The Epithelium-specific ETS Protein EHF/ESE-3 Is a Context-dependent Transcriptional Repressor Downstream of MAPK Signaling Cascades*

Antonio Tugoresa, Jennifer Le, Irina Sorokinab, A. J. Snijdersc, Mabel Duyaod, P. Sanjeeva Reddye, Leone Carléef, Mathew Ronshaugeng, Arcady Mushegianh, Tim Watanaskuli, Sunny Chud, Alan Bucklerd, Spencer Emtageb, and Mary Kay McCormickj

From Axys Pharmaceuticals, Inc., South San Francisco, CA 94080 and the g Department of Biology, University of California at San Diego, La Jolla, California 92093

Received for publication, December 4, 2000, and in revised form, February 23, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Exon trapping and cDNA selection procedures were used to search for novel genes at human chromosome 11p13, a region previously associated with loss of heterozygosity in epithelial carcinomas. Using these approaches, we found the ESE-2 and ESE-3 genes, coding for ETS domain-containing transcription factors. These genes lie in close proximity to the catalase gene within a ~200-kilobase genomic interval. ESE-3 mRNA is widely expressed in human tissues with high epithelial content, and immunohistochemical analysis with a newly generated monoclonal antibody revealed that ESE-3 is a nuclear protein expressed exclusively in differentiated epithelial cells and that it is absent in the epithelial carcinomas tested. In transient transfections, ESE-3 behaves as a repressor of the Ras- or phorbol ester-induced transcriptional activation of a subset of promoters that contain ETS and AP-1 binding sites. ESE-3-mediated repression is sequence- and context-dependent and depends both on the presence of high affinity ESE-3 binding sites in combination with AP-1 cis-elements and the arrangement of these sites within a given promoter. We propose that ESE-3 might be an important determinant in the control of epithelial differentiation, as a modulator of the nuclear response to mitogen-activated protein kinase signaling cascades.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Intracellular signaling through mitogen-activated protein (MAP)1 kinases is a universal mechanism controlling multiple aspects of cell division, migration, and differentiation (1). Among the known nuclear effectors downstream of MAP kinase signaling cascades are members of the ETS family of transcription factors, a group of proteins that share a conserved DNA binding domain, known as the ETS domain, and that are only found in metazoans. ETS transcription factors have been implicated as positive and negative regulators in signal transduction pathways that control a number of cellular processes, including differentiation, proliferation, cellular migration or invasion, and inflammation, and some of these factors are indeed direct targets of MAP kinases (2-5).

The biological role of ETS proteins in the integration of signals from MAP kinases has been best characterized in Drosophila melanogaster. Genetic and biochemical studies have led to the identification of two ETS proteins, Pointed and Anterior Open (Aop)/Yan, as pivotal components in the nuclear integration of MAPK signaling cascades (6-10). The activity of Pointed P2, a product of the pointed gene (11), and a putative ortholog of the vertebrate ETS-1 and ETS-2 proteins (12, 3), is dependent upon direct phosphorylation by MAP kinases. On the other hand, Aop/Yan has been described as a repressor of Pointed-responsive genes (6-10) and is also a downstream target of MAP kinase cascades. No mammalian epithelial ortholog of Aop/Yan has been identified yet. It has been proposed that phosphorylation of Aop/Yan by MAP kinases results in its inactivation and nuclear export, thus alleviating repression and allowing the transcriptional activation of Pointed target genes (6). The balance between the activity of these two transcription factors appears to modulate the specificity of the nuclear response to MAPK signaling cascades.

ETS proteins have also been shown to interact with each other and with other structurally unrelated transcription factors (see Refs. 3, 4, 13, and 14 and references therein), thereby providing additional complexity to their role as transcriptional regulators. In particular, the interaction of ETS proteins with Jun has significant relevance, since ETS binding sites appear in the vicinity of AP-1 cis-elements in the promoters of a number of genes involved in cell proliferation and migration, including those of extracellular proteases. This functional interaction results in the transcriptional activation of these genes in response to both extracellular signal-regulated kinase and Jun N-terminal kinase/stress-activated protein kinase MAP kinase signaling cascades (3, 15-17). Paradoxically, similar regulatory elements are present in the promoters of protease inhibitors and genes expressed during epithelial cell differentiation (18-23), raising the question of how specificity is achieved at the nuclear level. It is plausible that members of the ETS family may be important elements in the generation of target gene specificity. In this regard, transcription factors belonging to a specific group within the ETS family of proteins, such as ESE-1/ESX/JEN/ERT/ELF-3, ELF-5/ESE-2, and more recently EHF/ESE-3, have been implicated in the control of the transcription of epithelial differentiation genes (21, 24-32). The presence of various members with diverse binding specificities and the distinct expression patterns within the ESE group could be important for the selective control of the expression of epithelial target genes by these ETS transcription factors.

Elevated levels of ETS proteins have been also associated with cellular transformation (30, 33). Cancer is the result of a multistep process that is commonly associated with the loss of function of genes required for the maintenance of nonproliferative, differentiated cell fates and whose loss would facilitate tumor survival and/or metastasis. One of the mechanisms that may lead to the haploinsufficiency of such regulatory genes is the deletion of genomic regions containing these genes as a result of the increased genomic instability associated with the transformation process, a mechanism known as allelic loss of heterozygosity (LOH) (34).

We have performed an extensive search for genes at human chromosome 11p13, in a region that has been previously associated with allelic loss of heterozygosity in epithelial carcinomas (see Refs. 34-37 and references therein). In particular, LOH at this region is associated with increased metastatic potential, thus suggesting that this region contains genes required for the control of cell migration (36). As a result of this search, we have found the genes coding for two epithelium-specific ETS transcription factors, which we initially identified as I and J in our study (38) and have been later reported as ELF5/ESE-2 and EHF/ESE-3, respectively (26-28, 31). The analysis of EHF/ESE-3 revealed that the expression of this transcription factor is restricted to differentiated epithelial cells, and remarkably, it is absent in epithelial carcinomas originated from the same tissues where this factor is expressed normally. EHF/ESE-3 is a transcriptional repressor of a subset of ETS/AP-1-responsive genes, including the interstitial collagenase gene (matrix metalloproteinase 1; MMP-1) promoter. We suggest that EHF/ESE-3 might be an important regulatory factor in the maintenance of a differentiated phenotype in epithelial cells by modulating the nuclear response to MAP kinase signaling cascades.

