MOLECULAR AND CELLULAR BIOLOGY, May 1994, p. 3292-3309 Vol. 14, No. 5 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology ERP, a New Member of the ets /Oncoprotein Family: Cloning, Characterization, and Differential Expression during B-Lymphocyte Development MONICA LOPEZ, PETER OETTGEN, YASMIN AKBARALI, ULRICH DENDORFER, AND TOWIA A. LIBERMANN* Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215 Received 23 July 1993/Returned for modification 18 January 1994/Accepted 17 February 1994 The ets family encodes a group of which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, TT and ,uB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the w or the ,uB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related ), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 , a subgroup of the ets gene family that interacts with the . Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer wF site, and the kck ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH w site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

The different stages of B-cell development are characterized (for a review, see reference 46). Interestingly, each of these by differential expression of a whole set of genes, most regulatory regions contains several, at least partially redundant prominently the members of the immunoglobulin gene family, B-cell-specific enhancer elements, none of them being com- as well as by differential immunoglobulin gene rearrangement pletely essential for the regulation of the IgH gene (42, 49, 58). (for a review, see reference 7). Differential expression and/or We have recently identified two new enhancer elements, ir activation of transcription factors leading to stage-specific and RB, in the intronic IgH enhancer with characteristic expression of a certain set of genes appear to be crucial for cell functional differences (47, 49). Whereas the ,uB element is differentiation in general and B-cell development in particular active throughout B-cell development (49), the 'r enhancer (for a review, see reference 46). Transcriptional regulation of element functions primarily at early stages of B-cell develop- immunoglobulin genes is of special interest for B-cell differ- ment (47). Furthermore, whereas the pRB element does not act entiation, because different classes of immunoglobulin genes as an enhancer by itself, rr alone works as a very potent are switched on at different stages of B-cell development (7, enhancer in pre-B cells (47, 49). The DNA sequences of both 83). Thus, whereas immunoglobulin heavy-chain (IgH) gene enhancer elements show striking homology to binding sites for expression occurs at the earliest stages of B-cell development transcription factors of the ets transcription factor/oncoprotein prior to IgH gene rearrangement and continues throughout family, with a GGAA sequence in the core of the binding site B-cell development, immunoglobulin light-chain genes are (55, 61, 85, 94). All members of the ets family share a highly transcribed only upon maturation of B cells after IgH gene homologous 80- to 90-amino-acid DNA-binding domain, the rearrangement (7, 83). ETS domain, although the remainder of the proteins are Analysis of the regulatory elements involved in IgH gene strikingly divergent among different members of the family (for expression has resulted in the identification and characteriza- reviews, see references 55, 61, and 85). The ETS domain is tion of at least three regulatory regions including the promoter sufficient to interact specifically with DNA sequences contain- upstream of the IgH gene, an intronic enhancer, and an a in the center of the site enhancer 3' of the IgH gene (15, 51). Several positive and ing GGA(A/T) binding (20, 53, 61, within 94, 97). Members of the ets family have been recently shown to negative regulatory elements appear to be distributed a set a see reference Most play essential roles as transcriptional regulators of whole these regulatory regions (for review, 46). such as interleu- of these elements appear to be ubiquitously active; however, of genes including many T-cell-specific genes some B-cell-specific regulatory elements have been described kin 2 (IL-2) (88), CD3 (40, 88), T-cell receptors ot, ,, and 8 (26, 40, 88, 94), human immunodeficiency virus type 2 (HIV-2) long terminal repeat (LTR) (41, 57, 94), stromelysin (96), c-fos (14, * Corresponding author. Mailing address: Department of Medicine, 25), and urokinase (60) genes. Furthermore, several ets-related Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Phone: genes have been shown to be expressed in B cells (19, 31, 72). (617) 735-3393. Fax: (617) 735-3547. As a first step in identifying putative novel members of the 3292 VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3293 ets gene family which are expressed in pre-B cells, we have agarose gel. The relevant amplified DNA fragments were applied a PCR approach using degenerate oligonucleotides eluted with glass beads (Geneclean; Bio 101) as recommended encoding conserved domains of all known members of the ets by the manufacturer. Eluted DNA was digested with NotI and family and mRNA derived from a pre-B-cell line. We now Sall (for primers A to D) or NotI alone (for primers E and F), report the isolation and characterization of a cDNA clone repurified via glass beads, and subcloned into either NotI-SaI- encoding a novel member of the ets gene family, named ERP or NotI-digested pSL1 180 (Pharmacia) or Bluescript SK+ (ets-related protein). The ERP gene is most closely related to (Stratagene). Colonies of recombinant clones were randomly SAP-i (14) and ELK-I (68), two members of the ets gene picked, and minipreparations of DNA were carried out by the family that interact with the serum response factor (SRF) (14, Magic Miniprep (Promega) procedure as recommended by the 25). ERP mRNA is expressed to different extents in a variety manufacturer. The clones were digested with SalI and XhoI or of tissues but, in B cells, is much more abundant at the NotI and analyzed on a 2% agarose gel. Double-stranded pre-B-cell stage than at later stages of B-cell development. We sequencing of positive clones was performed with the Seque- demonstrate that carboxy terminus-deleted ERP interacts with nase II kit (U.S. Biochemical) according to the manufacturer's several ets-related binding sites including the E74 and the IgH instructions. Tr sites, suggesting a potential role in IgH . 5' RACE (rapid amplification of cDNA ends) primer exten- sion. First-strand cDNA synthesis and 5' tailing with dCTP was MATERIALS AND METHODS carried out with the 5' RACE system (Bethesda Research Laboratories) according to protocols recommended by the Cell culture. S194 (murine myeloma), NIH 3T3 (murine manufacturer. Briefly, 1 jig of poly(A)+ mRNA derived from fibroblast), and P19 (murine teratocarcinoma) cells were BASC6C2 cells was reverse transcribed for 30 min at 42°C by grown in Dulbecco modified Eagle medium containing 10% using 200 U of SuperScript reverse transcriptase and 200 ng of fetal calf serum. PD31 (murine pre-B), 230-238 (murine pre- ERP-specific oligonucleotide primer 5'-GCTAGCGGCCGCT B), NFS 5.3 (murine late pre-B), BASC6C2 (murine pre-B), GAGGAGCYT7GAACTCGCC-3' (primer G). The cDNA was WEHI231 (murine mature-B), A-20 (murine mature-B), J558 RNase H treated and purified over GlassMax spin cartridges (murine myeloma), MPC1 1 (murine myeloma), P3X63 AG8 (Bethesda Research Laboratories). The cDNA was tailed at the (murine myeloma), EL.4 (murine-T), and Pu5-1.8 (murine 5' end with 10 U of terminal deoxynucleotidyl transferase (TdT) monocyte) cells were maintained in RPMI 1640 supplemented and 200 nM dCTP for 10 min at 37°C. with 10% fetal calf serum and 50 ,uM 2-mercaptoethanol. Deoxycytidine-tailed cDNA was amplified by PCR using an HAFTL (murine early-pre-B) cells were grown in RPMI 1640 anchor primer (5'-CUACUACUACUAGGCCACGCGTCG medium containing 15% fetal calf serum and 50 ,uM 2-mer- ACTAGTACGGGIIGGGIIGGGIIG-3') provided by the captoethanol. manufacturer and a nested ERP-specific primer (5'-GCCACG Isolation of poly(A)+ mRNA. Poly(A)+ mRNAs were iso- GTCGACCTCGCCATCGTTCGATGTCC-3' [primer H]) up- lated from _108 cells as described by Libermann et al. (48). stream of primer G. cDNA synthesis. First-strand cDNA synthesis was carried 5' RACE PCR amplifications were carried out in a final out with the SuperScript Choice system (Bethesda Research volume of 50 ,lI containing 10% of the tailed cDNA, 50 mM Laboratories) according to protocols recommended by the KCl, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl2, 0.2 mM dNTPs, manufacturer. Briefly, 1 ,ug of poly(A)+ mRNA derived from 0.5 pl of AmpliTaq DNA polymerase (Perkin-Elmer Cetus), BASC6C2 cells was reverse transcribed for 1 h at 37°C by using 20 pmol of the anchor primer, and 25 pmol of primer H. 7.4-jig random hexamers as primers and 200 U of SuperScript Reaction mixtures were overlaid with mineral oil and amplified reverse transcriptase. The cDNA was precipitated with ethanol by using Perkin-Elmer Cetus thermal cycler 480 as follows: 2 and dissolved in 10 jil of water. cycles of 60s at 95°C, 60 s at 37°C, and 10 min at 68°C; then 40 PCR cloning. BASC6C2 cDNA (0.5 plI) was amplified with cycles of 60 s at 94°C, 60 s at 55°C, and 3 min at 72°C followed 100 pmol each of the following degenerate oligonucleotide by 7 min at 72°C. Ten microliters of the reaction products was primers: 5'-GCTAGCGGCCGCTGGCA(AIG)TT(TIC)(TIC) separated on a 2% agarose gel. Forty microliters of amplified T(A/G/C/T)(T/C)T(A/G/C/T)(C/G)A-3' (5' primer A), 5'-GC DNA was eluted with glass beads (Geneclean) as recom- TAGCGGCCGCTA(T/C)CA(A/G)TT(T/C)(T/C)T(A/G/C/T) mended by the manufacturer. Eluted DNA was digested with (T/C)T(A/G/C/T)(C/G)A-3' (5' primer B), 5'-GGAGTCGAC Sall, repurified via glass beads and subcloned into Sall- TT(A/G/C/T)T(C/G)(A/G)TA(A/G/C/T)GTCAT(A/G/C/T) digested Bluescript SK+ (Stratagene). Minipreparations of TT-3' (3' primer C), 5'-GGAGTCGACTT(AIGIC/T)T(CIG) recombinant clones were carried out by the Magic Miniprep (A/G)TA(A/G)TTCAT(A/G/C/T)TT-3' (3' primer D), 5'-GC procedure. The clones were digested with Sall and analyzed on TA GCGGCCGCAA(T/C)ATGAA(T/C)TA(T/C)GA(T/C) a 2% agarose gel. Double-stranded sequencing of positive AA-3' (5' primer E), and 5'-GCTAGCGGCCGCGT(A/G/CI clones was performed with a Sequenase II kit. T)(G/C)(T/A)CCA(A/G)AA(A/G)TG(A/G/T)AT-3' (3' prim- cDNA library screening. A murine lung cDNA library in er F). XgtlO was obtained from Clontech. The 900-bp ERP fragment Standard PCR amplifications were carried out in a final derived by PCR from BASC6C2 mRNA was labeled with volume of 50 pI containing 200 ng of cDNA, 50 mM KCl, 10 [a-32P]dCTP by random priming using a random priming kit mM Tris-HCl (pH 8.3), 3 mM MgCI2, 0.2 mM deoxynucleoside (Boehringer). Replica filters of -7.5 x 105 recombinant phage triphosphates (dNTPs), 5% dimethyl sulfoxide, and 0.2 [lI of were hybridized with -9 x 105 cpm of the ERP probe per ml. AmpliTaq DNA polymerase (Perkin-Elmer Cetus) and com- Hybridizations were performed for 1 h at 68°C with the binations of either 5' primer A or B with 3' primer C or D or Quickhyb solution (Stratagene) and 100 jig of sheared salmon 5' primer E with 3' primer F. Reaction mixtures were overlaid sperm DNA (Stratagene) per ml. Filters were washed twice for with mineral oil and amplified by using Perkin-Elmer Cetus 15 min each time at room temperature in 2 x SSC (1 x SSC is thermal cycler 480 as follows: 2 cycles of 60 s at 95°C, 60 s at 0.15 M NaCl plus 0.015 M sodium citrate)-0.2% sodium 32°C, and 150 s at 68°C; then 45 cycles of 60 s at 94°C, 60 s at dodecyl sulfate (SDS) and twice for 30 min each time at 68°C 55°C, and 45 s at 72°C followed by 7 min at 72°C. Ten in 0.2 x SSC-0.2% SDS. Six positive clones were rescreened microliters of the reaction products was separated on a 2% and used for further analysis. Inserts were cut out from the 3294 LOPEZ ET AL. MOL. CELL. BIOL.

