Gene Section Review. ELF4 (E74-like factor 4 (ets domain transcription factor)) Abstract DNA/RNA. Identity

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1 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL INIST-CNRS Gene Section Review ELF4 (E74-like factor 4 (ets domain transcription factor)) Stephen D Nimer, Yan Liu Sylvester Comprehensive Cancer Center Professor of Medicine, Biochemistry & Molecular Biology, University of Miami, USA (SDN), Indiana University School of Medicine, USA (YL) Published in Atlas Database: December 2013 Online updated version : DOI: /2042/53972 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence Atlas of Genetics and Cytogenetics in Oncology and Haematology Abstract Review on ELF4, with data on DNA/RNA, on the protein encoded and where the gene is implicated. Identity Other names: ELFR, MEF HGNC (Hugo): ELF4 Location: Xq26.1 Local order: The ELF4 gene maps on chomosome X at position (interpolated), at Xq26 according to Entrez Gene. In AceView, it covers 46,69 kb, from to , on the reverse strand. DNA/RNA Description The gene encompasses 45 kb of DNA; 9 exons. The ELF4 gene contains 12 distinct gt-ag introns. Transcription produces 5 alternatively spliced mrnas. There are 2 probable alternative promoters, 2 non overlapping alternative last exons and 2 validated alternative polyadenylation sites. The mrnas appear to differ by truncation of the 3' end, presence or absence of 3 cassette exons, overlapping exons with different boundaries, splicing versus retention of 2 introns. The ELF4 gene is located on the X-chromosome (Xq26) and is transcribed from telomere to centromere. The ELF4 gene contains 8 coding exons and 1 non-coding exon. Atlas Genet Cytogenet Oncol Haematol. 2014; 18(7) 511

2 The ELF4 protein contains the following domains: (A) transactivation domain, (B) AML1 interacting domain, (C) ETS domain, (D) serine/threonine rich region and (E) proline rich region. Transcription 4190 bp mrna; 1991 bp open reading frame. Pseudogene No observed pseudogenes. Protein Description 663 amino acids; Da protein; numerous posttranslational modifications: phosphorylation, sumoylation and ubiquitination (Miyazaki et al., 2001; Liu et al., 2006; Suico et al., 2006). ELF4 contains, from N-term to C-term, transactivation domain (53-86), AML1 interacting domain (87-206), ETS domain ( ), serine/threonine rich region ( ) and proline rich region ( ) (Miyazaki et al., 1996; Mao et al., 1999; Suico et al., 2002; Lacorazza and Nimer, 2003). Expression Abundantly expressed in the placenta and in a variety of myeloid leukemia cell lines. Moderate levels of expression in heart, lung, spleen, thymus, peripheral blood lymphocytes, ovary and colon. Lower levels of expression in Jurkat T-cells and other T-cell lines and no expression in brain (Lacorazza and Nimer, 2003). Localisation Nucleus, PML body. Accumulation into PML nuclear bodies is mediated by PML (Suico et al., 2004a; Suico et al., 2004b). Function ELF4 is a member of the ETS family of transcription factors (TF) with transcription activating properties (Lacorazza and Nimer, 2003). ELF4 binds to DNA sequences containing the consensus 5'-WGGA-3' and transactivates promoters of the hematopoietic growth factor genes CSF2, IL3, IL8, and of the bovine lysozyme gene (Miyazaki et al., 1996; Mao et al., 1999; Hedvat et al., 2004; Suico et al., 2004). ELF4 acts synergistically with RUNX1 to transactivate the IL3 promoter (Mao et al., 1999). It also transactivates the PRF1 promoter in natural killer (NK) cells (Lacorazza et al., 2002). ELF4 has important molecular functions, including protein binding, transcription activator activity, sequence-specific DNA binding, transcription factor activity. ELF4 interacts with multiple proteins, including Cyclin A/CDK2 kinase complex, FBXO4, FBXO7, PML, RUNX1, SKP2 and UBB (Miyazaki et al., 1996; Mao et al., 1999; Miyazaki et al., 2001; Liu et al., 2006; Suico et al., 2006). ELF4 interacts with multiple proteins and plays important role in development and oncogenesis. Atlas Genet Cytogenet Oncol Haematol. 2014; 18(7) 512