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Cloning of Genes in Chromosome 11p13-- To generate a physical map for the 11p13 region, we began with searches of publicly available information sources for yeast artificial chromosome (YAC) clones that best represented the region. Queries of the Whitehead Institute Genome Center's physical map data base yielded multiple CEPH mega-YAC clones that were detected by genetic markers from the region. Each clone was analyzed by STS content mapping with all available markers by pulsed field gel electrophoresis and blotting and by fluorescent in situ hybridization to metaphase chromosomes. This extensive analysis led to the establishment of a minimal tiling path of clones that appeared to be nonchimeric, stable, and free of deletions. The resulting ~4-Mb YAC contig encompasses an 8-9-centimorgan interval defined by D11S935 and D11S1776. To increase the map's resolution and utility for gene identification experiments, the minimal set of YACs was then used to identify plasmid-based genomic clones corresponding to the region. DNA samples from YACs purified by pulsed field gel electrophoresis were radiolabeled and used to screen high density filter grids of a human total genomic DNA BAC library (Research Genetics, Huntsville, AL), as well as a human chromosome 11-specific cosmid library (39). A subset of the detected clones could subsequently be ordered and oriented by STS content mapping. Overlap relationships between these clones were defined using DIRVISH, and gaps in the contigs were closed by end walking.

BAC clones covering the region were subjected to exon trapping (40) and cDNA selection rounds using a commercial kit (CLONTECH, Palo Alto, CA) to search for coding sequences. Full-length cDNAs corresponding to exon-trapped products or selected cDNAs were cloned, sequenced, and assigned letters as they were identified. cDNA clones containing the full-length J ORF were obtained from a human prostate pDR2 cDNA library (CLONTECH).

Computer-aided Sequence Analysis-- Partitioning of protein sequences into globular and nonglobular segments was performed using the local compositional complexity measures implemented in the SEG algorithm run with the following optimized parameters: window length, 45; trigger complexity, 3.4; extension complexity, 3.8 (41). Protein data base searches were performed using the PSI-BLAST program (42).

Phylogenetic analysis was performed on a continuous 127-amino acid data set derived by concatenating the conserved ETS and SAM/SPM domains. Positions containing gaps within these domains were deleted sitewise. Hypotheses of phylogenetic relationships were constructed using neighbor joining, a distance-based method, and the maximum likelihood methods. Support for neighbor joining topologies was evaluated using bootstrap analysis with 1000 replicates, and support for maximum likelihood tree interference utilized quartet puzzling reliability values from 10,000 puzzling steps (analogous to bootstrap replicates) (43). The distance matrix in the neighbor joining tree construction was calculated by two methods: the simple proportional difference and Lake's paralinear distance because of its robustness of interference on data sets having significantly unequal rate effects (44). The calculation of the distance matrices, neighbor joining interference of tree topology, and the subsequent bootstrap analysis were implemented in DAMBE (45). The neighbor joining tree derived from the maximum likelihood distance matrix was used to compute the parameters for the models of substitution and rate heterogeneity. A model of rate heterogeneity with one invariant and eight gamma -distributed rate categories was used.

DNA Clones-- A cDNA clone containing the full-length J (EHF/ESE-3b) ORF, was subjected to PCR to construct different clones containing a FLAG epitope either at the 5' (clone C7132) or at the 3' (clone F5) ends, that were subcloned into pRC/CMV (Invitrogen, Carlsbad, CA). To generate a full-length GST fusion bacterial expression construct (clone pGEX B4), a PCR fragment containing the full-length ORF was subcloned into the PGEX-20T vector, a derivative of PGEX-2T (Amersham Pharmacia Biotech). A similar approach was used to generate a His-tagged full-length construct in the pProEx-HTB vector (Life Technologies, Inc.). An ESE-1a/ESX expression vector was constructed by performing PCR on an expressed sequence tag containing the full-length ESE1a/ESX ORF (expressed sequence tag 78004; GenBankTM accession number AA 3667460), also inserting a FLAG epitope at the 5'-end, and subcloned into pRC/CMV. The murine ETS-2 and the oncogenic Ras 61L expression vectors, the human collagenase (MMP-1) (-1200 to +63), the (Py)2 Delta 56 dE Fos, (PyEts)2 Delta 56 dE Fos, and the (Py + 6F)2 Delta 56 dE Fos luciferase reporter constructs have been described previously (46-48). To make the TIMP-1 Delta 56 dE Fos luciferase reporter, the HT-1 sequence (see electrophoretic mobility shift assay probes below) was subcloned upstream of the Delta 56 dE Fos minimal promoter (48) driving the luciferase reporter gene. A -635 to +5 human matrilysin promoter fragment (49) was obtained through PCR amplification of total human genomic DNA and subcloned into a high copy number derivative of p19Luc (50).

Cell Culture, Transfections, and Luciferase Assays-- The colon HCT 15, COLO 205, prostate LNCaP FGC 10, and lung NCI H-1299 adenocarcinoma cell lines (ATCC, Manassas, VA) were cultured in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum, 10 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). COS-7 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented as above. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. Cells were kept at 37 °C, in a humidified atmosphere containing 5% CO2, 95% air.