AgtlO phage miniprep DNA by EcoRI and inserted into the buffer 0.5 x TBE (1 x TBE is 89 mM Tris-HCl, 89 mM sodium EcoRI site of Bluescript SK+. borate, and 2 mM EDTA [pH 8.3]). DNA sequencing. All cDNA clones were sequenced either Oligonucleotides used as probes and for competition studies with Sequenase II or by Lori Wirth (Molecular Biology Core are listed in Table 1. Facility, Dana-Farber Cancer Institute) using an Applied Northern and Southern blots. Northern (RNA) blots were Biosystems automatic DNA sequencer model 373A, the Taq made as described by Libermann et al. (48) with 3 pug of DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosys- poly(A)+ selected mRNA. A Northern blot containing tems), and a combination of specific oligonucleotide primers. poly(A)+ selected mRNA derived from different murine tis- DNA and protein sequence analysis. DNA and protein sues was obtained from Clontech. A Southern blot containing sequences were analyzed and aligned by using programs DNA genomic DNA derived from different species was obtained Strider 1.2, Gene Jockey, Blast, Seqapp, and Clustal V. from Bios Corp. (New Haven, Conn.). In vitro transcription and translation. Full-length ERP Northern and Southern blot analysis. Northern and South- cDNA encoding the whole open reading frame was inserted ern blots were hybridized with 2 x 106 cpm of random-prime- into the EcoRI site of the Bluescript SK+ plasmid (Stratagene) labeled 0.9-kb ERP cDNA fragment per ml at 42°C for 1 h in with the T7 promoter upstream of the initiator methionine. QuickHyb solution (Stratagene) containing 200 pug of salmon The plasmid was linearized either with SmaI to generate sperm DNA per ml. After being washed at 680C with 0.2 x full-length ERP or with Sacl (ERPAC396), BstXI (ERPLv SSC-0.2% SDS, the filters were autoradiographed for 2 days at C298), BglII (ERPt\C222), EcoNI (ERPAC106), or Eco47III - 70°C with intensifier screens. (ERPAC61) to generate various deletion mutants of ERP as Chromosomal localization. Southern blots containing described in Fig. 9 and used as a template for in vitro genomic DNAs derived from different hamster-human hybrid transcription. cell lines were obtained from Bios and hybridized as described In vitro transcription reactions were performed with the above. linearized plasmids by using an in vitro transcription kit and T7 Nucleotide sequence accession number. The GenBank nu- RNA polymerase (Promega) as recommended by the manu- cleotide sequence accession number for the sequence reported facturer. The RNA was extracted with phenol-chloroform, here is L19953. ethanol precipitated, and resuspended in 40 [lI of water. Part of the reaction was tested on an agarose gel for size and RESULTS quality. Wild-type and mutant ERP proteins were synthesized by in vitro translation of 2 ,ug of RNA in micrococcal nuclease- Isolation and characterization of the murine ets-related treated wheat germ extracts (Promega) in the presence of cDNA, ERP. Isolation of several genes related to the Ets-J either [35S]methionine (NEN) or unlabeled methionine as gene has recently led to the characterization of a novel specified by the manufacturer. A portion of the labeled in vitro DNA-binding motif, the ETS domain, which is highly con- translation products was analyzed on an SDS-20% polyacryl- served among all members of the ets family (for reviews, see amide gel. references 55, 61, and 85). Sequence comparison of the 80- to Renaturation of ERP. In vitro translation products were 85-amino-acid ETS domains of the different members of the denatured in SDS-sample buffer (36) containing 3.3% 2-mer- ets family revealed several regions of high homology between captoethanol and boiled for 5 min. After separation of proteins all ets-related proteins. Of these, two particular highly con- by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), gel served regions, as outlined in Fig. 1A, appeared to be suitable slices from different molecular weight regions were cut out and for designing degenerate oligonucleotides which would ground. Proteins were eluted, precipitated with acetone, dena- all genes of the ets family that had been cloned at that time and tured in 2.5 [lI of saturated urea, and renatured for >18 h at which could be used for PCR amplification of ets-related genes 4°C in 125 pl of a buffer containing twofold-concentrated from pre-B cells. The sequence (W/Y)QFLL(E/D/Q) defined electrophoretic mobility shift assay (EMSA) binding buffer (4), the 5' border of the region of cDNA to be amplified, and the 10% buffer D (17), and 0.1% (vol/vol) Nonidet P-40 as sequence (K/N)MN(Y/T)(D/E/Q)K defined the 3' border. The described elsewhere (4). To determine the molecular weights amplified product between these two sequences was expected of in vitro-translated proteins in a particular gel slice, 15 ,ul of to be 170 to 180 bp long including 21 bp derived from the renatured protein was reprecipitated with 4 volumes of ace- flanking NotI and SalI restriction sites. To identify members of tone, washed with methanol, resuspended in 10 ,ul of SDS- diverse subfamilies of the ets family which differ in one or two sample buffer, and boiled for 5 min. The samples were amino acids, we generated two different sets of each degener- separated by SDS-PAGE and analyzed by autoradiography. ate oligonucleotide primer. The sequences of the primers were For EMSAs, 10-pA renatured proteins were incubated with a based upon the DNA sequences encoding the two consensus 10-pA reaction mix containing 20 pKg bovine serum albumin amino acid sequences and differed in the codons for one amino (BSA), 0.5 pg poly(dI-dC), and the 32P-labeled double- acid as indicated in Fig. 1B. The oligonucleotide primers were stranded oligonucleotide probe for 15 min at room tempera- thus mixtures of 512 different oligonucleotides for primer A, ture and analyzed on 4% acrylamide gels as described below. 1,024 oligonucleotides for primer B, 128 oligonucleotides for EMSA. DNA-binding reactions and EMSAs were per- primer C, and 256 oligonucleotides for primer D. The pre- formed as described elsewhere (45, 47). Samples of 20 ,ul dicted thermal denaturation range of primers A and B was 42 containing 2 to 4 pA of the in vitro translation product were to 54°C, and that of primers C and D was 38 to 46°C. To ensure incubated with 0.1 to 0.2 ng of 32P-labeled double-stranded hybridization and extension in early amplification cycles, when oligonucleotide probes (5,000 to 10,000 cpm), 10% buffer D only the amino acid-encoding nucleotides hybridize, and to (17), 10 mM Tris-Cl (pH 7.5), 50 mM NaCl, 1 mM dithio- increase specificity later on, we annealed the primers at 32°C threitol, 1 mM EDTA, 5% glycerol, 20 pLg of bovine serum for two cycles and then increased the annealing temperature to albumin (BSA) (Boehringer), and 0.5 pug of poly(dI-dC) 55°C for the remaining cycles. PCRs were performed with (Pharmacia). Samples were incubated in the presence or random hexamer-primed cDNA derived from the murine absence of competitor oligonucleotides for 15 min at room pre-B-cell line BASC6C2 (27). This strategy enabled us to temperature and run on 4% polyacrylamide gels, containing as obtain one predominant ethidium bromide-stained amplifica- VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3295

TABLE 1. Oligonucleotides used as probes anid for competition studies Oligonucleotide Sequence

E74 VT ...... 5'-TCGAGTAACCGGAAGTAACTCAG-3' 3'-CATTGGCCTTCATTGAGTCAGCT-5'

E74 mutant ...... 5'-TCGAGTAACCTTTAGTAACTCAG-3' 3'-CATTGGAAATCATTGAGTCAGCT-5'

IgH enhancer 7r YT ...... 5'-TCGACTGGCAGGAAGCAGGTCATGC-3' 3'-GACCGTCCTTCGTCCAGTACGAGCT-5'

IgH enhancer ff mutant ...... 5'-TCGACTGGCATTAAGCAGGTCATGC-3' 3'-GACCGTAATTCGTCCAGTACGAGCT-5'

Ick promoter T...... 5'-TCGAGGTGGCAGGAAGCTTGG-3' 3'-CCACCGTCCTTCGAACCAGCT-5'

Ick promoter mutant ...... 5'-TCGAGGTGGCAATTAGCTTGG-3' 3'-CCACCGTTAATCGAACCAGCT-5'

HSV ICP4WT ...... 5'-TCGAGCGGAACGGAAGCGGAAACCG-3' 3'-CGCCTTGCCTTCGCCTTTGGCAGCT-5'

Polyomavirus PEA3 WT ...... 5'-TCGAGCAGGAAGTGACG-3' 3'-CGTCCTTCACTGCAGCT-5'

MSV LTRWT ...... 5'-TCGAGAGCGGAAGCGCGCG-33' 3'-CTCGCCTTCGCGCGCAGCT-5'

HIV-2 LTR VT ...... 5'-TCGAGTTAAAGACAGGAACAGCTATG-3' 3'-CAATTTCTGTCCTTGTCGATACAGCT-5'

HTLV-I LTRWT ...... 5'-TCGAGGGGAGGAAATGGGTG-3' 3'-CCCCTCCTTTACCCACAGCT-5'

Stromelysin promoter YT ...... 5'-TCGAGCAGGAAGCATTTCCTGG-3' 3 '-CGTCCTTCGTAAAGGACCAGCT-5'

Urokinase promoter YT...... 5'-TCGAGTCCAGGAGGAAATGAAGTCAG-3' 3'-CAGGTCCTCCTTTACTTCAGTCAGCT-5'

T-cell a enhancer Ta2 WT ...... 5'-TCCCGCAGAAGCCACATCCTCTG-3' 3'-AGGGCGTCTTCGGTGTAGGAGAC-5'

IL-2 NF-ATWT ...... 5'-TCGAGAAAGGAGGAAAAACTG-33' 3'-CTTTCCTCCTTTTTGACAGCT-5'

IL-2 IL-2B YT...... 5'-TCGAGAAGAGGAAAAATGAAG-3' 3'-CTTCTCCTTTTTACTTCAGCT-5' fos SRE WT...... 5'-TCGAGCTTACACAGGATGTCCATATTAGGACATCTG-3' 3'-CGAATGTGTCCTACAGGTATAATCCTGTAGACAGCT-5'

vpreB promoterWT ...... 5'-TCGAGGGAGGAAGCACCG-3' 3'-CCCTCCTTCGTGGCAGCT-5'

g(K) 3' enhancer T ...... 5'-TCGAGCTTTGAGGAACTGAAAACAG-3' 3'-CGAAACTCCTTGACTTTTGTCAGCT-5'

MHC class II promoter WT...... 5'-TCGAGAGTGAGGAACCAATCAG-3' 3'-CTCACTCCTTGGTTAGTCAGCT-5' aWT, wild type.

tion product of the expected size of -170 to 180 bp by using PCR product amplified by primers A and C was digested with primer A together with primer C and lower yields with primers NotI and Sall and subcloned into NotI-SalI-cleaved pSL1180 A and D (Fig. 1C). The combination of primer B with primer or Bluescript SK+ (see Materials and Methods). D or primer C resulted in much lower yields of the predicted In our preliminary analysis, 15 independent clones contain- DNA band. Additional larger products whose origins were not ing the amplified product were analyzed by DNA sequencing. further explored were observed with some combinations. The Of these 15 clones, 14 contained ets-related sequences. The 3296 LOPEZ ET AL. MOL. CELL. BIOL.