3 ELF4 has been implicated in widely divergent physiological and pathological processes (such as development and oncogenesis). ELF4 plays an important role in the development and function of NK and NK T-cells and in innate immunity (Lacorazza et al., 2002). It controls the proliferation and homing of CD8+ T- cells via the Kruppel-like factors KLF4 and KLF2 (Yamada et al., 2009; Yamada et al., 2010). ELF4 regulates hematopoietic stem cell quiescence and self-renewal (Lacorazza et al., 2006; Liu et al., 2009). ELF4 also regulates self-renewal of braintumor stem cells (Bazzoli et al., In addition, ELF4 plays an important role in osteogenic differentiation and bone development (Kim et al., 2007; Seul et al., 2011; Baek et al., It mediates DNA damage response (Morales et al., 2008; Sashida et al., 2011). ELF4 controls cell senescence in a p53-dependent manner and can also promote cellular transformation through inhibition of the p16 pathway as well (Sashida et al., 2009). ELF4, a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers (Lacorazza and Nimer, 2003; Sashida et al., 2010). In addition to its oncogenic potential, ELF4 has been proposed to be a candidate tumor suppressor gene on the X chromosome (Seki et al., 2002). Homology The transcription factor ELF4 belongs to the ETS family of transcription factors, named after the E26 (E twenty six) leukemogenic chicken virus which contains a gag-myb-ets fusion. These proteins are characterized by the presence of the ETS domain (a conserved 84 amino acid domain) that binds to a DNA consensus sequence containing a GGAA central core via a winged helix-turn-helix motif. Based on the homology of the ETS domain, these proteins have been classified in different subfamilies (e.g., Elf, Spi, and Yan). The ETS domain is highly conserved among family members with the greatest homology of ELF4 with other proteins in the Elf subfamily, which also includes ELF1, NERF1a, and NERF1b. ELF4 does not contain an HLH or pointed protein-protein interaction domain, which is present in members of the ETS, ERG, GABP, and Yan subfamilies (Lacorazza and Nimer, 2003). Mutations Note No known mutations. Fused in hepatocellular carcinoma with the BCORL1 gene. Implicated in Acute myeloid leukemia Acute myeloid leukemia (AML) is the most common acute leukemia in adults. It usually occurs around age 60 with no identifiable cause and it carries a very poor prognosis, with most patients living less than 18 months. AML is a devastating illness with over new diagnoses and patients dying annually in the United States. Although select molecular subpopulations of patients are long-term survivors, the overall fiveyear survival for adults is only 25%. ETS proteins (such as PU.1, Fli-1 and ETS-1) have been shown to play important roles in normal and abnormal hematopoiesis (Lacorazza and Nimer, 2003). ELF1 and ELF4 were expressed in all AML samples, whereas NERF was not. The relative expression of ELF4, but not ELF1, was significantly lower (P<0.0001) in AML with t(8;21) and t(15;17) compared with AML with normal karyotype (Fukushima et al., 2003; Ando et al., 2013). The low relative expression of ELF4, might be part of a gene expression profile characterizing AML with a good prognosis (Fukushima et al., 2003; Ando et al., 2013). Cytogenetics A chromosomal translocation has been reported in AML, t(x;21)(q25-26;q22), that generates a fusion transcript between two ETS family members, ELF4 (at Xq26) and ERG (at 21q22). The translocation occurs within intron 1 of ERG isoform 1 (ERG-1) and intron 2 of ELF4 resulting in an in-frame fusion joining exon 2 from ELF4 with exon 2 of ERG (Moore et al., 2006). Hybrid/Mutated gene ELF4-ERG. The fusion sequence includes exon 2 of ELF4 and exon 2 of ERG1, with the breakpoint occurring after exon 2 in ELF4 (intron 2) and before exon 2 of ERG1 (intron 1) (Moore et al., 2006). Abnormal protein ELF4-ERG (495 amino acids). Atlas Genet Cytogenet Oncol Haematol. 2014; 18(7) 513