Plasmids for transfections were isolated by alkaline lysis, RNase A treatment, and polyethyleneglycol precipitation (51), followed by a single centrifugation through a CsCl gradient. HCT-15 cells were transfected by using LipofectAMINE (Life Technologies) as recommended by the manufacturer. After 5 h, the transfection mixture was removed, and RPMI 1640 containing 0.5% fetal bovine serum was added to the cells. After 24 h, cells were treated with 10 ng/ml PMA for 12 h, and the luciferase activity of the cell extracts was analyzed in a 3010 Luminometer (Analytical Bioluminiscence, Ann Arbor, MI) using a commercially available reagent kit (Analytical Bioluminiscence). For the Ras 61L co-transfections, cell lysates were collected 24 h after the addition of fresh medium containing 0.5% fetal bovine serum. Relative luciferase units were normalized according to the protein concentration of the extracts. The transfection experiments shown were done in triplicate and repeated at least three different times.

Northern Analysis-- A full-length J cDNA fragment was radiolabeled with 32P using the Prime-It kit as recommended by the manufacturer (Stratagene, La Jolla, CA). Membranes containing polyadenylated human tissue mRNAs (CLONTECH) or 20 µg of total RNA extracted from epithelial cell lines were hybridized to the 32P-labeled DNA probe by using RapidHyb (Amersham Pharmacia Biotech) as recommended by the manufacturer. Filters were washed up to 0.1× SSC, 0.1% SDS at 65 °C (1× SSC: 0.15 M NaCl, 15 mM sodium citrate), analyzed in a Storm PhosphorImager using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA), and digital images were finally processed in Adobe Photoshop software (Adobe Systems, San Jose, CA) to elaborate the figures.

Recombinant Protein Production-- Bacterial expression constructs were transformed into the Escherichia coli strain BL21(DE3), and cultures at A600 = 0.5-0.6 were induced with 0.5 mM isopropyl-beta -thiogalactoside, for 4 h at 37 °C. His tag full-length ESE-3 was purified using the His·Bind resin and buffer kit, following the manufacturer's protocol (Novagen, Madison, WI). GST-full-length ESE-3 was purified on glutathione-Sepharose 4B resin according to the manufacturer's protocol (Amersham Pharmacia Biotech).

Generation of Monoclonal Antibodies-- Harlan Sprague-Dawley rats immunized with denatured purified His-tagged ESE-3 were tested for ESE-3-reactive antisera using Western blots. Rat 362, whose serum showed the highest immunoreactivity, was sacrificed, and spleen cells were fused to the SP2 mouse myeloma (ATCC) using standard procedures (52). Positive cloned hybridomas were selected by enzyme-linked immunosorbent assay and were further tested for specificity by performing sequential immunoprecipitations and Western blots (52) with the monoclonal antibodies (mAbs) and the anti-FLAG antibody (Sigma) on cell extracts from COS7 cells transfected with EHF/ESE-3b, ESE1a/ESX, and ETS-2 and by using immunocytochemical analysis on the prostate cell line LNCaP FGC 10. The mAb 5A5.5 was shown to detect EHF/ESE-3b with high specificity and did not show any cross-reactivity with the closest human homolog ESX/ESE-1. The epitope recognized by the 5A5.5 mAb is not included in either conserved domain (ETS or SAM) of the EHF/ESE-3b protein (not shown).

Immunohistochemistry-- Neutral buffered formalin-fixed, paraffin-embedded human tissues were sectioned (6 µm), baked on Superfrost glass slides (Fisher) at 65 °C for 2 h, rehydrated, and treated with antigen unmasking solution as recommended by the manufacturer (DAKO, Carpinteria, CA). Immunohistochemistry was carried out by using either the mAb 5A5.5-containing culture supernatant or the rat myeloma SP2 culture supernatant as a control, followed by incubation with a biotinylated mouse anti Rat mAb (DAKO) and a streptavidin-horseradish peroxidase conjugate (DAKO). Detection was performed with the Immunopure Metal Enhanced DAB Substrate Kit (Pierce), and after the detection reaction was completed (10 min to 2 h), slides were subjected to a light hematoxilin stain (Harris hematoxilin 1:10 in water for 20 s), rinsed in tap water, dehydrated, and mounted with DPX (Fluka, Buchs, Switzerland). Digital images were captured with a Polaroid DMC1 digital camera (Polaroid, Cambridge, MA) and imported into Adobe Photoshop software (Adobe Systems) to elaborate the figures.

Electrophoretic Mobility Shift Assays-- Nuclear protein extracts were prepared, and electrophoretic mobility shift assays were performed essentially as described (53). After electrophoresis, gels were dried and analyzed as described above for Northern blots. The sequences for the electrophoretic mobility shift assay probes containing the ETS sites on the ESE-3 promoter and their variants are shown in Fig. 5D. The full sequence of the ETS-AP-1 tandem on the human TIMP-1 gene promoter (HT1) is TGGGTGGATGAGTAATGCATCCAGGAAGCCTGGAGGCCTGTGGTTT (sites are underlined) (18), and the ESE1a/ESX binding site at the neu/erb2 gene (ESX) was as described (30).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of ETS-related Genes at Human Chromosome 11p13-- As part of a genomic mapping and sequencing study (38), we used a combination of exon trapping and cDNA selection to isolate novel genes at 11p13 within a genomic interval centered on the catalase gene and flanked proximally by CD44 and distally by CD59. Two of the genes identified, initially termed I and J (38), potentially coded for ETS domain proteins. Because of the role of ETS domain transcription factors in the regulation of multiple processes including cell division, migration, and transformation (2-5, 33) and the known association of this region to LOH in several adenocarcinomas (see Refs. 34-37 and references therein), these genes were investigated in more detail. During the course of this study, I has been reported as ELF5 (31) and as ESE-2 (26), and J has been reported as EHF and ESE-3b (27, 28). For simplicity, we will refer to I as ESE-2, and to J as ESE-3.

ESE-2 and ESE-3 are transcribed in opposite directions ("head-to-head") and are located ~110 Kb apart, with ESE-3 being more proximal (Fig. 1). Their close proximity and sequence similarity suggest that these two genes arose as a result of ancestral gene duplication. The 3'-end of the catalase gene is located ~7 kb from the 3'-end of ESE-2. The microsatellite markers that define the ESE-3 locus are D11S2008, situated ~2.5 kilobase pairs upstream of exon A, and D11S907, on the second intron and at 0.3 kilobase pairs from exon C. Our sequence differs slightly from those reported previously, and it is available from GenBankTM under accession number AF 212848. 