A) 34 - 38 AMINO ACIDS LWQFLLELL -- Y D SRTN T E MA G N G Q K Ct

W Q F L L E 13 K M N Y ) K N E Q B) PRIMER A S -GCTAGCGGCCGCTGGCAATTTTTNTTNCA- 3' PRIMER C 3 -TTNTACTTAATACTNTTCAGCTGAGG- 5' _G CC C G G GG SalI Y C F L L E D K M T Y D K Q N E Q

PRIMER B 5' -GCTAGCGGCCGCTATCAATTTTTNTTNCA-3r PRIMER D 3' -TTNTACTGNATACTNTTCAGCTGACG-5' GG Not 1 1G G SalI

C) PRIMERS AD AC BD BC DMSO + - + - + - + - MW -615 bp - 492

- 369 -246 -170-180 bp- -123

FIG. 1. Amplification of ets-related cDNAs with degenerate oligonucleotide primers. (A) Conserved regions within ETS domains. Blocks of amino acids (in one-letter code) conserved within the ETS domain of all members of the ets family are boxed. (B) Degenerate PCR primers derived from consensus sequences conserved within ETS domains. Degeneracies are indicated, with N denoting all four deoxynucleotides. In order to decrease degeneracy, two sets of primers differing in one amino acid each, as indicated, were derived. Restriction sites for NotI and Sall are flanking the amino acid-encoding nucleotides. (C) Gel electrophoresis of amplified ets-related sequences. Ethidium bromide-stained agarose gel of PCR products with the pre-B-cell, BASC6C2, cDNA as a template. Primer pairs are indicated above the gel. Dimethyl sulfoxide (DMSO) was used in the PCR where indicated (+). The expected 170- to 180-bp amplification product is indicated on the left. Molecular weight markers (MW) are indicated on the right.

remaining clone did not show any to Upon sequencing, this 900-bp DNA fragment gave us a ets-related genes and might represent a PCR artifact. Two predicted open reading frame which confirmed the similarity clones were identical to Ets-i (99), and two clones corre- to ELK-1 and SAP-1 in regions A to C as well as the striking sponded to the Ets-2 gene (99). However, 10 clones encoded divergence from ELK-1 and SAP-i in the remainder of the the same apparently novel ets-related protein with the highest cDNA (Fig. 2) (see Fig. 4). On the basis of the relatedness of homology to ELK-1 (68) and SAP-1 (14), a subgroup of the ets our gene to the ets family, we named it ERP (ets-related family (see Fig. 4 and 5). Since both ELK-1 and SAP-1 protein). sequences were derived from the human gene, whereas our To determine the 5' end of ERP, we performed the 5' gene was of murine origin, we had to make sure that our gene RACE method as described in Materials and Methods using was indeed a new gene and not the murine counterpart of primer G close to the 5' end of our cDNA for priming and ELK-1 or SAP-1. We designed two additional degenerate primer H further 5' of primer G together with an oligo(dG) oligonucleotide primers (Fig. 2) based on conserved regions A anchor primer for PCR amplification. We obtained several 5' and C present in both ELK-1 and SAP-1 (14). PCR amplifi- cDNA clones which upon sequencing were found to lie within cation using primers E and F and cDNA derived from 200 bp from each other, suggesting the possibility of multiple BASC6C2 cells resulted in a -900-bp amplification product. transcription start sites (Fig. 3). Primer extension analysis has

A B C A)

N n N Y D XC I H F W S T

PRIMER E 5 '-GCTAGCGGCCGCAATATGAATTATGATAA-3' PRIMER F 3 '-TAAGTAAAAACCTGNTGCGCCGGCGATCG-5' B) G G G AC C C C C T NotI NotI FIG. 2. (A) Homology regions within SAP-1 and ELK-1. Domains A to C denote regions of homology. Blocks of amino acids (in one-letter code) conserved within SAP-1 (14) and ELK-1 (68) are shown below. (B) Degenerate PCR primers derived from consensus sequences conserved within SAP-1 and ELK-1. Degeneracies are indicated, with N denoting all four deoxynucleotides. Restriction sites for Notl are flanking the amino acid-encoding nucleotides. VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 39

5-A,GCCTGTTTACACAGACTGCACACCGCCTGGGGAATAATGCAGTAAGGAAGTGAGCGGGCTCGGCCTGACTG,CTCCLACTTCCT 88

GCTC ~~~~~~~~~~~~~~~~~~~~~~~~~~191

GCAACAGACCGTCTGCAGACGCCGCGCTGA ::Z CTCCTAGAATCTCCCAAAAATCCCAAACGCTCCCCCACG 294 TCTGGGT ATG GAG AGT 1 H E S K~~~~~~~~~~~~~~~~~~__GCI~~~~~~~~~~~~...... I .....C.-G0CL? ...... -...C...... ? 373...... -...... W4W I 25~~~~~~~V T %SC4 LH W 8~~~~~~~~~~~...... 03?CTV...... GOC-4..... QL0 TTC 114 ---3)4-3-00....3 W..4...... L

TCGAGAGC CTC CTG G A G A TGTCCGGLGCCAGGGCCA G A G T C C 8 1031SLL C37A3AL Q D G D C K V SS~~~~~~~~~~~~~~~~~~~~~...... P 1 G R I V H K H G L S

CTCLAAAGTGCCIAGC COC~~~~~...... AG...T..C...... C.CT TIC....C A.LW.C.....AG.ACGCTC 76 129LK S AIB R N~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..K,:I%.xV B 4....B F S L Q CCAGAGCC ??C LG GCC A? AAG ACGGAG LAG TG GAG GG CCC TG GAT GACAGC CCC CT GTG 155P H A F K A I K T... GL.GALH K L I H P C D D G..AGG.84S P P V H H V R ACTGTGATC AGG ?T? GTG ACC ACC..AAT GAC .....LAG C.C..C..C.A.C. 04 1 181?V I K F V T~~~~~~~~~~~~~~~~~~~~~~...AAA~~~~~~~~...N.... K..DK..IT..PV..BCA.AC...GGCT...AG. L ICCOCT OCO CCL 4CC OCA~~~~~~...... CT... .TT C C V T C C A C .C....W.T C ATOT 9 LA S R 207T AL. A A I~~~~~~~~~~~~~~~~~~...... A- F...... B. V...... L N L P. . R C 4CChOCGTW TCQ TV? OCO TCACCI..T.C..W.T.C..CC..W.....CCC..(..CCC TV? TCL.CC..TCTTV? CQ~~~ WVC GAL.CAC. 1075 3 A V.S. .B...B... B...P...D.. -H AGA....AGC...CTC ...... C..CTG....GAG. GCA...... GCC.TG. CTG...... T...CTG.....TCA...... TCG...A A C A C T A C GGC.... TCC .LAA 259 K SF...... L L...... A...A..C..H...... S...... S...... P..L...N..L....S.S..G...S.K...T.ACC.1153 LAG?CTCCA?CT C?? CCC CCA LAA GGCTTG...... AAL AALGAL...... ATC..TCTCCC ALA GGC ..GCA..CCC...CAL. C ..G.T..G.C..C...CC..GGC..1231. 285KSPSL P P K G K K P K G L H I S A G~~~~~~~~~~~~~~~~~~~~~~~...... P Q L L L S 31TACCGACI A?CG GGC?CCI ATCAGCCCTC AAC AGCS CCAP GCC C?CL CCCP TCA GGGA ?CCS CTCL AC? CCAP ACCC T~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~C TTCF ACCT GCA CAG1309...... ACACCAAG? GGA C?G ?TT CTG T T C O T A T O O G T G C CC 337 P...... G .L.F...A.SS.P.L....PB...H.F....B.....P.V.CCC~~~~~~...... GC. GT... 5138 _-AC ...AG?.CC?.GCC.AGG....CAL...... G....G...... C... LA .W T?... .TC C...... CCC.. .CA.TG.CC.LC.GG.CACA.GCCG .TG.165...... L ...... P A..K...... G T.....F....F..P.T..L.L.....N.G...... H H P...... VE S CCG C G ? A G C C C C T T T CC 389P C.P...... P...... V. L.S.P.S....CC .G..Q.K.S.?CGAA...TATAGGCACAC 14 TGTCACTGCATCLACTAGGACTCAAACAGATGCGGATGATCCAGTTTGCCCCATGGCT.GTTTACTGTG.CAG.GALGGA.TTGTG..CCTG.164 GTTCTTTGTTGCATTTCGTAC..G..A.CAGA...G..GC.TT.AALC...... GTC?....A.TATTAT.ATTTCGC.175

CTCTATAAALGTCTGTTTCGCATTCAGTGALTTTTLATGTTTGTGT?rTTTTTTLATCTTGTTAGCTCTGGAGTGTTGALCACTGCCAGGGAGGACCCT??CT1855~~~~~~~~~~~~~~~~~~~~~~~~~~...... TLATG?TTTVLA?G?LACTLATGAGATGTGALGCC....A..GC...LACC.AC.TATGCTG...... G...... CC...... 195 C?TTGCATTTAG?G??A?A?GTACAGAL?TTA?G?A.....AG....G.GGGALC.G....LAGGA.A..T...... A 206 ?.T.C.C.C.T...... C...... A...... 2164 TATAAGALTGAL ....GAL.C.CA..A....AG.CLA..LA..C....G.?...G.....2211 FIG....3..Completenucleotide.....sequence...and.predicted.. amino..acid.. sequence...of..ERP...The.nucleotide....sequence...of.murine...ERP,..with..the..deduced. numberedamino.acid.onsequence.left...... the (one-letter.code).ofPeptide...... the.majorsequences...... uneriedwthadahd.ie.eot.he5.ad3'dgeeat.lioucetieprmopen.readingframe, .is.show. Nuclotids.ar.numeredon.te.riht;.minoacid.ar original .....PCR...amplification...... ais.Do (ETS dmain).B,.ad.C,.wich.ae.homrsuedfr.hto....SAP-i..logou andELK-.aswellas.a.alaine-erin-ric domin,areshaed infrae.wth.he.eadngfrae.ustram.nd.owntrem.ae.idictend abeed n te rght Te trmiatin cdon byasterisks.. A putative nuclear localization sequence is marked byfle circles..Th.ptaiv.plydeyltin.eqene,AAAA.loe.o.h polyadenylated...... 3'...end..of the.mRN.is.doublyunderline. Th.T.tf.ivle.i.Rtunvr.r.nerie.Aros.'A m A di fe en ...... DNA...... s...... nso

confirmed the presence of several transcription start sites (data brary with the 900-bp ERP PCR-amplified fragment. We not shown). isolated six positive clones, which were all sequenced in their To obtain the full-length cDNA encoding ERP, we screened entirety. One clone containing a -2-kb insert was almost full a random-primed-oligo(dT)-primed murine lung cDNA li- length starting with a 3' poly(A) tail and encoding the whole 3298 LOPEZ ET AL. MOL. CELL. BIOL.

