Proc. Natl. Acad. Sci. USA Vol. 94, pp. 7170–7175, July 1997 Biochemistry

ETS target : Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

LOIS ROBINSON*, ALEXANDRA PANAYIOTAKIS†,TAKIS S. PAPAS†,ISMAIL KOLA‡, AND ARUN SETH§¶

*Advanced BioScience Laboratories, National Cancer Institute–Frederick Cancer Research and Development Center, Frederick, MD 21702; †Center for Structural and Molecular Biology, Medical University of South Carolina, Charleston, SC; ‡Molecular Genetics and Development Group, Monash University, Melbourne 3168, Australia; and §Medical Research Council Group in Periodontal Physiology and Department of Pathology, University of Toronto, and Laboratory of Molecular Pathology, Women’s College Hospital, Toronto, Ontario, Canada M5S 1B2

Communicated by Ronald W. Davis, Stanford University School of Medicine, Stanford, CA, March 19, 1997 (received for review September 5, 1996)

ABSTRACT ETS transcription factors play important domain is comprised of 85 amino acids (4); the secondary roles in hematopoiesis, angiogenesis, and organogenesis dur- structures of ETS1 and FLI1 were determined recently by ing murine development. The ETS genes also have a role in NMR analyses and indicated the presence of three ␣-helices neoplasia, for example in Ewing’s sarcomas and retrovirally and a four-stranded ␤-sheet similar to structures of helix–turn– induced cancers. The ETS genes transcription factors helix motifs found in several mammalian and bacterial tran- that bind to specific DNA sequences and activate transcription scription factors (5–7). The ETS are an important of various cellular and viral genes. To isolate novel ETS target family of transcription factors that play roles in a number of genes, we used two approaches. In the first approach, we biological processes, such as organogenesis during murine isolated genes by the RNA differential display technique. development, hematopoiesis, B cell development, signal trans- Previously, we have shown that the overexpression of ETS1 duction, as well as maintenance of T cells in the resting state and ETS2 genes effects transformation of NIH 3T3 cells and and the subsequent activation of these T cells (8–11). specific transformants produce high levels of the ETS pro- The ETS family genes and their products also have been teins. To isolate ETS1 and ETS2 responsive genes in these implicated in several malignant diseases and pathological transformed cells, we prepared RNA from ETS1, ETS2 trans- genetic disorders. For instance, ETS1, ETS2, and ERG have formants, and normal NIH 3T3 cell lines and converted it into been shown to act as protooncogenes in that they can trans- cDNA. This cDNA was amplified by PCR and displayed on form NIH 3T3 cells in vitro, and the subsequent injection of sequencing gels. The differentially displayed bands were sub- these cells into nude mice results in tumor formation (12–14). cloned into plasmid vectors. By Northern blot analysis, several Furthermore, the FLI1 and ERG genes have been shown to be clones showed differential patterns of mRNA expression in the translocated and expresses chimeric fusion transcripts in al- NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen most all Ewing’s sarcomas as well as in a large number of other clones were analyzed by DNA sequence analysis, and 13 of primitive neuroectodermal tumors (15–17). Thus, these find- them appeared to be unique because their DNA sequences did ings are strongly suggestive of a basic role for these genes in the not match with any of the known genes present in the genesis of these types of tumors. Recently, the overexpression bank. Three known genes were found to be identical to the of ETS2 in transgenic mice has been shown to cause skeletal CArG box binding factor, phospholipase A2-activating pro- abnormalities phenotypically reminiscent of those seen in tein, and early growth response 1 (Egr1) genes. In the second Down syndrome, in which ETS2 genes are known to be present approach, to isolate ETS target promoters directly, we per- in triplicate (18). formed ETS1 binding with MboI-cleaved genomic DNA in the In view of the importance of the ETS family of transcription presence of a specific mAb followed by whole genome PCR. The factors to various biological and pathological processes, the immune complex-bound ETS binding sites containing DNA identification of downstream cellular target genes of the ETS fragments were amplified and subcloned into pBluescript and family of transcription factors is warranted. The methods for subjected to DNA sequence and computer analysis. We found elucidation of ETS targets used typically up to now have been that, of a large number of clones isolated, 43 represented mainly serendipitous or characterized by the purine-rich unique sequences not previously identified. Three clones GGAA͞T core sequences identified in the promoters͞ turned out to contain regulatory sequences derived from enhancers of various cellular or viral regulatory regions (1, 2). human serglycin, preproapolipoprotein C II, and Egr1 genes. Subsequently, synthetic oligonucleotides containing EBS were The ETS binding sites derived from these three regulatory used in electrophoretic mobility shift assays (EMSAs) and sequences showed specific binding with recombinant ETS transactivation assays using different ETS expression con- proteins. Of interest, Egr1 was identified by both of these structs together with reporter genes containing the minimum techniques, suggesting strongly that it is indeed an ETS target promoter linked to the prospective target genes EBS (19). gene. Although these approaches have had some success, they suffer from several disadvantages: (i) They are not comprehensive in that they rely on EMSAs or transfection–transactivation as- ETS genes have been cloned and characterized from a variety says; (ii) they often do not discriminate between the preferred of Metazoan species, ranging from human to Drosophila, that target genes that are transactivated by the different members retains a region of similarity with the v-ets oncogene (1, 2). ETS of the ETS family; and (iii) thus they may not adequately define family gene products bind specific purine-rich DNA sequences the biological and͞or pathological roles of specific members of and transcriptionally activate a number of genes that contain the ETS family of transcription factors. Therefore, for this ETS binding site(s) (EBS; refs. 1, 3). The ETS DNA binding study, we used two novel approaches using RNA differential display and whole genome PCR to identify putative cellular The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: CBF, CArG box binding factor; PLA P, phospholipase accordance with 18 U.S.C. §1734 solely to indicate this fact. 2 A2 activating ; EBS, ETS binding sites; EMSA, electrophoretic © 1997 by The National Academy of Sciences 0027-8424͞97͞947170-6$2.00͞0 mobility shift assays; CAT, chloramphenicol acetyltransferase. PNAS is available online at http:͞͞www.pnas.org. ¶To whom reprint requests should be addressed.