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Fig. 1.   Physical map of the ESE-3 genomic region. Microsatellite markers are listed across the top. The lines immediately below the markers depict YAC clones. Below the YAC clones are BAC and cosmid clones. Genes are shown at the bottom, and the arrows indicate the direction of transcription.

Expression of the ESE-3-specific mRNAs-- Northern blot analysis showed two major ESE-3 mRNA transcripts of 5.6 and 1.3 Kb that presumably arise from alternative utilization of two different consensus polyadenylation signals. An additional transcript of 4.6 kb could also be detected. ESE-3 mRNAs were strongly expressed in a variety of tissues with significant epithelial content including trachea, colon, pancreas, prostate, and lung and were absent or below the level of detection in nonepithelial tissues (Fig. 2A), showing a wider distribution than that previously reported for EHF or ESE-3 (27, 28). From the tissues analyzed, ESE-3 mRNAs appeared to be more widely expressed in tissues with high epithelial content than ESE-I/ELF5/ESE-2 (not shown).


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Fig. 2.   Expression of the ESE-3 mRNA. The expression of ESE-3 mRNAs was analyzed by Northern blot hybridization on human tissues (A) and the epithelial cell lines COLO 205, LNCaP FGC 10, HCT-15, and NCI-H1299 (B). The migration of the molecular size standards is shown on the left. The arrows indicate the three major mRNA transcripts observed.

Expression analysis in epithelial cell lines showed that the prostate carcinoma cell line LNCaP FGC 10 expressed high levels of ESE-3 mRNAs, followed by the colon adenocarcinoma cell lines COLO 205 and HCT 15. ESE-3 mRNA transcripts were absent or below the level of detection in the lung adenocarcinoma cell line NCI H-1299 (Fig. 2B).

ESE-3 Is a Nuclear Protein Expressed in Differentiated Epithelial Cells-- To evaluate the expression of the ESE-3 protein, a mAb was generated (see "Experimental Procedures" for details). Immunoprecipitation and Western blot analyses showed that the ESE-3 protein migrated as a Mr = 33,000 single polypeptide in both LNCaP FGC 10 and ESE-3-transfected COS-7 cell protein extracts, and it was absent in mock-transfected COS 7 cell extracts (not shown). Immunohistochemistry analysis on several human tissues revealed that ESE-3 expression was restricted to the nuclei of highly differentiated epithelial cells, particularly those with secretory functions (Fig. 3). Expression was highest in the serous and mucous glands, and secretory ducts of the airways (Fig. 3, A and B), the secretory epithelium of the seminal vesicle (Fig. 3C), the salivary glands (Fig. 3D), and the mucus-producing cells in the crypts of Lieberkuhn in the colon (Fig. 3, E and F). Moderately high levels of expression were also observed in the basal cells and to a lesser extent in the secretory epithelium of the prostate (Fig. 3G).


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Fig. 3.   ESE-3 is a nuclear protein expressed in differentiated epithelial cells. Human tissues were subjected to immunohistochemical analysis by using the ESE-3-specific monoclonal antibody 5A5.5. Brown staining reveals immunoreactivity, and blue staining corresponds to hematoxilin nuclear stain. A, bronchial columnar lining epithelium (top) and secretory ducts; B, bronchial secretory ducts and mucous and serous glands; C, seminal vesicle; D, salivary glands; E, colon lining and crypts of Lieberkuhn; F, crypts of Lieberkuhn (transversal section); G, prostate gland; H, esophagus stratified epithelium. Bar, 100 µm.

In the squamous nonkeratinized epithelium of the esophagus, the expression of ESE-3 was evident in the differentiating layers, while the basal cells, undergoing active proliferation, were negative (Fig. 3H). Expression of ESE-3 could be also detected in the columnar epithelial lining of the airways (Fig. 3A), and it was absent or below the level of detection in the lining epithelium of the colon (Fig. 3E).

ESE-3 Expression Is Lost during Epithelial Carcinogenesis-- Previous reports have shown an association between oncogenesis and elevated levels of expression of ETS proteins (see Refs. 3, 30, 33, and references therein). However, ESE-3 expression was shown to be restricted to differentiated epithelial cells and absent in proliferating cells. This was clearly evident in the expression pattern observed in the squamous epithelium of the esophagus (Fig. 3H). Additionally, expression of ESE-3 in the prostate was highest in the basal cells, a cell type that is lost in malignant prostate carcinomas (54). Consistently, we observed loss of ESE-3 positive cells in malignant prostate carcinomas (not shown).

Based these observations, ESE-3 immunoreactivity was monitored in other epithelial carcinomas. Using the ESE-3-specific mAb, we analyzed protein expression in samples of bladder adenocarcinoma (Fig. 4A), oral epithelium carcinoma (Fig. 4B), and breast ductal carcinoma (C and D). As shown, ESE-3 protein expression was restricted to the normal epithelium and was clearly excluded from the transformed epithelium. Both normal and transformed epithelium were evident in all samples, allowing us to exclude sample quality or processing as an explanation for the loss of immunoreactivity.


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Fig. 4.   ESE-3 immunoreactivity is lost during carcinogenesis. Sections from bladder (A) and oral epithelial (B) adenocarcinomas and breast ductal carcinoma (C and D) containing normal tissue adjacent to the carcinoma cells were subjected to immunohistochemical analysis by using the ESE-3-specific monoclonal antibody 5A5.5. The black arrows indicate the normal ESE-3-expressing epithelium, while the red arrows show the transformed epithelium, which does not show ESE-3 immunoreactivity. Bar, 100 µm in A and B and 50 µm in C and D.

