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Identification of a novel cis-acting element for fibroblast-specific transcription of the FSP1 gene HIROKAZU OKADA, THEODORE M. DANOFF, ANDREAS FISCHER, JESUS M. LOPEZ-GUISA, FRANK STRUTZ, AND ERIC G. NEILSON Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6144 FSP1; fibroblast; transcription; cis-acting element THE FIBROBLAST-SPECIFIC protein, FSP1, belongs to the S100 family of intracellular calcium-binding proteins (18, 41, 65). Members of this family have been implicated in microtubule dynamics (10, 18, 42, 54), cytoskeletal-membrane interactions (3, 18, 22, 30, 48, 50), calcium signal transduction (18, 25), cell-cycle regulation (41), and cellular growth and differentiation (6, 9, 37, 47, 48). The FSP1 gene or its corresponding protein (12, 26, 37) have been studied in various species (3, 15, 48, 74). The function of FSP1 is not completely understood, but its interaction with nonmuscle myosin II (20), nonmuscle tropomyosin (67), actin (24, 66, 75), or tubulin (42, 54), as well as its ability to facilitate movement when transfected into cultured cells (7, 19, 29, 55), suggest that FSP1 is involved in mesenchymal morphology and cell motility. Reports concerning the regulation of the FSP1 gene in normal cells are few (8), although FSP1 has been investigated as a possible F306 metastasis-related molecule in dedifferentiated or malignant cells (69–72). The S100 family of proteins reside in a gene cluster on human chromosome 1q21 called the epidermal differentiation complex (16, 46, 73) which is syntenic to chromosome 3 in the mouse (11). The pattern of expression of the S100 proteins, like FSP1, in normal tissue varies between the family members, but typically they are expressed in mesenchymal or interstitial-derived cells (3, 12, 15, 37, 65). We cloned FSP1 from a subtractive hybridization between renal fibroblasts and isogenic tubular epithelium and found that fibroblast cell lines from different tissues were positive for FSP1, whereas there was no or extremely low level expression of FSP1 in culture-normal, nonfibroblast cells (65). S100 genes (8, 16, 21, 41, 50, 65) are expressed in more than one tissue, although most are restricted to specific sets of cells. We anticipate selective regulatory processes control their individual expression (8, 21, 38). Tissue- (17, 57, 62) or cell-specific (5, 40, 43, 45, 51, 61) promoters for a growing number of genes are regulated by the modular assembly of cis-acting elements (17, 78) in open chromatin (49) following an interaction with lineage-specific trans-acting proteins (68). Cell-specific expression in fibroblasts suggests that the FSP1 gene may be controlled by mesenchymal-related transcriptional elements. MATERIAL AND METHODS Cell culture. The cells used in this study were derived from mice and passaged as continuous lines using standard conditions: NIH/3T3 fibroblasts, 3T3; renal tubulointerstitial fibroblasts, TFB (2); renal proximal tubular epithelial cells, MCT (31); and parietal yolk-sac cells, PYS-2 (63). Transcription analysis using luciferase reporter minigenes. Parts of the FSP1 gene have been reported (GenBank accession no. M88460) (69). Additional restriction enzyme mapping and sequencing were performed in this region. A series of luciferase reporter (L) minigenes were constructed bearing various 58 fragments of the FSP1 gene. The plasmids, pF2500.L, pF-1892.L, pF-1300.L, pF-970.L, pF-463.L, pF-263.L, pF-187.L, and pF-87.L contain genomic DNA upstream of the transcription start site, respectively, from approximately 22500, 21892, 21300, 2970, 2463, 2263, 2187, and 287 bp. These plasmids, as well as the first noncoding exon (167 bp 38), were placed 58 of the luciferase cDNA in pGL2b (Promega, Madison, WI). Fragments also containing the first intron (1159 bp) terminating immediately 58 of the translation start site (11222 bp 38) were inserted into pGL2b, yielding pF2500.IntL, pF-1892.IntL, pF-1300.IntL, pF-970.IntL, pF463.IntL, pF-263.IntL, pF-187.IntL, and pF-87.IntL. To characterize fragments containing possible regulatory elements, such fragments were inserted into the upstream or downstream multilinker sites of pF-263.IntL, pF-87.IntL, pGL2p, 0363-6127/98 $5.00 Copyright r 1998 the American Physiological Society Downloaded from http://ajprenal.physiology.org/ by 10.220.32.247 on August 3, 2017 Okada, Hirokazu, Theodore M. Danoff, Andreas Fischer, Jesus M. Lopez-Guisa, Frank Strutz, and Eric G. Neilson. Identification of a novel cis-acting element for fibroblast-specific transcription of the FSP1 gene. Am. J. Physiol. 275 (Renal Physiol. 44): F306–F314, 1998.