4 Horizontal lines represent amino acid residues for ELF4 and ERG-1. Numbers above each line indicate the number of amino acids at the corresponding position. Boxes below indicate locations of the conserved domains within both protein sequences. The predicted fusion protein would include the first 25 amino acid residues from ELF4 encoded by exon 2 and the last 470 amino acid residues encoded by ERG exons 2-10 resulting in a protein of 495 amino acids. The conserved SAM-PNT and ETS domains from ERG would be included in the fusion while the ETS domain from ELF4 would be excluded. ERG has been associated with other fusion partners, specifically FUS and EWSR1, and implicated in both AML and Ewing's sarcoma. The ELF4-ERG fusion suggests a new role for ELF4 in leukemogenesis and human cancer (Moore et al., 2006), as it provides a strong promoter to express ERG. In addition, wild-type nucleophosmin (NPM1) inhibits the DNA binding and transcriptional activity of ELF4 on the HDM2 promoter, while a common, leukemia-associated NPM1 mutant protein (Mt-NPM1) enhances these activities of ELF4 (Ando et al., 2013). Moreover, clinical leukemia samples with NPM1 mutations have higher human MDM2 (HDM2) mrna expression. These data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in ELF4-dependent leukemogenesis (Ando et al., 2013). Ovarian cancer Ovarian cancer is the seventh most common cancer in women worldwide and the second leading cause of death among the gynecological malignancies. ELF4 is expressed in a significant proportion of ovarian carcinomas, and in the CAOV3 and SKOV3 ovarian cancer cell lines, but not in normal ovarian surface epithelium (Yao et al., 2007). High levels of ELF4 expression in ovarian cancer are associated with a poor prognosis (Yao et al., 2007). Manipulating MEF levels in ovarian cancer cell lines alters their behavior; reducing ELF4 levels, using short hairpin RNA expressing vectors, significantly inhibited the proliferation of SKOV3 and CAOV3 cells in culture, and impaired the anchorage-independent growth of CAOV3 cells. Overexpression of ELF4 in SKOV3 cells significantly increased their growth rate, enhanced colony formation in soft agar and promoted tumor formation in nude mice. The oncogenic activity of MEF was further shown by the ability of ELF4 to transform NIH3T3 cells, and induce their tumor formation in nude mice (Yao et al., 2007). Thus, ELF4 is an important regulator of the tumorigenic properties of ovarian cancer cells and could be used a therapeutic target in ovarian cancer (Yao et al., 2007). Gliomas Malignant gliomas represent the most prevalent primary brain tumor in adults and inevitably have a poor prognosis. Despite the implementation of new therapeutic strategies, the median survival of patients with glioblastoma multiforme (GBM), the most aggressive glioma variant, is only months and these tumors remain rapidly and uniformly fatal. High-grade gliomas are composed of a heterogeneous population of cells that include many with stem-cell-like properties. The acquisition of stem-like traits might contribute to glioma initiation, growth, and recurrence. ELF4 is highly expressed in both human and mouse glioblastomas and its absence impairs gliomagenesis in a PDGF-driven glioma mouse model (Bazzoli et al., High levels of ELF4 expression in gliomas are associated with a poor prognosis (Bazzoli et al., Modulation of ELF4 levels in both mouse neural stem cells and human glioblastoma cells has a significant impact on neurosphere formation. Thus, ELF4 is a gatekeeper gene that promotes stemness in the pathogenesis of gliomas (Bazzoli et al., Atlas Genet Cytogenet Oncol Haematol. 2014; 18(7) 514

5 References Miyazaki Y, Sun X, Uchida H, Zhang J, Nimer S. MEF, a novel transcription factor with an Elf-1 like DNA binding domain but distinct transcriptional activating properties. Oncogene Oct 17;13(8): Mao S, Frank RC, Zhang J, Miyazaki Y, Nimer SD. Functional and physical interactions between AML1 proteins and an ETS protein, MEF: implications for the pathogenesis of t(8;21)-positive leukemias. Mol Cell Biol May;19(5): Miyazaki Y, Boccuni P, Mao S, Zhang J, Erdjument- Bromage H, Tempst P, Kiyokawa H, Nimer SD. Cyclin A- dependent phosphorylation of the ETS-related protein, MEF, restricts its activity to the G1 phase of the cell cycle. J Biol Chem Nov 2;276(44): Lacorazza HD, Miyazaki Y, Di Cristofano A et al.. The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells. Immunity Oct;17(4): Seki Y, Suico MA, Uto A, Hisatsune A, Shuto T, Isohama Y, Kai H. The ETS transcription factor MEF is a candidate tumor suppressor gene on the X chromosome. Cancer Res Nov 15;62(22): Suico MA, Koyanagi T, Ise S, Lu Z, Hisatsune A, Seki Y, Shuto T, Isohama Y, Miyata T, Kai H. Functional dissection of the ETS transcription factor MEF. Biochim Biophys Acta Aug 19;1577(1): Fukushima T, Miyazaki Y, Tsushima H et al.. The level of MEF but not ELF-1 