The ESE-3 Protein Binds Selectively to a Small Subset of ETS Binding Sites-- Since a number of transcription factors regulate their own transcription, we examined the ESE-3 promoter for putative ETS binding sites. Two potential ETS binding sequences were found: one at -240 and another at -730 nucleotides from the major transcription start site.2 Both His tag (Fig. 5A, lane 1) and GST-full-length ESE-3 (Fig. 5A, lane 3) could bind the -730 site with high affinity, as evidenced by a competition profile using cold oligonucleotide as competitor (not shown). The mobility of these nucleoprotein complexes was affected by the addition of an anti-ESE-3 polyclonal antiserum (alpha 362; see "Experimental Procedures" for details) (Fig. 5A, lanes 2 and 4).


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Fig. 5.   DNA binding specificity of the ESE-3 protein. A, electrophoretic mobility shift assays were used to monitor the binding of E. coli expressed His-tagged full-length ESE-3 (His-ESE-3; lanes 1 and 2) or GST-full-length ESE-3 (GST-ESE-3; lanes 3 and 4) fusion proteins (10-100 fmol) to a 32P-labeled double-stranded oligonucleotide (40 fmol; 3 nM final concentration) containing the ETS consensus binding site present at position -730 on the ESE-3 gene promoter. Specificity of the nucleoprotein complexes was determined by using the 362 rat antiserum against ESE-3 (362 alpha S; lanes 2 and 4). B, binding of E. coli expressed and purified GST-ESE-3 to double-stranded oligonucleotides containing the following: the ETS consensus sites at -240 (240; lane 1) and at -730 (730; lanes 2 and 6) on the ESE-3 gene promoter; a variant of the -730 site (730 m1; lane 3); the ETS and AP-1 sites on the human TIMP-1 gene promoter (HT-1; lane 4); the ESE1a/ESX binding site at the human NEU gene (ESX; lane 5); and the variants of the 730 sequence (730 m2 (lane 7), 730 m3 (lane 8), 730 m4 (lane 9), and 730 m5 (lane 10)). C, detection of ESE-3 binding activity in the prostate LNCaP FGC 10 epithelial cell line. Twenty µg of nuclear proteins were incubated with the 730 m2 32P-labeled oligonucleotide in the presence of the 362 rat anti ESE-3 antiserum (362 alpha S, lane 2), preimmune serum (362 PI; lane 1), or a 50-fold molar excess of cold competitor oligonucleotide (lane 3). The arrows show the nucleoprotein complexes that are reactive to the anti-ESE-3 antiserum. D, sequences of the probes used, relative binding affinities, and proposed consensus.

A number of natural known ETS binding sites were compared with the -730 site and with several sequences derived from the -730 site by single nucleotide substitutions. Full-length GST-ESE-3 displayed different relative affinities for the sites tested (Fig. 5B), suggesting the consensus A > G:C > G:A:G:G:A:A:G:T, as a high affinity binding site for ESE-3 (Fig. 5D).

To assess the binding of native ESE-3 protein, nuclear extracts from the LNCaP FGC 10 prostate epithelial adenocarcinoma were tested for binding to the -730 m2 probe (see below) and showed a number of nucleoprotein complexes (Fig. 5C, lane 1). The addition of the anti-ESE-3 polyclonal serum to the binding reactions selectively altered the migration of two nucleoprotein complexes (Fig. 5C, lane 2). The migration profile of these DNA-protein complexes was similar to that of purified His-tagged ESE-3 (Fig. 5A, lane 1), indicating that both bands arose from a single polypeptide. The slower migrating complexes, which were not recognized by the anti ESE-3 serum, were shown to bind specifically to the -730 m2 sequence, and they did not appear when the -730 m1 oligonucleotide was used as a probe in the binding reactions (not shown). Thus, these complexes may result from the binding of other ETS proteins present in the extracts. The band near the top of the gel only appeared in the lanes that contained preimmune serum and not in those containing antiserum or no serum at all (not shown), indicating that it is an artifact caused by the preimmune serum.

ESE-3 Is a Context-dependent Transcriptional Repressor-- The polyoma virus enhancer ETS site contains a very similar sequence to the one that we experimentally determined as a high affinity binding site for ESE-3 and is contained within a regulatory cis module termed the oncogene response element (ORE). Transcription from the ORE is synergistically stimulated through the cooperation of ETS transcription factors and AP-1 in response to Ras signaling (48). To analyze the transcriptional role of ESE-3 in the ORE-mediated transcription, HCT-15 colon adenocarcinoma cells were transfected with an ESE-3 expression vector and the (Py)2 Delta 56 dE Fos luciferase reporter, either in the presence or the absence of an oncogenic Ras (Ras 61L) expression construct. The (Py)2 Delta 56 dE Fos luciferase reporter contains two ORE modules upstream of a minimal Fos promoter driving the luciferase gene (48). HCT-15 cells were used in this study due to their low, albeit detectable, endogenous levels of ESE-3 mRNA expression (see Fig. 2B). As shown (Fig. 6A), co-expression of ESE-3 repressed the oncogenic Ras-mediated transcriptional activation of the polyoma virus ORE-driven reporter. Similar results were obtained when a constitutively active membrane-targeted form of the nucleotide exchange factor SOS (55) was co-transfected instead of the oncogenic Ras mutant (not shown).