ETS DOMAIN (DOMAIN A)

SAP-1 ERP ELK-1

SAP-1 I- --- *MT- -G3II---E-- ESLNFSE--* SSSKDRENGgKDKPPQPG ERP _--- AHAI EOLLLQD30 ----PEGR--HRH-LSS ELK-1 ITEDC PQP VVT-VTMPNVAPAAIHAAPGDT"GRPGTPXGAGM--AGPGG DOMAIN B SAP-i AT D I L SSNVKLE1I% JPAKLAEKKS*QAPTPS,ZK.WV"TPSKEPPVEWVA g R#NKTUiHIlRg ERP "SAS-EAX#SNA-A)A XL T;LSNRXtRP-CUDS**VjEVRTl4.. ELK-1 ZAR MRPPPQPPP------HPRPAVVLPNAA$AGAAAP------PSGSRS S SP

SAP-1 ATIEIGPSISPSSEETIQALETLPZLPEA TS NVMTAFATTVPIeSIW0QEPPRTPSP LSA-5PD ERP --MSPSTSETAAAASAF'ASSYAEISiLNL*§A-S--VNSSASASSSjRSViZ-SiDS#- Pi RS ELK-1 LEAC E-----AEEAGLPLQVILTPPEAPNRKSEELNVEPGLGRALPPEVKVEGPKEELEVAGERGFVP*TTK DOMAIN D SAP-1 DT 30ISVAQPMi L*EXL X.LEPDQD XLERDRVNSSS i l- TVVITSS P--SPLGILP S ERP LFL*AACH**nS Lti--JL:i SGSZTKSPS ----- PEg SA#Q1:LLSOTDif GSIA LNEPA ELK-1 AEP3VPPQ*GVPARLiAVVVMDTAGQAGGHAASSPEIS QPLSPSLLGGPGPERTPGSGSGSG DOMAIN C

SAP-1 V--PTAi i V5VSEGPFTLS ERP PSG* SGLF-A r LI GMP- ---- ELK-1 tQA*GPA SLLP4HTLtP--VLtT SSGSAQ------

SAP-1 GWMIDLPPAHFPQTYRRHNLCTCGMREPRNEETDIQHDCI ERP - -VP*4DRAPSP------VL*ESS&ES ELK-1 --VHIP8ISV------DGLSTPVVZ8 GP P FIG. 4. Comparison of the amino acid sequence of ERP with those of SAP-1 and ELK-i. The optimal alignment of the predicted amino acid sequences of ERP, SAP-1 (14), and ELK-I (68), using the Seqapp and Clustal programs, is shown. The four major homology regions-the ETS domain (domain A) and domains B to D-are boxed. Regions of amino acid identity with ERP are shaded. open reading frame. The other clones between 900 bp and 1.3 initiation site. In addition, deletion of all the sequences kb in length contained overlapping portions of either the 3' upstream of the ATG does not alter the size of the protein end or the 5' end of the ERP cDNA. Interestingly, when the product, when the product is translated in vitro (62). However, sequence of the lung ERP cDNA was compared with the we cannot exclude the possibility that translation of ERP starts BASC6C2 pre-B-cell line ERP cDNA, two nucleotide differ- at a codon different from ATG. The ERP cDNA contains a ences (C-466-T-466 and C-469-->T-469) were observed with- long poly(A) tract starting at position 2211 (Fig. 3) which is out resulting in a change of the encoded amino acids. This preceded by a classical polyadenylation site (Fig. 3) at an suggests a polymorphism for these two nucleotides in the appropriate distance. Three ATITA motifs (Fig. 3), thought to murine ERP gene rather than a PCR artifact, since sequencing be involved in rapid mRNA turnover (1, 73, 82), are found in of several independent PCR amplification products resulted in the 3' untranslated region, suggesting a low stability of ERP the same sequence. The combined length of the cDNA clones mRNA. equaled 2,211 bp (Fig. 3). A hydropathicity plot of the predicted amino acid sequence Predicted amino acid sequence of ERP. Both strands of the reveals a hydrophobic amino terminus (-15 amino acids) 2.2-kb ERP cDNA were sequenced entirely by double- followed by a -285-amino-acid primarily hydrophilic domain stranded dideoxy sequencing using M13 sequencing primers, and a carboxy-terminal hydrophobic domain of -110 amino T7 and T3 polymerase sequencing primers, and ERP-specific acids. The deduced amino acid sequence of ERP predicts a primers based on partial DNA sequencing (Fig. 3). Sequence protein rich in proline (9%), serine (16%), leucine (14%), and analysis of the ERP cDNA revealed a 1,227-nucleotide open lysine (7%). The middle of ERP, spanning from around amino reading frame encoding a 409-amino-acid protein with a acid 200 down to around amino acid 250, is characterized by predicted molecular mass of 44.5 kDa (Fig. 3), starting with an the high abundance of alanine and serine residues (Fig. 3). Of ATG at position 302 and terminating with a TGA at position 57 amino acids, 21 (37%) represent serine and 11 (19%) 1529. The ATG initiator codon partially conforms to the represent alanine. Interestingly, a cluster of five alanines is consensus eukaryotic translation initiation sequence (33) with found in amino acids 208 to 212. Similarly, the serines appear a G at position +4 and a G at position - 3. There are several to form short clusters. In contrast to the amino-terminal half, reasons to believe that this ATG is the translation initiation which contains most of the basic and acidic amino acids, the codon. No additional ATG is found in frame, and an in-frame carboxy-terminal half contains very few acidic or basic amino termination codon is found 228 bp upstream of the ATG. acid residues but is proline rich, with 27 (17%) prolines out of Furthermore, when ERP is compared with ELK-1 and SAP-1 163 amino acids. Serine- and proline-rich domains have been (Fig. 4), all three contain the initiator methionine at the same observed in a variety of transcription factors and have been position, indicating that this ATG is the true translation implicated previously in transactivation (67, 84, 101). Interest- VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3299

IDENTITY

ZAP VPWF L K(EM--ZWSN[- R ERALG1 NTvKNYDRL~SxAI.Ry,vYYK -v I QKFVYKV SAPLE-1w N- -K WV L XYL -V -QXKvYK ei ELK-i _ QftiWSLQFLS Q Ta R ONNsGI - I S DG_ P-F xVV EWLR -TSPXRYLS: XXLRT TYYDK0 - S - KRYVYK 78 YAM GR L1¶FQQL RNQKYS a K GV -F IlVDPAG ZZdE IRLINDj~NA.Y!iN L-KQ.RHO 48 MRG It1LVF 13 SSNSS--CT EQ-TNO - -F KTDP NVARRW A -S P KYDKL8JtkLRYYXDX* K-TXKH-KRYA YRV 59 FLI-i Q WF S 3SANAS- -CT EG-TNG --F KTDP NVARRW ERX-Sg NKYDRZ,;SRJ%LR'YY N K- Tk -KRYAIYR 59 D-ZLG QV LQ- TLr CEi TD- - VIZ VG-TZG.._XTDPEDR-FR DPD LWC z -N P lN4RL6AlR GD -S XV -KR YRI 56 GAP- a Q a tL. KD A R D C S VG-jG --FX NQ P _ixwV QN- PT JNllILBRLRYYTI GDQ M-C Q -R FVYRI 7 60 Z4TFl-60 QIIIQL F E T KDARD--C cs VG-G - Fr NQ .Q X - KR&LEt yK 60 ZR7i Pl QilpFTAUE GARSS-C- G-NSR - Z1 CDP NL L KR -R PG MMYTX LtRY RR L- S R[gYRYTT 56 ZTS- KSCQS-FW- s G-0 N --FE SDP E RR -R P N T S-L J4RNtlJ? D X T -KR 62 62 N K - 62 CETS-2NT-3Q TQaFAFL E A NNSAN ---ACGQS G-- --F TDPKDP NV K KK -S- PP NYDELS GLYTYDNI - TS -K RIRWYVIR ETS-2 T>A S KSQS-NQ - GAS EG-TNw - -FEplX-F IzDPSA VNVARKR]NXL } N -II P MVINDEiLERARLDS;80;3 ;GLXTVRW -i TS -K8RTrQ 63 plX>LVQFZ 59 48 ZTS- P L KTELLASPQVNG-T- S IDRSKGI - -FRF IEDV SRW K - RP WY;ED I Q - T GRYVI DTS-2ECTS- P IAQYT LgtLi KTiAf4QHRzYCQS8NSMSN - mS WG-QNRzIRG-S v TVDPIDPSZVCV RliN RVINMSN -EiA- P MIIN3XEYDK1aLSRLSNjfaYYDX3RXRYYYDR19-N K-- s;TDQ TS --KiRKRYYVY:KI 59 ZTSA3E74 LQTTYL~ILT-FA z SDPNAS--C2TSHREYCPR-Fw VGR-TNG-NREKGV- -R :I:PIXVDSKA S RIQrLWUNG Z: -7S P 9N47XNYAMGLkrVYQRn t L-A D -[gR VQf1 47 ELF- Tfj LIFir," ~ KTP -YIQRKI -FE VDSKA S LW K I-I PD MNITNGIR&trlRYT QRG L-A N ~-GRV1j 46 ERSi SL QLW#F VA L DHPSNS 1--Fil GRC; - --FT ZEP ENYV K XQX- RP IENYDE it SLAY?f liqN K-Q :Z-R VYRF 58 PEA.3 [gLQ LWa_rV L PTN AM~--F1,IN GR-0K~- F IN NAV LW IQX- RP UNY:DXLSRSL.RYV K-QE A-R VY 60

-- 39 1 IP - VDXG GTFQSSKS A IQ RK L Spi-i/Pu. RSGDNKD- sH PQIKRELN G- TN VK-WK Q Spi-B KLR FLLG TRGDKRE- -CV VZPGAGVFQ SSKH L R QQ RKR Q ASL A- TGEIRKK- - NIL 36