correlates with FAB subtype of acute myeloid leukemia and is low in good prognosis cases. Leuk Res May;27(5): Lacorazza HD, Nimer SD. The emerging role of the myeloid Elf-1 like transcription factor in hematopoiesis. Blood Cells Mol Dis Nov-Dec;31(3): Hedvat CV, Yao J, Sokolic RA, Nimer SD. Myeloid ELF1- like factor is a potent activator of interleukin-8 expression in hematopoietic cells. J Biol Chem Feb 20;279(8): Suico MA, Lu Z, Shuto T, Koga T, Uchikawa T et al.. The regulation of human beta-defensin 2 by the ETS transcription factor MEF (myeloid Elf-1-like factor) is enhanced by promyelocytic leukemia protein. J Pharmacol Sci. 2004a Aug;95(4): Suico MA, Yoshida H, Seki Y, Uchikawa T et al.. Myeloid Elf-1-like factor, an ETS transcription factor, up-regulates lysozyme transcription in epithelial cells through interaction with promyelocytic leukemia protein. J Biol Chem. 2004b Apr 30;279(18): Lacorazza HD, Yamada T, Liu Y, Miyata Y, Sivina M, Nunes J, Nimer SD. The transcription factor MEF/ELF4 regulates the quiescence of primitive hematopoietic cells. Cancer Cell Mar;9(3): Liu Y, Hedvat CV, Mao S, Zhu XH, Yao J, Nguyen H, Koff A, Nimer SD. The ETS protein MEF is regulated by phosphorylation-dependent proteolysis via the proteinubiquitin ligase SCFSkp2. Mol Cell Biol Apr;26(8): Moore SD, Offor O, Ferry JA, Amrein PC, Morton CC, Dal Cin P. ELF4 is fused to ERG in a case of acute myeloid leukemia with a t(x;21)(q25-26;q22). Leuk Res Aug;30(8): Suico MA, Nakamura H, Lu Z, Saitoh H, Shuto T, Nakao M, Kai H. SUMO down-regulates the activity of Elf4/myeloid Elf-1-like factor. Biochem Biophys Res Commun Sep 29;348(3):880-8 Kim YJ, Kim BG, Lee SJ, Lee HK, Lee SH, Ryoo HM, Cho JY. The suppressive effect of myeloid Elf-1-like factor (MEF) in osteogenic differentiation. J Cell Physiol Apr;211(1): Yao JJ, Liu Y, Lacorazza HD, Soslow RA, Scandura JM, Nimer SD, Hedvat CV. Tumor promoting properties of the ETS protein MEF in ovarian cancer. Oncogene Jun 7;26(27): Morales M, Liu Y, Laiakis EC, Morgan WF, Nimer SD, Petrini JH. DNA damage signaling in hematopoietic cells: a role for Mre11 complex repair of topoisomerase lesions. Cancer Res Apr 1;68(7): Liu Y, Elf SE, Miyata Y, Sashida G, Liu Y et al.. p53 regulates hematopoietic stem cell quiescence. Cell Stem Cell Jan 9;4(1):37-48 Sashida G, Liu Y, Elf S, Miyata Y, Ohyashiki K, Izumi M, Menendez S, Nimer SD. ELF4/MEF activates MDM2 expression and blocks oncogene-induced p16 activation to promote transformation. Mol Cell Biol Jul;29(13): Yamada T, Park CS, Mamonkin M, Lacorazza HD. Transcription factor ELF4 controls the proliferation and homing of CD8+ T cells via the Krüppel-like factors KLF4 and KLF2. Nat Immunol Jun;10(6): Sashida G, Bazzoli E, Menendez S, Liu Y, Nimer SD. The oncogenic role of the ETS transcription factors MEF and ERG. Cell Cycle Sep 1;9(17): Yamada T, Gierach K, Lee PH, Wang X, Lacorazza HD. Cutting edge: Expression of the transcription factor E74- like factor 4 is regulated by the mammalian target of rapamycin pathway in CD8+ T cells. J Immunol Oct 1;185(7): Sashida G, Bae N, Di Giandomenico S, Asai T, Gurvich N, Bazzoli E, Liu Y, Huang G, Zhao X, Menendez S, Nimer SD. The mef/elf4 transcription factor fine tunes the DNA damage response. Cancer Res Jul 15;71(14): Seul KJ, Cho HS, Heo SH, Baek WY, Kim JE, Park EK, Choi JY, Ryoo HM, Cho JY. Osteoblast-specific expression of MEF induces osteopenia through downregulation of osteoblastogenesis and upregulation of osteoclastogenesis. J Bone Miner Res Feb;26(2): Baek K, Cho JY, Hwang HR, Kwon A et al.. Myeloid Elf-1- like factor stimulates adipogenic differentiation through the induction of peroxisome proliferator-activated receptor γ expression in bone marrow. J Cell Physiol Nov;227(11): Bazzoli E, Pulvirenti T, Oberstadt MC et al.. MEF promotes stemness in the pathogenesis of gliomas. Cell Stem Cell Dec 7;11(6): Ando K, Tsushima H, Matsuo E, Horio K et al.. Mutations in the nucleolar phosphoprotein, nucleophosmin, promote the expression of the oncogenic transcription factor MEF/ELF4 in leukemia cells and potentiates transformation. J Biol Chem Mar 29;288(13): This article should be referenced as such:. ELF4 (E74-like factor 4 (ets domain transcription factor)). Atlas Genet Cytogenet Oncol Haematol. 2014; 18(7): Atlas Genet Cytogenet Oncol Haematol. 2014; 18(7) 515