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target genes downstream of the ETS1 gene product (20–22). DNA sequence analysis. The DNA sequences were evaluated Of 59 clones identified by these combined techniques, 53 of by FASTA and BLAST computer programs. them turned out to be unique and not previously identified or EMSA. Nuclear extracts of recombinant proteins prepared characterized. Of the five known genes, Egr1 was identified by in a baculovirus system were incubated with 32P-labeled oli- both methods. The CArG binding factor (CBF) and the gonucleotides (50,000 cpm) in the presence or absence of mAb. phospholipase A2 activating protein (PLA2P) were cloned and Samples were electrophoresed on a 4% polyacrylamide gel in characterized by differential display. The other ETS gene 0.25 ϫ TBE buffer (90 mM Tris͞64.6 mM boric acid͞2.5 mM targets, human serglycin and preproapolipoprotein C II were EDTA, pH 8.3) for 1.5 h at 250 V. The gel was dried and cloned and identified by whole genome PCR. The CBF gene autoradiographed overnight (26, 27). product was found to be expressed in both ETS1- and ETS2- Chloramphenical Acetyltransferase (CAT) Assays. The co- expressing cell lines using Northern blot analysis. Similar transfection experiments were done using CAT reporter plas- analysis found PLA2P expressed only in the ETS2-transformed mids containing Egr1–EBS and ETS expression vectors (ETS1 cell line and Egr1 expressed only in the ETS1-transformed or FLI1) or CAT reporter plasmids containing the entire Egr1 cells. None of these gene products was detected in any of the promoter and specific deletion variants. The cells were har- control NIH 3T3 cell lines. We also confirmed that the vested after 48–72 h and the cell extracts were normalized for promoter regions of human serglycin, preproapolipoprotein, the protein concentrations and ␤-galactosidase activity and and Egr1 contain consensus EBS and are able to bind to the assayed for CAT activity by thin layer chromatography (28, ETS proteins. Collectively, these findings (i) demonstrate the 29). Quantitation was performed by scanning TLC plates on effective deployment of RNA differential display and whole the AMBIS radioisotopic imaging system (28, 29). All trans- genome PCR as novel approaches for discovering downstream fection experiments were repeated three times. cellular ETS target genes, and (ii), in this report, identify the Northern Blot Analysis. Total RNA was fractionated on Egr1 as an ETS target gene by both techniques. 1.2% agarose gels containing formaldehyde followed by trans- fer on to Nytran membrane (23). Blots were hybridized with MATERIALS AND METHODS Egr1, PLA2P, and CBF probes (30).