CONSNSUS - IQLWQ_LLELL-D ------I-WTG-DG-E--RKL-DP-EVAR-WG-RK-NKP-NNYDKLSRALRYYYDKNI --- XV-G-KR-VY-F FIG. 5. Comparison of the ETS domain of ERP with those of all known members of the ets gene family. Percent identity with ERP is indicated on the right. Amino acids identical to those in ERP are shaded. Gaps are introduced to optimize alignment. A derived consensus sequence is shown at the bottom. Amino acids conserved among all members of the ets family are underlined. References: SAP-1, 14; ELK-1, 68; YAN, 37; ERG, 76; Fli-1, 65; D-ELG, 66; GABPex, 38; E4TF1-60, 98; ER71, 10; Ets-1, 12, 23, 43, 74, 99; Ets-2, 99; C-Ets-2, 8; D-Ets-2, 66; Ets-3, 13; Ets-4, 13; Ets-6, 13; E74, 11; ELF-1, 41; ER81, 10; PEA3, 103; Spi-i/Pu.1, 30; Spi-B, 72. ingly, alanine-rich regions appear to be involved in transcrip- amino terminus. Spacing between regions A and B, as well as tional repression functions (50). A potential nuclear localiza- between regions B and C, appears not to be conserved. The tion sequence, KGKKPK, is located between amino acids 292 regions in between the conserved domains show only limited and 297 (Fig. 3). Several potential glycosylation sites, NKTN, homology to each other. NKTD, and NLSS, as well as seven putative protein kinase C Alignment of the ETS domain of ERP with that of other phosphorylation sites, five casein kinase II phosphorylation members of the ets family revealed highest homology to SAP-1 sites, a cdc2 kinase phosphorylation site, and a tyrosine kinase (81%) and ELK-1 (78%) (Fig. 5). Sequence identity to most phosphorylation site are present in ERP (29, 35, 81, 92). The other members of the ets family was -60%. ERP is least predicted ERP protein sequence reveals, furthermore, 15 related to Pu.i/Spi-I (39%) (31) and Spi-B (36%) (72). potential mitogen-activated protein (MAP) kinase phosphor- Homology to other members of the ets family is restricted to ylation sites (S/TP), two of them containing the optimal the ETS domain which is involved in sequence-specific DNA PX(S/T)P sequence (3, 81). Several of the putative MAP binding (Fig. 5). However, short stretches of similarity between kinase phosphorylation sites are present in ELK-1 and SAP-1 ERP and E74A or E74B (11) exist in the alanine-serine-rich as well (14, 56, 68). The importance of MAP kinase phosphor- region of ERP, which is divergent from ELK-1 and SAP-1 ylation sites for the biological function of ELK-1 has recently (data not shown), as well as between ERP and Ets-I (99) in a been demonstrated (56), suggesting that at least some of these region homologous to SAP-1 (data not shown). putative sites might be functionally relevant for ERP as well. Expression pattern of ERP in murine tissues. To determine We observed also that the antisense DNA strand to ERP the expression pattern of ERP and the sizes of the ERP contains another 209-amino-acid open reading frame (data not transcripts, poly(A)+ mRNAs derived from various mouse shown). No homology to any known protein was found when tissues were analyzed by Northern blot hybridization using the data base was searched for this potential protein. We have ERP cDNA as a probe. To control for RNA quality and no evidence as to whether the complementary DNA strand is quantity, we rehybridized the Northern blots with a ,- indeed transcribed. probe. The results indicate the presence of at least three Sequence comparison of ERP with other members of the ets predominant transcripts of approximately 4.4, 2.2, and 1.9 kb family. Comparison of the deduced amino acid sequence of and a minor transcript of 5.9 kb (Fig. 6). We found that the ERP with other members of the ets family revealed the highest ERP gene was expressed in all tissues tested with significant homology to SAP-1 (14) and ELK-1 (68), a subgroup of the ets variation in abundance. Lung, spleen, and heart tissues ex- family (Fig. 4 and 5). Overall, the amino acid homology of ERP pressed the largest amounts of ERP. Moderate levels of ERP with SAP-1 is 46%, and that of ERP with ELK-1 is 36%. were found in kidney and . Very low levels of Homologies are clustered in three primary regions, the amino- ERP mRNA were observed in brain, liver, and testis tissues. terminal domain A, a smaller domain B slightly further down- The 5.9-kb transcript was observed only in mRNA derived stream, and domain C close to the carboxy terminus. An from the heart. The 4.4- and 2.2-kb transcripts were expressed additional short stretch of homology, domain D, contains in all tissues in approximately equivalent amounts. The results potential nuclear localization sequences of these proteins. All suggest that ERP is expressed to various degrees in many three proteins apparently start at the same methionine, with tissues. Our cDNA appears to be derived from the 2.2-kb the highly conserved ETS domain (domain A) directly at the transcript. The different transcripts might be due to either 3300 LOPEZ ET AL. MOL. CELL. BIOL.

w -J E C')en 0 0 U~~~~~~~~~) -J 4 U)E_ E z w CC LL C: (. Z F co D i0 Z90 Q cn X LLJCC O.IL U>> Ye0L- < D) mL c. ODM < I muz *iJ D e C F CL l CLZ I m 0 CL z < n E

9.5 Kb-

7.5 - -5.9 Kb ERP 4.4 - - 4.4 _^__0 2.4 - _ _.AkA * -* - 4.4Kb -2.2 ERP 1.9 'WI_ I 2.2=1 1.35 - -....

-2 -1.8RA8 R-ACTIN 1 2 3 4 5 6 7 8 -2 FIG. 6. Expression of ERP in different mouse tissues. Northern f-ACTIN blot analysis of poly(A)+ mRNAs from the indicated mouse tissues. The blot was sequentially probed with a 900-bp ERP cDNA probe (upper panel) and a ,B-actin cDNA probe (lower panel) under stringent 1 2 3 4 5 6 7 8 9 1011 12 1314 15 16 conditions as described in Materials and Methods. The sizes of major FIG. 7. Expression of ERP in various mouse cell lines. Poly(A)+ mRNA bands are indicated on the right; the sizes of molecular weight mRNAs from various mouse cell lines were analyzed by Northern blot markers (RNA ladder; Bethesda Research Laboratories) are indicated hybridization as for Fig. 6. The blot was sequentially probed with a on the left. 900-bp ERP cDNA probe (upper panel) and a B-actin cDNA probe (lower panel) under stringent conditions as described in Materials and Methods. The sizes of major mRNA bands are indicated on the right. RA, retinoic acid. alternative splicing, cross-hybridization with transcripts de- rived from related genes, or transcription from the DNA strand opposite to that encoding the ERP transcripts. In by retinoic acid into glial and neuronal cells (63) low levels of particular, the 1.9-kb transcript is similar in size to the SAP-1 ERP transcripts became visible. These results suggest that transcript. Further detailed analysis will clarify the origins of ERP expression is regulated upon B-cell and teratocarcinoma the different transcripts. differentiation. Southern blot hybridization analysis of genomic DNAs Chromosomal localization of the human ERP gene. To isolated from different species suggests that the ERP gene is determine the location of the ERP locus in the highly conserved during evolution but might be absent in flies we performed Southern blot hybridization and yeasts (data not shown). analysis using PvuII-digested genomic DNA derived from a Expression pattern of ERP in murine B-cell lines represent- panel of hamster-human somatic cell hybrids carrying overlap- ing different developmental stages. To gain further insight into ping subsets of human . As shown in Fig. 8, the the expression of the ERP gene during B-cell differentiation, presence of the human ERP-specific band correlates only with we performed Northern blot hybridization analysis of hybrids containing human chromosome 12. These data provide poly(A)+ mRNAs derived from a variety of murine B-cell lines evidence that the ERP gene is located on human chromosome representing different stages of B-cell development (Fig. 7). 12, where no other known member of the ets family has been High levels of ERP mRNAs were detected in all pre-B-cell found. lines tested (Fig. 7, lanes 1 to 5). Cell lines representing later ERP contains a carboxy-terminal negative regulatory do- stages of B-cell differentiation, including mature B-cell lines main which inhibits autonomous DNA binding. To determine (Fig. 7, lanes 6 and 7) and myeloma cell lines (Fig. 7, lanes 8 to whether ERP can bind sequence specifically to DNA and to 11), in contrast, expressed only very low levels of ERP. Thus, delineate the domains of ERP responsible for DNA binding, the ERP gene appears to be expressed primarily at early stages we created a series of different carboxy-terminal-deletion of B-cell development and, therefore, might play a role in early mutants of ERP as outlined in Fig. 9a. In vitro-transcribed B-cell development and gene expression. Interestingly, approx- RNAs encoding either full-length ERP or the different dele- imately equal amounts of the 4.4- and 2.2-kb ERP transcripts tion mutants were translated into protein in a wheat germ were detected in all pre-B-cell lines, whereas the 1.9-kb ERP extract. SDS-PAGE analysis of the [ 5S]methionine-labeled in transcript was detectable only in some of the pre-B-cell lines vitro translation reaction mixtures revealed as the major (Fig. 7, lanes 1, 2, and 5). Significant expression of the ERP products proteins of the expected molecular weights (Fig. 9b, gene was also observed in the EL.4 T-cell line, Pu5-1.8 lower panel). Small amounts of additional faster-migrating monocyte/macrophage cell line, and NIH 3T3 fibroblasts. proteins were visible as well in some of the reaction mixtures However, no expression of ERP was detected in the embryonal because of either partial proteolysis, premature translational carcinoma cell line P19, while upon differentiation of P19 cells termination, or alternative internal initiation codons. VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3301 a and the ERPAC396 deletion mutant formed much weaker protein-DNA complexes (Fig. 9b, upper panel, lanes 2 and 3) than more extensive carboxy-terminal-deletion mutants of ERP (ERPAC298, ERPAC222, and ERPAC106) (Fig. 9b, upper panel, lanes 4 to 6). These data suggested the existence -HAMSTER ERP of a potential negative regulatory domain at the carboxy terminus of ERP located between amino acids 298 and 396 -HUMAN ERP which encompasses domain C and contains several putative MAP kinase phosphorylation sites. DNA binding was the strongest in ERP AC106, which contains primarily the amino- 1 2 3 4 5 6 7 8 9 10 11 terminal ETS domain. Further deletion to amino acid 61 (ERPAC61) within the ETS domain completely abolished the DNA-binding capacity of ERP, demonstrating that the DNA- 1 2 ) FRPFRP HYRDI 3 lRl1i -12311415 j~9:l 20 binding domain of ERP maps indeed to the ETS domain and is sufficient for specific DNA binding. No protein-DNA com- plex formation was observed when a mutant E74 oligonucleo- tide was used as a probe in EMSAs (Fig. 9c), verifying the sequence specificity of ERP interaction with the E74 DNA motif. ERPAC298, ERPAC222, and ERPAC106 formed protein- DNA complexes with mobilities in the EMSA consistent with their relative molecular weights. However, the most slowly migrating complexes formed with full-length ERP or ERPAC396 did not correlate with their expected relative molecular masses and appeared to be of similar size as ERPAC298, which is 100 amino acids smaller, raising the question of whether these bands may be due to complex formation with smaller translation products rather than the full-length proteins. There was a weak smear (Cl) above this complex which was not observed in ERPAC298 and most likely represents a protein-DNA complex formed by full-length ERP FIG. 8. Chromosomal localization of the human ERP gene. (a) Southern blot hybridization analysis of 26 hamster-human somatic cell with lower affinity than the other complexes. hybrids. Genomic DNAs of hamster-human somatic cell hybrids (lanes To analyze in more detail which protein-DNA complex is 2 to 10), as well as human (lane 1) and hamster (lane 11) control formed by which translation product, in vitro translation DNAs, were digested with PvuII and subjected to Southern blot reaction mixtures were separated by SDS-PAGE. Slices repre- analysis. The panel blots were obtained from Bios Corp. The blot was senting different molecular weight regions were cut out of the probed with the ERP cDNA probe under stringent conditions. Infor- gel, and proteins were eluted, denatured, and renatured in the mative hybrids and controls are shown. (b) Chromosomal localization presence of urea. Aliquots of the renatured proteins were analysis of 26 hamster-human somatic cell hybrids. The number of the analyzed by SDS-PAGE and autoradiography for the presence hamster-human somatic cell hybrid line is indicated on the left. Shaded and the size of in vitro-translated ERP proteins as shown in box, presence of the chromosome indicated at the top; open box, proteins absence of the chromosome; black box, presence of both the chromo- Fig. 9d, lower panel. A second equivalent of renatured some and the human ERP gene. The pattern of retention (+) of the was used for EMSAs using the E74 site as a probe (Fig. 9d, human ERP gene in the hybrids is shown on the right. upper panel). Single, strong protein-DNA complexes were formed by renatured proteins recovered from the ERPAC298, ERPAC222, and ERPAC106 in vitro translations (Fig. 9d, lanes 5, 8, and 10), which correlated with the presence of single To evaluate whether ERP can specifically interact with [35S]methionine-labeled proteins in SDS-PAGE and corre- canonical ets-related binding sites, we tested the ability of in sponded to the molecular weights expected for the particular vitro-translated full-length or truncated ERP to bind specifi- deletion mutants. Renatured protein recovered from full- cally to an oligonucleotide containing the Drosophila E74 length ERP translations, however, gave rise to only very weak binding site (11) which has previously been demonstrated to complexes (Fig. 9d, lanes 1 to 3). In particular, the fraction bind several members of the ets family including the highly containing the apparent full-length 44-kDa ERP protein (Fig. related ELK-1 and SAP-1 (28, 70, 71, 90). We compared the 9d, lane 2) formed only a very weak complex. This protein- abilities of wheat germ extracts containing the different-length DNA complex (Fig. 9d, lane 2) comigrated with a complex ERP proteins and of unprimed wheat germ extracts to form formed by renatured protein from the equivalent molecular complexes with the E74 DNA motif in EMSAs. To ensure weight region of the ERPAC298 translation (Fig. 9d, lane 4), equivalent amounts of in vitro-translated protein in each despite the fact that no [35S]methionine-labeled protein was EMSA reaction, we measured the amount of [35S]methionine present in this fraction of the ERPAC298 translation. These incorporation and adjusted for the number of methionines denaturation-renaturation experiments most vividly demon- present in the different ERP proteins. Molar equivalents of strate that the full-length ERP protein expresses negligible each ERP protein were used for EMSAs. The E74 oligonucle- DNA-binding activity by itself and that, upon removal of an otide formed several complexes with proteins present in the apparent carboxy-terminal negative regulatory domain, DNA- ERP extracts (Fig. 9b, upper panel) which were not formed by binding capacity is recovered. the control extract (Fig. 9b, upper panel, lane 1). Some ERP binds to functionally important ets-related binding additional weaker complexes were formed by the control as sites in a variety of genes including the pre-B-specific IgH well as all the other extracts. Strikingly, both full-length ERP enhancer ir site. To analyze the DNA sequence requirements 3302 LOPEZ ET AL. MOL. CELL. BIOL. a MUTANT PROTEIN DNA BINDING A B ALA/SER D C ERP K +1- 1 409

