Vol. 3, 705-713, October 1992 Cell Growth & Differentiation 705 The ERGB/Fli-1 Gene: Isolation and Characterization of a New Member of the Family of Human ETS Transcription Factors’ Dennis K. Watson,2 Fiona E. Smyth, Delores M. Thompson, Jin Quan Cheng, Joseph R. Testa, Takis S. Papas, and Arun Seth Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21702-1201 [D. K. W., D. M. T., T. S. P., A. S.]; Program Resources, Inc., Frederick, Maryland 21702-1201 [F. E. S.]; and Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [J. Q. C., J. R. T.] Abstract All cellular ets proteins contain a region of high amino acid identity to those found in the last two exons of the ets-i gene (C domain). We have identified and characterized a new member of the human ETS gene family, ERGB. The ERGB gene shows extensive amino acid identity to the human ERG and the mouse FIi-i genes. The ERGB gene is found to be transcriptionally active in a variety of human cell lines and tissues, in contrast to the more restrictive expression pattern of the ERG gene. The ERGB gene encodes for a 3.2- kilobase mRNA containing an open reading frame of 451 amino acids. The ERGB gene, like human ETS1, is located on chromosome 1 1 and is transposed to chromosome 4 as a result of the translocation t(4;i 1) associated with leukemia. Pulse-field gel analysis suggests that ETS1 and ERGB are more than 200 kilobases apart. Similar to the other members of the ets family (etsi, ets2), this new member is also able to trans-activate transcription of a reporter gene linked to the ETS-binding sequences derived from either the GATA-i promoter or an optimal Ets-binding site. Introduction The v-ets oncogene was originally identified in the avian leukemia virus E26, and it encodes, along with gag and v-myb, the tripartite nuclear protein p135 (gag-myb-ets). This replication-deficient retrovirus transforms cells of erythroid and myeloid lineages (for a review, see Ref. 1). Many cellular genes have been cloned and sequenced from species ranging from Drosophila to human and have been placed into the ets gene family by their extensive amino acid identity to the carboxy-terminal portion (C domain) of p1 35, a region localized to the last two exons of the c-ets-1 gene (2). The human ETS1 and ETS2 genes were the first identified and molecularly characterized members of the ets family (3). Comparative nucleotide sequence homology identified additional human genes: ERG (4, 5), ELK1 (6), SAP1 (7), and ELF-i (8). The presence of other ets-related genes-GABPa (9), PU.i/Spi-i (10, i 1), Fli-i (12), PEA3 (13) in mammals, and D-elg (14) and E74A (15, 16) from invertebrates-suggests that the hu- man genome may contain additional distinct ets genes. A number of properties of the ets gene products, including their nuclear localization and rapid response to external signals, as well as their posttranscriptional regu- lation, suggest that they may serve important roles in gene regulation. Sequence-specific DNA binding for the ets-1 proteins was demonstrated for a number of target sites (1 7, 18). Comparison of target sequences (i.e., mu- rine sarcoma virus long terminal repeat, polyoma en- hancer, and T-cell receptor a) revealed that ets-i binds to a purine-rich core sequence (containing GGAA, i.e., the EBS3 (19). We have found that the COOH-terminal etsi or v-ets protein containing only the DNA-binding domain is sufficient for this specific DNA-binding activity, as demonstrated by gel-shift and Southwestern blot as- says.4 Recently, the DNA-binding consensus sequences for the Drosophila E74A (20) and human ETS1 (21) pro- teins have been determined. Since these divergent pro- teins are able to bind the same core sequence, CCGGAAGT, it is likely that other members of the ets family may recognize this preferred nucleotide target. Recent evidence further suggests that members of the ets family are transcriptional regulatory factors, able to trans-activate transcription through target sites in a num- ber of genes (22-25). In tumor viral enhancers, the ETS1 product was shown to trans-activate the human T-cell leukemia virus long terminal repeat and the Py enhancer via their interaction with the EBS (22, 23). It has been shown in a cotransfection assay with EBS containing CAT and etsl and ets2 expression vectors, that the etsi and ets2 proteins trans-activate transcription. It has recently been shown that the human ERG gene products (ERG-i and ERG-2) and Elki protein can also trans-activate re- porter plasmids containing three copies of the E74 EBS (24, 25). In light of this, we have continued to character- ize human ets-related genes. A XgtlO cDNA library made from CEM RNA was screened with a v-ets probe. Multiple clones were obtained, and the DNA was sequenced after subcloning into Bluescript vectors. One of the clones, designated XT2O, was found to be highly related to human ERG and appears to be the human homologue of the mouse Fli-1 gene. Because of this homology, we have designated this gene ERGB/Fli-1 . In this study, we present the molecular characterization, chromosomal localiza- Received 6/i/92. 1 Presented in part at the Seventh Annual Meeting on Oncogenes, Fred- erick, MD, June 24-29, 1991, and at the Cold Spring Harbor Meeting on Cancer Cells, Cold Spring Harbor, NY, August 28-September 1, 1991. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: EBS, ETS-binding sequence; CAT, chlor- amphenicol acetyltransferase; TK, thymidine kinase; cDNA, complemen- tary DNA; SDS, sodium dodecyl sulfate; kb, kilobase(s). 4 R. Ascione, D. M. Thompson, R. Thomas, A. Panayiotakis, R. Ramsay, M. Tymms, I. Kola, and A. Seth. ETS1 and ETS2 DNA-binding and mechanism of ETS1 autoregulation, submitted for publication.