RNA Differential Display. The method used for differential RESULTS display was essentially as described by Liang and Pardee (21). Total RNA was prepared from cells by RNazol method and Identification of Egr1 as a Putative ETS1 Target Gene converted to cDNA by oligo(dT) and reverse transcriptase. Using RNA Differential Display. To identify genes that are cDNAs were amplified using T12MN and 5Ј arbitrary primers downstream targets of the ETS1 , we used provided in the RNA map differential display kit (GenHunter, RNA differential display using RNA from NIH 3T3 cell line Boston). After PCR, the samples were displayed on a 6% that has been transfected with ETS1 cDNA and produces high acrylamide DNA sequencing gels. The differentially displayed levels of ETS1 protein (12). The RNA differential display bands were recovered and subcloned into pBluescript (Strat- pattern of this ETS1-expressing cell line was compared with agene), and the DNA sequence was analyzed by FASTA and the differential display pattern from both the nontransfected BLAST computer programs. The isolated cDNA fragments parental NIH 3T3 and an ETS2-expressing cell line generated were used as probes to determine the differential expression by by transfection of ETS2 cDNA (14). More than 80 bands were Northern blot analysis (23). found to be differentially expressed using eight different Whole Genome PCR. HTB-166 genomic DNA digested with primer sets (see Material and Methods and ref. 21). From a total MboI was ligated to double stranded 43͞39 unphosphorylated of 82 differentially expressed cDNA bands, 16 were found to linkers using T4 DNA ligase (24): GATCCGGCAACGAAG- be differentially expressed in reproducible fashion. These 16 GTACCATGGCCGCATAGGCCACTAGTGCGC- bands were subcloned and subsequently analyzed by DNA CGTTGCTTCCATGGTACCGGCGTATCCGGT- sequencing and Northern blot analysis. GATCACG. The linker ligated genomic DNA was incubated DNA sequence and FASTA analyses revealed that three of with recombinant ETS1 protein followed by immunoprecipi- the 16 clones represented sequences that had significant tation with specific mAb (25). The ETS1 protein-bound DNA identity to other genes in the database; the other 13 clones may was released and amplified by PCR using primer I:GCAC- represent novel sequences not previously identified. The three TAGTGGCCTATGCGG in the first two rounds of PCR specific clones identified by us corresponded to: PLA2P, CBF, amplification (22). Primer II:GTACCTTCGTTGCCGGATC and the Egr1) (Table 1; and refs. 31–33). was used in the fourth PCR round to minimize nonspecific Identification of Egr1 as a Downstream Target Gene of amplifications. PCR amplification products were labeled with ETS1 Using Whole Genome PCR. To isolate and identify ETS 32P using polynucleotide kinase and separated on a 6% poly- target genes by whole genome PCR, the MboI-digested acrylamide gel. The DNA bands in the range of 500 bp were genomic DNA was ligated to unphosphorylated linkers to recovered and digested with MboI and subcloned at the facilitate PCR amplification of the selected genomic fragments BamHI site of pBS (23). Recombinant clones were analyzed by (24). Protein–DNA binding was carried out using linker-