ERP \C396 +1- 1 396

ERPAC298 - 1 298

ERPAC222 ++ 1 222

ERPAC1 06 ++t 1 106

ERP-C61 1 61

N ow o_ b tD Cm N 0 w ERP 0 0) N 0 0000_'c i d ERPAC298 ERP.XC222 ERP_AC1o6 C) N CrN -0 -i -1 < - -< <11,-i < i t: cl: CC D: m D: E74 E74 E74 E74 I 1 MUTL w I W WW WW W E74 MUT I I E74 WT

Cl so _rn S C2 - C3- --o 4

k.

kDa p 105.1- 1 2 3 4 5 6 7 69.8- kDa 43.3- S0 69.8 - 28.3- _ 43 3- 18.1- 28.3- 15.4- 18 1- .I's i _ 15.4- lb 1 2 3 4 5 6 7 9 10 11 12

1 2 3 4 5 6 7 FIG. 9. Delineation of protein domains involved in DNA binding of ERP. (a) Schematic representation of the structures and DNA-binding abilities of full-length and deletion mutants of ERP. Carboxy-terminal-deletion mutants of ERP are denoted by AC and the number of the last amino acid. The 409-amino-acid open reading frame is represented by a box. Amino-terminal and carboxy-terminal residues are indicated for each translation product. The SAP-1/ELK-1 homology domains A to C, as well as the alanine-serine-rich region, are indicated. The relative abilities of the full-length protein and deletion mutants to bind to the E74-binding site in EMSA as shown in panels b and d are indicated on the right. (b) (Upper panel) DNA binding of full-length ERP and deletion mutants in an EMSA using synthetic oligonucleotides coding for the E74 site. The full-length and carboxy terminus-truncated ERP constructs in panel a were transcribed in vitro and translated in vitro in a wheat germ extract. VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3303 for the binding of ERP, we designed oligonucleotides encoding but divergent from the Pu.1 and ELF-1 consensus sequences a whole spectrum of different functionally relevant binding (64, 94). sites for ets-related factors including sites of several B- or To analyze in more detail the interaction of ERP with some T-lymphoid-specific genes (see Materials and Methods) (Fig. of the ets-related binding sites, we performed EMSAs with 10a). The relative binding affinity and specificity of ERP for ERPAC222 and the control translation using several of the these sites were compared with its affinity for the E74 site in ets-related binding sites as probes (Fig. lla). The E74 oligo- competition experiments. Equivalent amounts of wild-type nucleotide formed the strongest protein-DNA complex, which oligonucleotides were used as competitors in EMSA with comigrated with a similar strong complex formed by the HSV equal amounts of ERPAC222 in vitro-translated protein and ICP4 oligonucleotide and was absent in the control reaction. A the E74 oligonucleotide as a probe (Fig. 10b). The wild-type specific protein-DNA complex with similar migration was E74 oligonucleotide competed effectively with complexes obtained with the ERPAC222 but not the control translation formed by ERPAC222, whereas the mutant E74 oligonucleo- when the IgH enhancer rr site, the MSV LTR ets site, or the Ick tide was unable to inhibit binding of ERPAC222 even at high promoter ets site was used as a probe, even though the quantity concentrations (Fig. 10b, lanes 1 to 5). Ten nanograms of wild- was much less than with the E74 probe. Virtually no specific type competitor completely abolished binding of ERPAC222 complex formation was observed with the HIV-2 LTR CD3R to the E74 probe. None of the other ets-related-binding-site site and the MHC class II ets site. A strong, slowly migrating oligonucleotides was as effective as the E74 oligonucleotide in nonspecific complex which was much weaker in experiments competing for ERPAC222. Only the herpes simplex virus for Fig. 9 and 10 was visible in all reactions in Fig. 11. The (HSV) ICP4 oligonucleotide encoding the binding site for EMSA of Fig. 11 was carried out with a different batch of in GA-binding protein (GABP) in the HSV ICP4 gene expressed vitro transcription-in vitro translation reaction mixture and had an affinity similar to that of the E74 site for ERPAC222 (Fig. to be exposed for longer periods than those of Fig. 9 and 10, 10b, lanes 6 and 7), but it might contain actually two ets- suggesting that the fresh in vitro translation reaction was not as binding sites. Oligonucleotides encoding the IgH enhancer ir efficient as the previous one. The results correlate relatively site (Fig. 10b, lanes 34 and 35), the polyomavirus PEA3 site well with the affinities determined in the competition analysis. (Fig. 10b, lanes 28 and 29), and the Ick promoter ets site (Fig. Sequence specificities of the complexes formed by the IgH IT 10b, lanes 26 and 27) still competed efficiently with site and the Ick ets site were, furthermore, confirmed by ERPAC222, showing at least partial competition with 10 ng of competition analysis with either wild-type or mutant IgH ir site oligonucleotide. Other sites including the urokinase promoter and Ick ets site oligonucleotides, (Fig. 1lb). Wild-type but not ets site (Fig. 10b, lanes 8 and 9), the HIV-2 LTR CD3R site mutant IgH rr site and Ick ets site oligonucleotides competed (Fig. 10b, lanes 10 and 11), the human T-cell leukemia virus efficiently with the specific complex. An additional complex type I (HTLV-I) LTR ets site (Fig. 10b, lanes 14 and 15), the which was formed with both the ERPAC222 and the control murine sarcoma virus (MSV) LTR ets site (Fig. 10b, lanes 18 translation product competed with both the wild-type and the and 19), the c-fos promoter serum (SRE) site mutant oligonucleotides, suggesting that this protein binds to (Fig. 10b, lanes 20 and 21), the stromelysin promoter ets site sequences not directly overlapping the IgH 'r or the Ick ets site. (Fig. 10b, lanes 30 and 31), and the vpreB promoter ets site Interestingly, competition with the mutant oligonucleotides (data not shown) competed only weakly, leading to partial actually increased formation of the specific complex, most competition only at 100 ng of oligonucleotide. Interestingly, likely because of competition with the other unrelated protein virtually no competition was observed with the Ig(K) 3' en- which might bind with higher affinity to an overlapping site. hancer ets site (Fig. 10b, lanes 12 and 13) and the major Results from these studies suggest that ERP interacts with histocompatibility complex (MHC) class promoter ets site (Fig. various, but not all functionally relevant, ets-related binding 1Ob, lanes 16 and 17), which bind Pu.1, as well as with the IL-2 sites, although with different affinities. In this context it is promoter IL-2B and NF-AT sites (Fig. 10b, lanes 22 to 25), particularly interesting that ERP binds to the IgH enhancer 1r which interact with ELF-1, and the T-cell receptor a enhancer site which is active primarily during early stages of B-cell TaL2 site (Fig. 10b, lanes 32 and 33), which interacts with Ets-1 development coinciding with the expression of ERP in the or ELF-1. Figure 10a summarizes the results obtained in this B-cell lineage. Future experiments will clarify whether ERP is competition analysis, indicating the relative binding affinities of indeed the natural activator of the IgH ir site. the different sites for ERPAC222 and the DNA sequence of the binding core. On the basis of this experiment, we have DISCUSSION compiled a putative consensus binding site for ERP (Fig. 10a) which is very similar to the consensus recognition sequences In our search for transcriptional regulators of B-cell differ- for Ets-1, Ets-2, GABPot, Fli-1, and E74 (10, 19, 30, 61, 91, 102) entiation, we have identified and cloned a gene for a novel