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Fig. 6.   ESE-3 represses the Ras- and the phorbol ester-dependent trans-activation of ETS/AP-1-responsive genes. HCT-15 colon adenocarcinoma cells were transfected with either (Py)2 Delta 56 dE Fos luciferase (A) or the human collagenase Luc (B) luciferase reporter constructs (1 µg/30 mm dish) and with eucaryotic expression constructs encoding murine ETS-2 (hatched bar) (0.5 µg/30-mm dish) or human ESE-3 (clone pRC/C7132; 0.5 µg/30-mm dish) (gray bar) in the presence of a Ras 61L expression construct (0.5 µg/30-mm dish) where indicated. HCT 15 cells were also transfected with increasing amounts of the ESE-3 (C-I) or ESE-1/ESX expression construct (J) (0.1 µg, hatched bar, and 0.5 µg, gray bar) as indicated, together with the (Py)2 Delta 56 dE Fos luciferase (C), human intersticial collagenase (MMP-1) (D), TIMP-1 Delta 56 dE Fos luciferase (E), matrilysin (F), (Py + 6F)2 Delta 56 dE Fos luciferase (G), Py + 6F Delta 56 dE Fos luciferase (H), and (PyEts)2 Delta 56 dE Fos luciferase (I and J) luciferase reporter constructs and treated with PMA where indicated. Luciferase activity was measured from cell lysates, and values were normalized to the protein concentration of the extracts. Experiments shown are representative of three independent experiments, all of them performed in triplicate. Error bars, S.D.

The human interstitial collagenase (MMP-1) promoter has been also shown to be a Ras-responsive promoter by virtue of the presence of ETS and AP-1 cis-elements, which act synergistically to activate Ras-dependent transcription of the gene (15, 16). Similarly to what was observed previously with the synthetic ORE reporter, ESE-3 was able to repress the -1200 to +63 collagenase promoter response to Ras (Fig. 6B). In both cases, expression of ETS-2 resulted in enhanced expression of the reporter in these cells (Fig. 6, A and B).

Similar results were obtained when the tumor promoter PMA was used as an activator of the transcription of the reporter constructs. Phorbol esters activate protein kinase C, which in turn is able to phosphorylate Raf leading to activation of the MAPK cascade (56). ESE-3 repressed, in a dose-dependent manner, the PMA-induced transcriptional activation of both reporters (Fig. 6, C and D). Co-transfection of ETS-2 in this setting resulted neither in an increase nor in a decrease of luciferase activity (not shown).

To determine the specificity of the transcriptional repression mediated by ESE-3, other ETS/AP-1-responsive promoters were analyzed. Expression of ESE-3 did not repress transcription stimulated by the tandem ETS/AP-1 from the human TIMP-1 gene promoter cloned upstream of the minimal Delta 56 dE Fos promoter (Fig. 6E). This is consistent with the low affinity of ESE-3 for the ETS cis-element present in the TIMP-1 promoter (see Fig. 5).

The epithelium-specific metalloprotease matrilysin (MMP-7) also contains ETS and AP-1 cis-elements in its promoter, and its expression, like that of MMP-1, is also induced by PMA, tumor necrosis factor-alpha , interleukin-1, and epidermal growth factor (16, 49). The activity of matrilysin has been associated with normal epithelial cell migration, although it is also expressed at high levels in nonmigrating glandular epithelial cells (57), which also express ESE-3 (this study). In agreement with their tissue co-localization, we found that ESE-3 was unable to repress transcription from the matrilysin promoter (Fig. 6F) despite the presence of a high affinity binding site for ESE-3, suggesting that repression might be context-dependent.

The context-dependent repression by ESE-3 is further confirmed by deletion of one tandem of ETS/AP-1 sites or by mutation of the AP-1 cis-elements in the context of the ORE reporter. Because it is impossible to eliminate the AP-1 site in the polyoma ORE without affecting the ETS site, since they overlap, we used the (Py + 6F)2 reporter containing the ETS/AP-1 tandem of the polyoma ORE, where the ETS and AP-1 binding sites are separated by 6 nucleotides (48). As shown, ESE-3 was still able to repress the PMA-mediated trans-activation of the (Py + 6F)2 reporter (Fig. 6G). Although (Py + 6F)2 reporter is, on average, 10-20-fold less active than the parental(Py)2 Delta 56 dE Fos luciferase reporter, we feel that, qualitatively, they both reflect similar results. It is also possible that ESE-3 repressed less efficiently in this setting. In any case, ESE-3 was unable to repress a reporter that contained a single copy of the Py + 6F module (Fig. 6H). Mutation of the AP-1 cis-elements in this context, represented by the (PyEts)2 Delta 56 dE Fos luciferase reporter (48), showed that repression was dependent on the presence of functional ESE-3 binding sites in combination with AP-1 cis-elements. In fact, ESE-3 then behaved as an activator at low concentrations (Fig. 6I), although this activity was very moderate in comparison with the related polypeptide ESE-1/ESX (Fig. 6J). On the other hand, synthetic promoters where the ETS binding sites were eliminated in this context, leaving multimers of the AP-1 cis-elements (3×AP-1, and 6×AP-1; see Ref. 48), were not repressed by ESE-3 (not shown), indicating that DNA binding to cognate cis-elements is essential to mediate this activity.

Previous reports indicate that overexpression of ETS-2 may lead to transcriptional repression (58). In our experiments, co-transfection of ETS-2 did not lead to a decrease of transcription in any of the cases analyzed (not shown). Western analysis also revealed that both the ETS-2 and the ESE-3 expression vectors were expressed at comparable levels (not shown), and therefore we excluded the possibility that the repression observed upon co-transfection with ESE-3 is due to nonspecific repression.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The genes coding for ELF5/ESE-2 and EHF/ESE-3 were found during an extensive search for genes localized at human chromosome 11p13 (38), in a region that has been previously associated with LOH in a subset of epithelial carcinomas (see Refs. 34-37 and references therein). In the case of some lung carcinomas analyzed, LOH at this locus has been associated with a higher patient mortality rate, a direct measurement of the tumors' metastatic potential (36), thus suggesting that this region may contain genes required for the control of cellular migration. Genetic mapping of this LOH has been mostly associated with the Catalase locus (35-37), which we found in close proximity to ESE-2, and ESE-3.