Table 1. ETS target genes identified by differential display and whole genome PCR RNA expression Clone Strategy Insert size, bp ETS–DNA binding NIH 3T3 ETS1 ETS2 AE112 DD* 240 CBF ND Ϫϩϩϩϩ AE134 DD* 206 PLA2P (rat) ND ϪϪϩϩ AE117 DD* 258 Egr1 ϩϪϩϩϪ L510 WG† 500 Serglycin ϩ ND ND ND L45 WG† 500 EGR1 ϩϪϩϪ L29 WG† 500 Preproapolipoprotein C II ϩ ND ND ND ND, not determined. *Differential display: From 82 cDNA bands, three known and 13 unknown clones were isolated. †Whole genome PCR: Of 43 clones, three are known and 40 are unknown. Downloaded by guest on October 2, 2021 7172 Biochemistry: Robinson et al. Proc. Natl. Acad. Sci. USA 94 (1997)

ligated DNA and recombinant ETS1 protein in the presence of ETS1 mAb E44 (25, 34). Recovery of the immunocomplexed ETS1 protein-bound DNA fragments was achieved with pro- tein A-Sepharose. PCR amplification of the antibody-selected, ETS1-bound DNA was accomplished using primer I. The immunoprecipitation and PCR amplification were repeated twice using primer I. The selected fragments were subjected to a final round of amplification using primer II (see Materials and Methods). The resultant PCR-amplified DNA fragments were subcloned into the pBluescript vector at the BamHI site. Forty-three clones were sequenced and subjected to computer analysis using the FASTA DNA sequence comparison program. Forty clones were found to be unlike any in the database although some showed partial homology with various known DNA sequences. Three clones that were found to be homol- ogous to genes in the database were derived from the regu- latory regions of the human serglycin, the preproapolipopro- tein C II, and the Egr1 genes (Table 1) (33–36). The pre- proapolipoprotein gene promoter contains an EBS located 10 bp upstream of the TATA box (Fig. 1). The human serglycin promoter also contains an EBS at a position Ϫ75 to Ϫ71 from the RNA transcription start site (Fig. 1). Of interest, the Egr1 promoter contains multiple EBSs, two of those flank a CArG box, and a third site is flanked by two CArG boxes (Fig. 1). DNA sequences derived from these regulatory regions show specific binding with recombinant ETS1 and FLI1 proteins (see below). Significantly, the Egr1 gene was identified as a potential downstream cellular target gene of the ETS1 tran- scription factor using these two different methods, RNA differential display, and whole genome PCR. Consequently, in the next series of experiments, we focused our attention on the regulation of Egr1 promoter by ETS transcription factors. The EBSs in the Egr1, Human Serglycin, and Preproapo- lipoprotein Promoters Are Able to Bind Recombinant ETS Proteins. To validate the findings of our novel approaches to elucidate ETS targets, we tested the ability of recombinant ETS proteins to bind to the EBS located within the promoters FIG. 2. Interaction of ETS proteins with target promoters. (A) isolated by whole genome PCR. EMSAs were carried out using EMSA of ETS1 and FLI1 with Egr1 SREI and SREII. The ETS1 and synthetic oligonucleotides corresponding to the EBSs located FLI1 proteins were expressed in insect cells, and the cell extracts were in the Egr1, preproapolipoprotein, and human serglycin pro- prepared as described (19). Cell extracts (1 ␮g) contained ETS1 (lanes moters (Fig. 1). The results demonstrate that recombinant 1–8) and FLI1 (lanes 9–16) and SREI oligonucleotides (lanes 1–4 and ETS1 and FLI1 proteins bind to the ETS sites in Egr1, as well 9–12) and SREII oligonucleotides (lanes 5–8 and 13–16) (19). The binding was performed in the presence of excess oligonucleotides as serglycin and preproapolipoprotein promoters (Fig. 2). (lanes 2, 4, 6, 8, 10, 12, 14, and 16) and specific mAb (lanes 3, 7, 11, Although the ETS1 protein bound to both the Egr1–SREI and and 15). Arrows indicate protein–DNA complexes in the absence (I) Egr1–SREII with equal efficiency, the FLI1 protein showed or presence (II) of mAb. (B) EMSA of FLI1 with preproapolipopro- tein C II serglycin promoters. 32P-labeled EBS containing oligonucle- otides were incubated with FLI1 protein in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of competitor. Arrow indicates the binding of FLI1 to preproapolipoprotein C II and serglycin promoter EBSs.