Equal amounts of the translation products as assayed by SDS-PAGE were incubated with the labeled E74 oligonucleotide. Lane 1, unprogrammed ( -) wheat germ extract; lanes 2 to 7, wheat germ extracts programmed with the ERP RNAs indicated at the top and illustrated in panel a. DNA-protein complexes are indicated on the left. (Lower panel) SDS-PAGE analysis of the corresponding [35S]methionine-labeled in vitro translation products. The apparent sizes of molecular weight markers are indicated on the left. WT, wild type. (c) DNA binding of full-length ERP and deletion mutants in an EMSA using synthetic oligonucleotides coding for a mutant (MUT) E74 site. Equal amounts of the full-length and carboxy terminus-truncated ERP translation products as assayed by SDS-PAGE were incubated with the labeled mutant E74 oligonucleotide. Lane 1, unprogrammed (-) wheat germ extract; lanes 2 to 7, wheat germ extracts programmed with the ERP RNAs indicated at the top and illustrated in panel a. (d) DNA binding of renatured translation products of ERP in an EMSA using oligonucleotides coding for the E74 site. The full-length and carboxy terminus-truncated ERP constructs in panel a were translated in vitro in a wheat germ extract. The translation products were subjected to SDS-PAGE, followed by excision of different molecular weight regions from the gel, elution, and renaturation. Renatured proteins from different molecular weight fractions were analyzed by SDS-PAGE (lower panel) and in an EMSA (upper panel) using the labeled E74 oligonucleotide. Lanes I to 3, renatured proteins from the full-length ERP translation; lanes 4 to 12, renatured proteins from the deletion mutant translations as indicated at the top. The apparent sizes of molecular weight markers are indicated. 3304 LOPEZ ET AL. MOL. CELL. BIOL.

a FIG. 10. Interaction of ERP with functionally relevant regulatory ENHANCER BINDING SITE ERP,xc222 sites in different genes. (a) Comparison of the relative binding affinities E74 AACCGGAAGTAA of ERP for ets-binding sites in the transcriptional regulatory regions of HSV ICP4 GAACGGAAGCGG different genes. Sequences present in the regulatory regions of various LCK Prom. GGCAGGAAGCTT ++F Polyoma PEA3 AGCAGGAAGTGA genes that have been shown to bind ets-related factors or that IgH Enh. iT site GGCAGGAAGCAG ++ correspond to the consensus ets-binding site including E74 (91), HSV UROKINASE Prom. AGGAGGAAATGA ICP4 (89), Ick promoter (Prom.) (44), polyomavirus PEA3 (95), IgH STROMELYSIN Prom. AGCAGGAAGCAT ++ vpreB Prom. GGGAGGAAGCAC + enhancer (Enh.) nr site (47), urokinase promoter (60), stromelysin HIV-2 LTR CD3R GACAGGAACAGC promoter (96), vpreB promoter (34), HIV-2 LTR CD3R (41), HTLV-I HTLV-1 LTR GGGAGGAAATGG LTR (21), MSV LTR (23), c-fos SRE (14, 25), IL-2 NF-AT (88), T-cell MSV LTR GAGCGGAAGCGC FOS SRE CACAGGATGTCC + receptor a enhancer Ta2 (26), Ig(K) 3' enhancer (64), IL-2 IL-2B (88), IL-2 NF-AT AGGAGGA.AAAAC and MHC class II promoter (31) are shown. The relative DNA-binding TCR If Enh. Tut2 CAGAGGATGTGG +1- affinity of the ERPAC222 deletion mutant towards each site as Ig- 3' Enh. TTGAGGAACTGA IL-2 IL-2B AAGAGGAAAAAT determined by EMSA (see panel b) is shown on the right. A potential MHC claSs II PrOM. GTGAGGAACCAA consensus binding site for ERP based on this analysis is summarized at the bottom. Nucleotides at high-affinity binding sites are uppercased; CONSENSUS GGCCGGAAGCNN nucleotides at lower-affinity binding sites are lowercased. (b) Relative AAAA taT DNA-binding activities of ERP for different ets-binding sites in an c g ca EMSA using synthetic oligonucleotides coding for the E74 site. ERPAC222 was incubated with the labeled E74 oligonucleotide probe in the presence or absence of unlabeled competitor oligonucleotides. Lane 1, no competitor. MUT, mutant; HSV, HSV ICP4; URO, urokinase promoter; HIV-2, HIV-2 LTR CD3R; IgK, Ig(K) 3' en- hancer; HTLV-1, HTLV-I LTR; MHC II, MHC class II promoter; MSV, MSV LTR; STROM, stromelysin promoter; TCR a, T-cell receptor enhancer Ta2. Arrow on the left, specific complex.

N b 0.14 IN VITRO 0L TRANSLATED PROTEIN w

PROBE

- COMPETITOR E74 E74 MUT HSV URO HIV-2 IgK HTLV-1 MHC i1 MSV FOS SRE IL-2B NF.AT LCK PEA3 STROM TCR ii IgH (ng) 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100

member of the ets transcription factor/oncoprotein family, unique for particular members of the ets family (for reviews, ERP. ERP is expressed in a variety of tissues; however, within see references 55, 61, 85, and 94). Amino acid sequence the B-cell lineage, ERP is transcribed primarily at the pre-B- comparison demonstrates that ERP shows the highest homol- cell stage, and expression drastically declines upon B-cell ogy to a particular subgroup of the ets family with distinctive maturation. This expression pattern correlates with the en- features which includes SAP-1 (14) and ELK-1 (68). Both hancer activity of the IgH enhancer site, and ERP is indeed SAP-1 and ELK-1 are able to form a ternary complex with the able to bind specifically to this enhancer element. Our results SRF and bind to the SRE of the c-fos promoter in an suggest an involvement of ERP in the regulation of B-cell SRF-dependent manner (14, 25, 28, 71, 90). Protein-protein differentiation and, possibly, in IgH gene expression as well. interactions appear to be common mechanisms for the func- ERP interaction with DNA appears to be inhibited by a tion of most, if not all, ets-related proteins. Pu.1, for example, carboxy-terminal negative regulatory domain, indicating the cooperates with a second factor, NF-EM5 (64), and GABPot role of putative posttranslational modifications and/or protein- binds to GABP,B (38, 89). Similarly to SAP-1 and ELK-1, ERP protein interactions in the control of DNA binding of ERP. contains the three homology regions A to C. Domain A, the ERP was isolated on the basis of its homology to the ETS ETS domain, appears to be involved in DNA binding and is the domain which is highly conserved among all ets-related pro- region which is highly conserved among all members of the ets teins and which is responsible for DNA binding (55, 61, 85, 94). family (55, 61, 85, 94). Domain B has been shown to be ets-related proteins can be further grouped into subclasses on involved in the interaction of SAP-1 and ELK-1 with SRF and the basis of additional homologous protein domains which are has been found only in SAP-1 and ELK-1 (14, 28, 71, 90). Since VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3305

CM I cm N CS N a N cm CY e es N 1.) U U V.. U U 1X 0C4 IN VITRO 0. 0. .' a.' CL I CL' INN VITRO tr 0 X TIa 7RANSLATED PROTEIN TRANSLATED PROTEIN LU . LU I LUI I Mw pi'ROBE E74 LCK IgHX10m -10010100 PROBE E74 IgH x HSV HIV-2 MSV LCK MHC II cl'OMPETITOR EWT MUT WT M1UT WT MUTI (ng) 10 100 10 100 10a 100 10 l1|0 10 100 10 100

1 2 3 4 5 6 7 8 9 10 11 12 13 14

FIG. 11. ERP interacts specifically with the Ick promoter ets site and the IgH enhancer rr site. (a) DNA binding of ERP to various ets-binding sites. Equal amounts of the unprogrammed (-) and ERPAC222 in vitro translation products were incubated with labeled oligonucleotide probes encoding the E74, IgH enhancer iT, HSV ICP4, HIV-2 LTR CD3R, MSV LTR, Ick promoter ets, and the MHC class II promoter sites. (b) ERP binds specifically to the lck ets site and the IgH enhancer ar site, as determined by EMSA. ERPAC222 was incubated with the labeled E74, Ick, or IgH enhancer -r site oligonucleotide probe in the presence or absence of unlabeled competitor oligonucleotides. Lanes 1, 7, and 13, DNA-binding reactions with unprogrammed (-) in vitro translation products and E74, Ick, or IgH enhancer rr site oligonucleotide probes. Lanes 2, 8, and 14, no competitor. WT, wild type; MUT, mutant. Arrow on the left, specific complex.

