ETV-1, E1AF, or FEV, are critically involved in the development of Ewing tumors. The fusion protein presumably acts as a transcription factor that binds through the Ets domain to Ets-responsive genes. In addition, EWS–Ets proteins have been suggested to interfere with RNA splicing. Ets fusion proteins, as found in leukemias, harbor either Tel or Erg2. Erg2 is fused to TLS, a protein structurally related to EWS. Hence, TLS-Erg2 chimeric proteins are supposed to have similar functions as EWS–Ets proteins. Tel is frequently fused to tyrosine kinases, such as PDGFRβ (Platelet-Derived Growth Factor), Abl (BCR-ABL1), or Jak2 (Signal Transducer and Activators of Transcription in Oncogenesis). The Pointed domain of Tel mediates homo-dimerization resulting in constitutively active kinases. In another fusion protein, Tel is linked to the DNA binding factor AML-1 (runx) which, together with CBFβ, forms the transcription factor CBF. CBF activity is often inhibited in leukemic cells. As a result of Tel-dependent dimerization, CBF function is also blocked when AML-1 is fused to Tel.