much higher affinity for the dual EBS-containing Egr1–SREI (Fig. 2a). The ETS1 and FLI1 DNA interactions are specific because the complexes can be supershifted with specific mAb (lanes 3, 7, 11, and 15) and can be competed out in the presence of excess unlabeled oligonucleotides (lanes 2, 4, 6, 8, 10, 12, 14, and 16). Similarly, the FLI1 (Fig. 2b) and the ETS1 (data not shown) proteins also bound specifically to EBSs derived from preproapolipoprotein and serglycin promoters because the protein DNA interactions are effectively blocked with excess unlabeled oligonucleotides. These data illustrate that ETS proteins are able to bind to the EBSs present in Egr1, preproapolipoprotein, and serglycin promoter sequences. FIG. 1. Schematic representation of ETS target gene promoters. ETS Proteins Transactivate the Egr1 Promoter. Having The ETS target gene promoters (Egr1, preproapolipoprotein C II, and shown that ETS proteins bind the EBSs in the Egr1 promoter, serglycin) were identified by the whole genome PCR technique. Open we analyzed whether the ETS proteins are able to transactivate rectangles indicate the promoter regions. TATA boxes and EBSs are indicated. The preproapolipoprotein C II and human serglycin pro- the Egr1 promoter. Cells were cotransfected with the ETS1 or moters contain single EBSs whereas the Egr1 promoter contains FLI1 expression vector and the CAT construct containing multiple EBSs. Three of the EBS in Egr1 are part of serum response Egr1 SREI (Fig. 3a) or with the ETS expression vectors and elements SREI and SREII as indicated. a control CAT construct lacking Egr1 EBSs (28, 29). We Downloaded by guest on October 2, 2021 Biochemistry: Robinson et al. Proc. Natl. Acad. Sci. USA 94 (1997) 7173

FIG. 3. Transient transactivation of CAT reporter via binding to Egr1 EBS by ETS1 and FLI1 proteins. (A) Map of the CAT reporter plasmids. The pBLCAT2 was used to construct reporter plasmid that contained the Egr1 SREI. (B) CAT assays were carried out with plasmids containing CAT reporter gene linked to Egr1 SREI and either ETS1 or FLI1 expression vectors. The CAT activity was examined by thin layer chromatography. The reaction products were quantified by analyzing the TLC plates on an AMBIS radioisotopic imaging system. FIG. 4. Transactivation of Egr1 promoter linked to CAT gene. (A) Schematic representation of the CAT constructs containing serial observed more than a 5-fold greater transactivation of the deletions of promoter sequences (30). (B) Relative activity of each construct also has been shown. CAT assays were performed as CAT gene from the Egr1 promoter construct compared with described in Materials and Methods. The CAT activities were analyzed the control plasmid lacking EBS (Fig. 3b). Thus, the ETS by thin layer chromatography as described in legend of Fig. 3. Three proteins bound sequences within the Egr1 SREI and are able different concentrations of reporter vector were used, as indicated to transactivate transcription of the CAT reporter gene, fur- below each lane. CAT vectors p662 and p663 that lack the SREI are ther verifying our unique approach. inactive in promoter activation assays. The Egr1 Promoter Contains Multiple EBSs, One of Which Is in a Region Essential for Promoter Functionality. Mouse 14). Egr1 expression was induced in three independent clones Egr1 