ERP contains a region of similarity to domain B, it is possible only upon deletion of the carboxy terminus of ERP, which that ERP binds to SRF as well. We are currently testing this contains domain C, suggesting the existence of a negative possibility, but we were so far unable to detect any ternary regulatory domain in that region. A protein-DNA complex complex formation with SRF (2). Domain C contains phos- with full-length ERP is observed in our experiments, but phorylation sites for MAP kinase and has been demonstrated deletion mutant analysis and denaturation-renaturation stud- to be involved in regulation by growth factors and transactiva- ies indicate that this complex does not correspond to the tion (56, 90). ERP contains putative MAP kinase phosphory- expected mobility and most likely represents an early termina- lation sites homologous to the sites in SAP-1 and ELK-1, tion or degradation product of ERP. ERP might have to suggesting a potential role in the regulation of ERP activity. interact with other proteins in vivo and/or might have to be We have identified an additional shorter region of homology, posttranslationally modified, leading to derepression and in- domain D, that might encode a nuclear localization signal for creased affinity of ERP for DNA. Negative regulatory domains ERP, ELK-1, and SAP-1. While domains A to D are highly have been found in several other members of the ets family conserved, the remainder of the ERP protein reveals only including ELK-1 and Ets-1 (52, 71, 97) as well. We demon- limited similarity to SAP-1 or ELK-1, even though there are strate here that carboxy-terminal truncation of ERP which some short stretches with similarity to SAP-1, while other eliminates the conserved domain C reconstitutes DNA-binding regions are similar to ELK-1. The relevance of these homolo- activity. Domain C and the surrounding regions contain several gies for the biological function of ERP, SAP-1, or ELK-1 has putative MAP kinase phosphorylation sites which are con- to be determined. The small stretches of similarity and the served in SAP-1 and ELK-1 as well and which have been shown divergent regions in the remainder of these proteins suggest to be crucial for growth factor-regulated transactivation by that each of these ets-related proteins has unique features ELK-1 (56). A possible model predicts that the carboxy whether or not it is through interaction with particular binding terminus represses DNA binding of ERP by an intramolecular sites, interaction with particular secondary factors, unique mechanism in which the carboxy terminus masks the amino- posttranslational modifications, or other undetermined func- terminal ETS domain. Phosphorylation of certain amino acid tions. It is likely that these unique domains play crucial roles in residues in the carboxy-terminal region of ERP might affect the diverse functions of ERP, SAP-1, and ELK-1. Overall, the DNA-binding ability of ERP by conformational modifica- ERP has a slightly higher sequence identity to SAP-1 (46%) tion of the carboxy terminus, as has been observed for other than to ELK-1 (36%). A few short amino acid stretches in ERP transcription factors (9, 54). Another possible mechanism which are not conserved in SAP-1 or ELK-1 are conserved in might involve the interaction of ERP with a second protein another member of the ets family, E74 (11), which otherwise either at the carboxy terminus or at domain B which might shows very little homology to ERP, SAP-1, or ELK-1 outside relieve the negative constraint on DNA binding. Our deletion the ETS domain. The relevance of this region for the function analysis of ERP, furthermore, confirms that the highly con- of E74 is not known. served ETS domain constitutes the DNA-binding domain. This Both ELK-1 and SAP-1 are able to interact with DNA domain A is sufficient to bind sequence specifically to DNA. independently of SRF as well (28, 71, 90). The E74A-binding The DNA-binding domains of ERP, ELK-1, and SAP-1 are site of the Drosophila ecdysone-inducible E74A gene, for remarkably similar. Nevertheless, a region of 6 amino acids example, binds to ELK-1 and SAP-1 (28, 70, 71, 90), as well as close to the amino terminus is unique in each of these three to the ets-related protein E74 (11), and can be transactivated proteins, and there are several other amino acid differences by ELK-1 and SAP-1 (28, 70, 71, 90). We show here by EMSAs which could affect the DNA-binding specificity. All members that ERP interacts with the E74A site as well. However, of the ets family share a common recognition sequence, significant sequence-specific DNA-binding activity is detected 5'-GGA(A/T)-3' (55, 61, 85, 94), which is located in the middle 3306 LOPEZ ET AL. MOL. CELL. BIOL. of the DNA-binding site, whereas the flanking sequences are important for B-cell-specific activation of the Ig(K) enhancer, divergent for different members of the ets family. Differences and Pu.1 interacts with a second B-cell-specific nuclear factor in the ETS domain between ERP and SAP-1 or ELK-1 could which binds to an adjacent site (64). Another member of the ets therefore influence the precise recognition sequence for ERP, family, Ets-1, is expressed in B cells as well, and ligation of which might be slightly different from those of SAP-1 and membrane Ig leads to calcium-dependent phosphorylation of ELK-1. Whereas full-length ERP expresses only negligible Ets-1, suggesting that Ets-1 may play a role in B-cell gene DNA-binding activity towards a variety of ets-binding sites, regulation (19). A third member of the ets family, Spi-B, was carboxy terminus-deleted ERP interacts with a variety of recently shown to be expressed in B cells and might bind to functionally relevant ets-binding sites with different affinities similar sites as Pu.1 (72). In addition, Fli-1 and ERG-3 were including the HSV ICP4 GABP site (89), the IgH enhancer fT shown to be differentially expressed in B cells and suggested to site (47), the polyomavirus PEA3 site (95), the Ick promoter ets be involved in IgH gene regulation (80). Thus, ERP and site (44), the urokinase promoter ets site (60), the HIV-2 LTR several other ets-related genes are expressed in B cells, sug- CD3R site (94), the HTLV-I LTR ets site (21), the MSV LTR gesting that, analogously to the apparent role of ets-related ets site (61), the c-fos promoter SRE site (14, 25), the strome- factors in the control of T-cell-specific gene expression, ets- lysin promoter ets site (96), and the vpreB promoter ets site related proteins might regulate B-cell-specific gene expression (34). Thus, ERP is another candidate factor for the transcrip- as well. tional regulation of these genes. An approximate consensus The expression pattern of ERP suggests a widespread tissue high-affinity binding site for ERP is similar to recognition distribution of ERP, though to different degrees in the tissues sequences for Ets-1, Ets-2, ERG, Fli-1, PEA3, GABP, E74, analyzed. Nevertheless, in the B-cell lineage it appears that and ELK-1 (10, 19, 25, 30, 61, 64, 70, 71, 75, 91, 94, 102, 103). ERP is highly expressed primarily at the pre-B-cell stage and However, ERP displays very low or no affinity towards binding expression is down-regulated upon maturation of B cells. We sites for two other members of the ets family, Pu.1 and ELF-1, do not know the function of ERP in B-cell development or which recognize more divergent DNA motifs than the rest of gene regulation. However, we speculate that ERP plays a role the family (31, 64, 88, 94). It is therefore unlikely that the slight in early B-cell development and gene regulation, since expres- differences in DNA-binding affinities among the majority of sion of ERP drastically decreases upon B-cell maturation. ets-related factors towards highly related recognition se- Interestingly, expression of ERP in these B-cell lines correlates quences will determine which member of the ets family will well with the activity of the aT enhancer element which we have interact with a particular site in different genes. We anticipate identified in the IgH enhancer and which we have shown to be that the specific arrangement and combination of different primarily active at the pre-B-cell stage (47). The ar enhancer regulatory sites surrounding an ets-binding site in a particular element contains a functionally crucial 5'-GGAA-3' in the gene will favor the selection of a specific member of the ets center of the site similar to other recognition sites for ets- family due to specific protein-protein interactions and poten- related factors. Recently, several members of the ets family tial posttranslational modifications. including Ets-1, Fli-1, and ERG-3 were shown to bind to the At least four mRNA transcripts are detected on Northern IgH -r site and to activate it when overexpressed (59, 80). We blots hybridizing to ERP cDNA probes. The levels of the demonstrate here that ERP is also able to bind specifically to transcripts, however, vary in different tissues and cell lines. The the IgH IT site. Expression of ERG-3 and ERP, but not Ets-1 2.2-kb transcript appears to encode the ERP cDNA which we or Fli-1, correlates with the activity of the rr site, suggesting a have cloned. It contains a poly(A) tail at the 3' end, and possible role of either of these two factors in IgH gene preliminary primer extension analysis indicates the existence of regulation (47, 59, 80). Several other putative target genes for two main transcription start sites within 100 nucleotides from ERP which are transcriptionally active exclusively at the pre- each other, similar or identical to the 5' ends of our cDNAs. B-cell stage include vpreB (5), X5 (34), TdT (53, 78), and mb-1 The other mRNA species might encode either alternative (39). The promoter regions of all these pre-B-cell-specific splice products of ERP, transcripts of ERP-related genes, or genes contain DNA sequences related to ets-binding sites and antisense transcripts. Indeed, we have observed that the anti- might represent targets for ERP. Furthermore, several other sense strand of the ERP cDNA encodes another long open B-cell-specific genes including IgH (2, 22), Ig(K) (64), and reading frame for a putative polypeptide of 209 amino acids. FcsRIIa (77) contain apparent binding sites for ets-related No homology to any known protein is apparent. Therefore, we proteins in their regulatory regions. It is likely that some of do not know at the moment whether this protein really exists. these genes are regulated by ERP. Preliminary results pre- Nevertheless, at least one other ets-related gene isolated from sented here indicate that ERP is indeed able to interact with drosophila, D-elg, encodes an antisense transcript for an uni- the vpreB ets-binding site but not with the Ig(K) ets site. Since dentified protein whose expression is coregulated with the protein-protein interactions and posttranslational modifica- D-elg gene (66). We are now in the process of verifying, using tions play a crucial role in the DNA-binding capacity and cRNA probes, whether any of the other transcripts is derived transactivation of most members of the ets family including the from the antisense strand of ERP. highly related ELK-1 and SAP-1 and since ERP binds to DNA Recent data from several laboratories suggest that ets- only upon deletion of the carboxy terminus, we cannot rule out related proteins are essential in the regulation of many T-cell- that the actual target sites for ERP are different from the specific genes including T-cell receptors a, ,B, and 8, IL-2, sequences determined in our analysis. granulocyte-macrophage colony-stimulating factor, and CD3 Southern blot analysis revealed that the ERP gene is highly (26, 40, 57, 88, 94), and there is evidence that ets-related conserved during evolution, since we can detect specifically proteins play a role in B-cell gene regulation and differentia- hybridizing bands in all mammalian, bird, and frog DNAs and tion as well (18, 24, 31, 53, 64, 72, 80). In particular, the Pu.1 weaker bands in nematode DNA. However, we were unable to factor appears to be involved in B-cell-specific expression of detect hybridization to fly or yeast DNA, suggesting either that both IgH (2) and Ig(K) genes (64). Binding sites for Pu.1 were these organisms do not contain any gene closely related to identified in the IgH intronic enhancer (2, 59) and shown to be ERP or that our hybridization conditions were too stringent. critical for B-cell-specific activation of the IgH enhancer (2,49, We have mapped the gene for ERP to human chromosome 12. 58). Similarly, a Pu.1-binding site in the 3' Ig(K) enhancer is No other member of the ets family has been located to this VOL. 14, 1994 ERP, A NEW MEMBER OF THE ets FAMILY 3307 chromosome yet. However, there are clusters of ets-related Karin, and T. Hunter. 1991. Activation of protein kinase C genes located on human chromosomes 11 and 21 (6, 16, 65, 69, decreases phosphorylation of c-Jun at sites that negatively regu- late its DNA binding activity. Cell 64:573-584. 100). 10. Brown, T. A., and S. L. McKnight. 1992. Specificities of protein- The involvement of transcription factors in tumorigenesis protein and protein-DNA interaction of GABPct and two newly has only recently received more attention, when it was ob- defined ets related proteins. Genes Dev. 6:2502-2512. served that many of the oncogenes and tumor suppressor genes 11. Burtis, K. C., C. S. Thummel, C. W. Jones, F. D. Karim, and D. S. indeed encode transcription factors (87). Chromosomal trans- Hogness. 1990. The Drosophila 74EF early puff contains E74, a locations involving transcription factor genes appear to be complex ecdysone-inducible gene that encodes two ets related especially frequent in human leukemias and lymphomas (32, proteins. Cell 61:85-99. 87, 93). At least two ets-related genes, Fli-] (65) and ERG (86), 12. Chen, J. H. 1990. Cloning, sequencing, and expression of mouse have been directly implicated in human tumor formation. c-ets-1 cDNA in baculovirus expression system. Oncogene Res. Human chromosome 12 is involved in translocations in acute 5:277-285. 13. Chen, T., M. Bunting, F. D. Karim, and C. S. Thummel. 1992. myeloid and lymphoblastic leukemia (79) and in several other Isolation and characterization of five Drosophila genes that neoplasms. Further analysis will determine whether the ERP encode an ets related DNA binding domain. Dev. Biol. 151:176- gene is involved in translocations of chromosome 12 and 191. whether ERP may act as an oncogene. 14. Dalton, S., and R. Treisman. 1992. Characterization of SAP-1, a In conclusion, we have established ERP as a new member of protein recruited by serum response factor to the c-fos serum the ets family with significant sequence homology to SAP-1 and response element. 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Accurate regulation. transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475- ACKNOWLEDGMENTS 1489. 18. Feldhaus, A. L., D. Mbangkollo, K. L. Arvin, C. A. Klug, and H. We thank Lori Wirth for help in DNA sequencing and Dave Singh. 1992. BLyF, a novel cell-type- and stage-specific regulator Gonzalez for synthesis of the oligonucleotides. We acknowledge of the B-lymphocyte gene mb-i. Mol. Cell. Biol. 12:1126-1133. fruitful discussions with T. B. Strom, R. Kapeller, and K. LeClair. 19. Fisher, C. L., J. Ghysdael, and J. C. Cambier. 1991. Ligation of This study was supported by National Institutes of Health grant membrane Ig leads to calcium-mediated phosphorylation of the AI/CA3321 1-01 ALY to T.A.L. proto-oncogene product, Ets-1. J. Immunol. 146:1743-1749. M.L. and P.O. contributed equally to this study. 20. Gegonne, A., B. Punyammalee, B. Rabault, R. Bosselut, S. Seneca, M. Crabeel, and J. Ghysdael. 1992. 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