The expression of ESE-3 is widespread in tissues with high epithelial content. We show that ESE-3 mRNA has a much wider expression profile than previously reported (27, 28), and contains an additional 1.3-Kb mRNA species. We did not isolate the short splice variant reported by others, indicating that it was underrepresented in the library used. Using a newly generated specific monoclonal antibody, we show that the ESE-3 protein is specifically expressed in differentiated epithelial cells. This is most evident in the stratified squamous epithelium of the esophagus, where ESE-3 is absent in the basal proliferating stem cells, and appears as soon as they differentiate. Importantly, ESE-3 expression is lost in a number of epithelial carcinomas, including bladder, oral squamous, breast ductal (Fig. 4), and prostate epithelial carcinomas (not shown). Certainly, it can be argued that the decreased expression of ESE-3 is not an essential pathogenic event and that other oncogenic mechanisms lead to the loss of ESE-3 expression. Indeed, although our data are suggestive altogether, we do not have direct evidence correlating the loss of ESE-3 expression with cancer progression. Nonetheless, the restricted association of ESE-3 with differentiated epithelial cells indicates that, at least, could be a marker for malignancy, since the cell fate associated with ESE-3 expression appears to be no longer determined. Others have reported variable EHF/ESE-3 mRNA expression in several epithelial carcinomas analyzed (27), although increased mRNA expression does not necessarily have to correlate with elevated protein levels. This seems to be the case of the prostate LNCaP FGC 10 adenocarcinoma cell line, in which the ESE-3 mRNA levels are the highest among the cell lines tested, but the protein is barely detectable both in Western blots (not shown) and in mobility shift assays (Fig. 5). More tumor samples will have to be analyzed to clarify this issue.

Functionally, ESE-3 is a transcriptional repressor of a subset of genes that contain ETS and AP-1 cis-elements within their promoters and have been shown to be downstream targets that are positively regulated by MAP kinase signaling cascades. ESE-3-mediated repression of these promoters is dependent on the presence of high affinity binding sites, which we have defined in good agreement with those described by others (28). The restricted binding affinity of this repressor to a limited number of ETS cis-elements, combined with specific promoter requirements, indicates that ESE-3 is only a repressor for a specific subset of ETS/AP-1-responsive genes. These include the interstitial collagenase gene (MMP-1) promoter, also observed by others (27), and a dimer of the oncogene response element of the polyoma virus enhancer, both well characterized Ras-responsive genes. Alternatively, ESE-3 may also modulate the transcription of genes downstream of other pathways that are activated via phorbol esters and that are only secondarily linked to the main Ras pathway.

The restricted expression pattern of ESE-3 in the squamous stratified epithelium of the esophagus may provide insights into the physiological role of this repressor. Stratified epithelia have served as a paradigm for the study of epithelial differentiation (59). In this dynamic system, a population of self-renewing basal stem cells give rise to committed, nonproliferating precursors that undergo progressive differentiation as they are pushed toward the lumen, where they finally delaminate (60). The molecular mechanisms that govern the transition from a proliferating basal cell to a committed nondividing precursor are of great importance for deciphering the etiology of cancer. A failure to adopt the committed fate by the basal cell may lead to the development of adenomas or carcinomas, giving rise to most solid tumors.

Both autocrine and paracrine factors are known to maintain the proliferative potential of the basal cells. Some of these factors, such as transforming growth factor-alpha , keratinocyte growth factor, and fibroblast growth factors, are ligands for tyrosine kinase receptors and thus trigger the activation of signaling cascades involving MAP kinases. These signaling cascades are reiteratively used throughout the differentiation of these cells, since they activate the expression of differentiation markers in the upper layers of the epidermis (59). This implies that additional factors are required to modulate appropriate response (e.g. proliferation versus differentiation) to MAP kinase signaling cascades.

As mentioned earlier, transcription factors of the ETS protein family are good candidates as modulators of the nuclear response to MAP kinase signaling cascades. Among these, factors belonging to the ESE subgroup seem to play an important role in the activation of epithelium-specific genes (21, 24-29). Indeed, a number of genes whose expression is associated with epithelial cell differentiation contain ETS cis-elements within their promoters, thereby supporting this view (19-23). Both ESE-1 and ESE-2 are expressed at later stages of keratinocyte differentiation (21, 26), indicating that they are unlikely to play a role in the determination of the early differentiated spinous precursor in stratified squamous epithelia. Thus, it appears that, among the ESE family members described to date, ESE-3 is expressed the earliest in the differentiated precursors. Based on the functional analysis and the expression profile for this gene, as presented herein, we hypothesize that ESE-3 could play a role in the determination of the early differentiated fate in stratified squamous epithelia. Since ESE-3-mediated repression may only occur at a specific set of MAP kinase target promoters, while allowing the transcription of others, we conclude that ESE-3 might be an important factor in the nuclear specific response to MAP kinase signaling cascades.

The molecular mechanisms underlying the differentiation of secretory epithelial cells is less known, although recent reports indicate that epidermal growth factor receptor signaling is also involved in the determination of goblet cells in the airways and subsequent mucus production (61). The epithelium-specific ETS factors ESE-1/ESX/JEN/ERT/ELF3 and ELF5/ESE-2 and more recently also EHF/ESE-3 have also been implicated in the selective transcriptional activation of genes specific to glandular epithelia (26, 28). Further work will be needed to understand how the interplay between these genes orchestrates gene expression in secretory epithelia and their role (if any) in the induction of secretory fates.

The role of ESE-3 as a repressor resembles that described for Aop/Yan, an ETS protein in D. melanogaster (6). Additionally, ESE-3 and Aop/Yan not only share a similar structure but also recognize the same DNA sequences, since the polyomavirus enhancer in the context of the interstitial collagenase gene promoter has been used to demonstrate repression by Aop/Yan (62). However, ESE-3 does not have any consensus extracellular signal-regulated kinase phosphorylation sites; nor is it phosphorylated by extracellular signal-regulated kinase or Jun N-terminal kinase 1 in vitro,3 suggesting that, unlike Aop/Yan, ESE-3 may not be regulated post-translationally by direct MAP kinase phosphorylation.