promoter-linked CAT reporter gene constructs, and of NIH 3T3 cells that had been transfected with ETS1 (Fig. 5). similar constructs with several deletions in the promoter This effect was specific for ETS1 because Egr1 expression was region (Fig. 4a), were tested for expression in NIH 3T3 and either undetectable or weakly detectable in clones transfected COS cells (28). The largest promoter fragment (p903) and one with ETS2 (Fig. 5). Thus, these data demonstrate that ETS1 that contained 667-bp sequences 5Ј of the transcription initi- expression is sufficient to effect induction of Egr1 expression ation site (p667) were able to strongly effect CAT reporter in NIH 3T3 cells. In contrast to Egr1, however, elevated expression (Fig. 4b). Both of these fragments contained two expression of CBF occurs in both ETS1 and ETS2 transfec- intact, serum response elements [note that the serum response tants (Fig. 5). On the other hand, the PLA2P gene product was elements contained EBS(s) together with a CArG box(es)]. Of found to be expressed only in the ETS2-expressing cell lines, interest, a promoter fragment (p666) that had one SRE that is, neither in the ETS1-expressing cells nor in nontrans- disrupted by a deletion of the CArG box, together with the 5Ј fected control NIH 3T3 cells, where endogenous ETS gene EBS, resulted in a dramatic reduction in promoter function- expression is low or absent. Thus, in the ETS1- and ETS2- ality (Fig. 4). Further deletion of the promoter resulted in a expressing cells, these data consistently identify Egr1 promoter total loss of promoter functionality. These data demonstrate as a putative downstream cellular target gene for the ETS1 that SREI (a CArG box flanked by two EBSs, compare to. Fig. transcription factor, PLA2P as a target for the ETS2 transcrip- 1) is necessary for significant transactivational activity of the tion factor, and the CBF gene as a target for both of these ETS Egr1 promoter and that deletion of these site(s) results in a loss proteins. of most of the promoter activity. The SREII site, on the other hand, appears to be insufficient by itself to effect promoter DISCUSSION activity function because the Egr1 promoter shows no effect using a construct that contained only an intact and presumably The aim of this study was to reliably identify downstream functional SREII site. cellular target genes that are either directly or indirectly ETS1 and ETS2 Induce the Expression of ETS Target Genes regulated by the ETS transcription factors. Our initial ap- Egr1, CBF, and PLA2P in NIH 3T3 Cells. To study the proach applied the novel use of RNA differential display regulation of candidate ETS target genes identified by differ- analyses of NIH 3T3 cells transfected with vectors expressing ential display PCR and whole genome PCR techniques, we ETS1 or ETS2 transcription factors, respectively, and com- compared, by Northern blot analyses, the expression of Egr1, pared these to appropriate controls. The up-regulation of CBF, and PLA2P in ETS1-transfected NIH 3T3 cells to ones genes specifically in ETS1 transfectants was indicative of bona that had been transfected with ETS2 and parental controls (12, fide downstream cellular targets rather than genes whose Downloaded by guest on October 2, 2021 7174 Biochemistry: Robinson et al. Proc. Natl. Acad. Sci. USA 94 (1997)

and FLI1 are also found to be expressed in hematopoietic cells, including activated T cells (41). Taken together, these obser- vations suggest that the ETS1 and FLI1 transcription factors play an essential role in the regulation of human and mouse serglycin genes. The promoter of the preproapolipoprotein C II gene has an optimal EBS, containing the seven residues that are identical to the murine sarcoma virus–long terminal re- peat; these sequences were originally used to establish the ETS1 as a sequence-specific DNA binding protein (42). Analysis of the Egr1 promoter revealed two SREs (SREI and SREII), each containing CArG box(es) contiguous with EBS(s). The two SREs had different configurations; the distal one contained a CArG box flanked by two EBSs (SREI) whereas the proximal one contained a single EBS flanked by two CArG boxes (SREII). Deletion analysis demonstrated that the SREI is necessary for promoter function because removal of just this SRE resulted in a dramatic loss of promoter activity. It is important to note that this dramatic loss in promoter activity of p666 construct was achieved solely by the deletion of the most 5Ј EBS and the CArG box of the SREI element. These findings provided further evidence for the Egr1 as a cellular target of an ETS family transcription factor. The finding that ETS1 binds to and transactivates transcription from the Egr1-SREI suggests that Egr1 is a cellular target of ETS1. Further support for this comes from the data showing that Egr1 expression is up-regulated only in ETS1-expressing, and not in ETS2-expressing, NIH 3T3 cells. SREs regulate the expression of various immediate early FIG. 5. Northern blot analysis of ETS target . RNA genes, including c-fos, Egr1, and pip92 (32, 43, 44). EMSAs was prepared from a control NIH 3T3 cell line and from two ETS2- demonstrated the ability of ETS1 and FLI1 to bind the EBSs and three ETS1-derived soft agar clone cell lines. Total RNA (10 ␮g) was loaded in each lane. For RNA integrity, the ethidium bromide located within the Egr1 SREs. This finding is intriguing staining of the gel before transfer onto nylon membrane is shown in because data currently in the literature suggest that the ETS the lowest panel. The blot was probed sequentially with Egr1, CBF, proteins (ELK1 and SAP1a) do not form binary complexes and PLA2P probes. with c-fos SRE and require SRF to form ternary complexes (3, 43). In this study and recently, we have shown that ETS expressions change as a consequence of transformation be- proteins such as ETS1, ETS2, FLI1, EWS-FLI1, ELK1, and cause the latter would also be up-regulated in both ETS1 and SAP1a can form binary complexes with the Egr1 SREs and ETS2 transfectants. The differential display strategy allowed that ELK1, SAP1a, FLI, and EWS-FLI1 also can form ternary us to identify target genes that are either directly or indirectly complexes with the Egr1 SREs (45). It is possible that all ETS regulated by ETS1 or ETS2 transcription factors. By this proteins may be capable of binding to specific SREs; however, method, we isolated 16 previously unknown and three known some of these binding interactions will be dependent and genes (Egr1, CBF, and PLA2P). The second whole genome modulated by SRF, and others will be independent of inter- PCR approach used the binding of genomic DNA fragments actions with SRF. However, the issue of which specific ETS with the recombinant ETS1 protein to isolate target promoters family member binds to specific SRE depends, perhaps, on the that are directly regulated by ETS transcription factors. By this context of specific sites (EBS or CArG) located within a given method, we were able to clone 43 gene regulatory fragments. promoter. This is supported by our previous finding that spatial Three of those clones contained inserts that corresponded to configurations of EBSs within the promoter influence the the promoter regions of Egr1, preproapolipoprotein C II, and specificity with which individual ETS family proteins bind human serglycin genes. Important to note, Egr1 was identified these sequences (27). In addition, our data demonstrate that by both the methods independently, indicating by its consis- FLI1 can form ternary complex on the Egr1 SRE but not on tency that it is a bona fide ETS transcription factor target gene. the c-fos SRE (45). However, ELK1, SAP1a, and EWS-FLI1 Human serglycin is a proteoglycan that is stored in secretory can form ternary complexes on both the Egr1 and c-fos SREs. granules of many hematopoietic cells and is involved in its These findings suggest that the Egr1 promoter is regulated differentiation (37). The 5Ј flanking regions of human and stringently and, depending on the cell types, it may be regu- mouse serglycin genes have been characterized and show 96% lated by different ETS proteins in a SRF-dependent or -inde- identity in the 119-bp portion just upstream of the transcrip- pendent manner. tion start sites (38). Both of the promoters lack TATA boxes, Similar to SRF, CBF binds to CArG boxes found in different but the GC-rich areas, however, contain cAMP response promoters, acting as a transcriptional repressor on smooth element binding͞ATF (residues Ϫ70 to Ϫ65) and glucocorti- muscle ␣-actin genes (32). Significantly, there is an EBS site coid (residues Ϫ63 to Ϫ58) binding sites. By whole adjacent to the CArG box in the regulatory region of smooth genome PCR approach, we identified an EBS in the 5Ј flanking muscle ␣-actin gene. It would be interesting therefore to region (residues Ϫ75 to Ϫ80) of the human serglycin gene, explore the possibility of protein–protein interaction between which also is conserved in the mouse serglycin promoter CBF and various ETS factors. region, suggesting that this site is important for transcriptional In this paper, we demonstrate that ETS1 is able to regulate regulation. The expression of the human serglycin gene was expression of EGR1, which has been shown to bind to defined shown to be up-regulated in a number of human leukemic cell DNA sequences, thereby regulating protooncogenes, genes lines, ones that coincidentally have been shown to express high encoding mitogens, and mitogen receptors that are involved in levels of ETS1 and FLI1 (39, 40). In addition, we demonstrated cell growth and transformation (46). Up-regulation of EGR1 that this site is functional in EMSAs with ETS1 and FLI1 may therefore be an important step in transformation of NIH proteins. Furthermore, similar to human serglycin, the ETS1 3T3 cells transfected with ETS1. Taken together, our results Downloaded by guest on October 2, 2021 Biochemistry: Robinson et al. Proc. Natl. Acad. Sci. USA 94 (1997) 7175

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