The ESE family has been shown to contain two distinguishable domains, namely the ETS DNA binding domain at the carboxyl terminus and an additional domain that has been recognized in some ETS-domain proteins as the A domain, or the Pointed domain (after the Drosophila ETS protein Pointed; reviewed in Ref. 3), located at the amino terminus. Recent detailed analysis of conserved motifs and comparison of three-dimensional structures established the similarity of the A domain to the SAM/SPM (sterile alpha  motif/scm-polyhomeotic motif) domains, also involved in protein-protein interactions (3, 63). In the case of the ETS proteins belonging to the TEL subgroup, this domain has been shown to mediate homotypic interactions (14, 64), which are required for repression (65-67), but we have not observed such a role in the case of ESE-3.3

The semiautonomous nature of the SAM/SPM domain and its apparent rapid and diverse evolution (68) impose a challenge in defining the phylogenetic relationship between ESE-3 and related ETS proteins in other species. In particular, the ETS domain sequence in Drosophila with highest similarity to that of ESE-3 is found in E-74 (GenBankTM accession number AAA28493), which does not contain a SAM/SPM domain. It is for this reason that we do not favor the ELF nomenclature (E-74-like factor) for this group. Phylogenetic analysis of the ETS family members that contain ETS and SAM/SPM domains using both domains in the analysis clearly indicates that Aop/Yan and the TEL proteins are members of a monophyletic group (66, 28) also containing the ESE and the PDEF/ETS 98B families. This clade of ETS proteins clusters to the exclusion of a second monophyletic group containing Drosophila Pointed P2 and its vertebrate orthologs c-ETS-1 and c-ETS-2 (Fig. 7).


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Fig. 7.   Evolutionary relationship among ETS transcription factors structurally related to ESE-3. Phylogenetic analysis on ETS family members that contain both ETS and SAM/SPM domains was performed, including both domains as described under "Experimental Procedures." A star phylogeny for the maximum likelihood tree is shown. All three methods of phylogenetic interference used (see "Experimental Procedures" for details) recovered the same topology. The numbers close to the internal nodes represent the quartet puzzling support values for the branching relationships. The human sequences used were as follows: ESE-1a/ESX/JEN/ERT/ELF3 (AAB65824), ESE-2a/ELF5 (AAD22960.1), TEL-1 (NP 001978), TEL-2b (AAD43251), c-ETS-1 p54 (P 27577), c-ETS-2 (P 15036), FLI-1 (Q 01543), human GABP alpha  (Q 06546), and the human epithelial prostate-specific PDEF (AAC 95296). Our ESE-3/EHF cDNA sequence data are available (as ESE-J) from GenBankTM under accession number AF 212848. The tree also includes all the Drosophila ETS proteins that contain both the ETS and SAM domains: Aop/Yan/Pok (Q 01842), Pointed P2 (P 51023), ETS 21C (AAF51484.1), ELG/ETS 97D (Q 04688), and ETS 98B (AAF 56746).

Functionally, TEL-1, TEL-2b, ESE-1, ESE-2, and EHF/ESE-3 may all behave as repressors (Refs. 14, 26-28, 32, and 65-67 and this study). It is tempting to speculate that the regulatory role of Aop/Yan may have diversified in vertebrates and could be represented by these genes. This regulatory role may have acquired additional functions during evolution, since some of these proteins also behave as activators, although to date, it has not been reported whether Aop/Yan is only a repressor. Since TEL-1 appears to be only expressed in mesoderm, where it appears to have an important role during hematopoiesis and angiogenesis (69), it seems plausible that the ESE subgroup could represent the function of Aop/Yan in vertebrate epithelia. Furthermore, we hypothesize that the function of these genes could be partially redundant, as evidenced by the fact that deletion of ESE-3 coding sequences in mice did not result in any clearly apparent phenotype.4

As Aop/Yan, transcription factors belonging to the ESE subgroup might be also involved in the election of alternative cell fates, such as the choice between proliferation and migration versus differentiation, in response to complex environmental cues. We propose that the role of these proteins has been conserved throughout evolution as a nuclear switch necessary for the modulation of MAPK signaling cascades in epithelial cells.

    ACKNOWLEDGEMENTS

The contributions of the Physical Mapping, Sequencing, and Bioinformatics groups at Sequana were fundamental to this work. We thank Javier Fraga for advice and Michael Karin, William McGinnis, John K. Westwick, José Javier García Ramírez, and David A. Brenner for valuable suggestions on the manuscript. J. Fraga, Craig Hauser, and M. Karin kindly provided materials used in this study.

    FOOTNOTES

* This work was supported by Boehringer Ingelheim Pharma KG, and Axys Pharmaceuticals Inc. (formerly Sequana Therapeutics, Inc.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF212848.

a To whom correspondence should be addressed: Dept. of Biology, 0349, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 858-822-0461; Fax: 858-822-0460; E-mail: atugores@yahoo.com.

b Present address: Structural Genomics, Inc., 10505 Roselle St., San Diego, CA 92121.

c Present address: Highwire Press, 1454 Page Mill Rd., Palo Alto, CA 94304-1124.

d Present address: Ardais Corp., 128 Spring St., Lexington, MA 02421.

e Present address: Maxim Pharmaceuticals, 6650 Nancy Ridge Dr., San Diego, CA 92121.

f Present address: Division of Molecular Genetics/H5, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

h Present address: Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110.

i Present address: Biocept Inc., 2151 Las Palmas Dr., Suite C, Carlsbad, CA 92009.

j Present address: GenProbe Inc., 10210 Genetic Center Dr., San Diego, CA 92121.

Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M010930200

2 A. Tugores, unpublished observations.

3 P. S. Reddy, unpublished results.

4 L. Carlée, and M. Duyao, unpublished results.

    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; MAPK, MAP kinase; AP-1, activator protein 1; BAC, bacterial artificial chromosome; LOH, loss of heterozygosity; mAb, monoclonal antibody; ORE, oncogene response element; PCR, polymerase chain reaction; PMA, phorbol myristate acetate; YAC, yeast artificial chromosome; MMP, matrix metalloproteinase; ORF, open reading frame; kb, kilobase; contig, group of overlapping clones.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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