—The FSP1 gene encodes a filament-binding S100 protein with paired EF hands that is specifically expressed in fibroblasts. This led us to look for cis-acting elements in the FSP1 promoter that might engage nuclear transcription factors unique to fibroblasts. The first exon of FSP1 is noncoding, therefore, a series of luciferase reporter minigenes were created containing varying lengths of 58-flanking sequence, the first intron, and the noncoding region of the second exon. A position and promoter-dependent proximal element between 2187 and 288 bp was shown to be active in fibroblasts but not in epithelium. Sequence in the first intron from 1777 to 1964 had an enhancing effect that was not cell type specific. Hsv TK reporter constructs driven by this promoter/ intron cassette in transgenic mice were coexpressed appropriately with FSP1 in tissue fibroblasts. Gel mobility shift competitor assays identified a novel domain, FTS-1 (fibroblast transcription site-1; TTGAT from 2177 to 2173 bp), that specifically interacts with nuclear extracts from fibroblasts. The necessity of this binding site was confirmed by site-specific mutagenesis. Database searches also turned up putative FTS-1 sites in the early promoter regions of other fibroblast expressed proteins, including the a1 and a2(I), and a1(III) collagens and the aSM-actin gene. We hypothesize that the selective engagement of FTS-1 elements may contribute to the mesenchymal phenotype of fibroblasts and perhaps other dedifferentiated cells. A REGULATORY ELEMENT IN THE FSP1 GENE and pGl2b with various promoters: RSV from pREP4 (Invitrogen, San Diego, CA), or minimal promoters E1B and murine alkaline phosphatase/pAP-44 (Gifts of Dr. Thomas Kadesch, Howard Hughes Medical Institute, University of Pennsylvania). In addition, pF-187M1.IntL and pF-187M2.IntL were similar constructs to pF-187.IntL except that the sites 2177/ 2173 bp and 2151/2146 bp were mutated, respectively. pGL2c (Promega, Madison, WI) served as a positive control. The accuracy of all constructed plasmids were verified by restriction enzyme mapping or sequencing. Transient transfections were carried out using CaPO4 (1). Six micrograms of pGL2c or isomolar amounts of sample luciferase constructs were cotransfected with 1.5 µg of pCH110 (Pharmacia), a vector expressing b-galactosidase, into 1.0 3 105 cells plated on each well of the 6-multiwell plate. Medium was changed 24 h later, and cells were harvested 48 h after F307 transfection by lysis in KPO4-DTT with 1% Triton X-100. Supernatants were assayed for luciferase activity by Lumat LB 9501 luminometer. Each luciferase activity was normalized for b-galactosidase activity and then expressed as relative percentage of control pGL2c activity. The final values of the luciferase activity represent the average of at least three independent transfections 6 SE. Mobility gel shift and competitor assay. Nuclear extracts were prepared from 3T3, TFB, MCT, and PYS-2 cells (1). Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce), and nuclear extracts were divided into aliquots, and stored at 270°C. The probe 100–58 (2187 to 288) was created by PCR amplification of ,100 bp region from an FSP1 genomic fragments using the flanking oligomers as primers; 58 acgcgtCACTCACTACTTGATTGT 38 and 58 gtcgacTGTTGGTTGATGTAGTAA 38. The lower case Downloaded from http://ajprenal.physiology.org/ by 10.220.32.247 on August 3, 2017 Fig. 1. Transient transfection of FSP1 minigenes in fibroblasts and tubular epithelium. Minigene reporters were prepared using the murine FSP1 gene. The 1st exon is noncoding, and 1st intron is ,1100 bp. A: luciferase reporters only bearing various 58 fragments of the FSP1 gene were transiently transfected into 3T3 fibroblasts and MCT epithelium. All numbering of constructs is referenced to the number of base pairs upstream of the putative transcription start site (11, arrows). All the constructs contain the 1st exon and end at 167 bp. B: constructs here are similar to those in A except these contain the 1st intron in native orientation in addition to the 58 promoter sequences; all constructs ended at 11222 bp. In contrast to the constructs lacking the 1st intron, the 1st intron-containing constructs (pF-2500.IntL, pF-263.IntL, and pF-187.IntL) showed strong luciferase activities in fibroblasts and weaker expression in tubular epithelium. The strong luciferase activity of these intron-containing reporters drops off in fibroblasts with the deletion of 2187/288 bp (pF-187.IntL vs. pF-87.IntL; P # 0.001). Activities of each construct in MCT tubular epithelium were similar [P 5 not significant (NS)]. Luciferase activity of each reporter in all experiments was normalized for transfection efficiency using b-galactosidase activity and then expressed as relative percentage of control pGL2c activity. F308 A REGULATORY ELEMENT IN THE FSP1 GENE M sodium acetate, 0.05 M magnesium acetate, 1 mM EDTA, and 0.1 mg/ml yeast tRNA. Modified probes were precipitated with ethanol, washed, dried, and resuspended in 10 mM Tris · HCl, pH 7.5, and 1 mM EDTA. Nuclear extracts (0 and 10 µg) were incubated with 105 cpm of probe in the presence of poly-d(I-C) for 30 min at 4°C in a 25 µl of binding buffer. The samples were electrophoresed through a 5% polyacrylamide gel in low ionic running buffer. The wet gels were exposed to X-ray film for overnight, and the free probe and the proteinbound probes were recovered by DEAE membrane method (39). The recovered samples were cleaved at the positions of the modifications. To display methylated purines, DNA was heated at 90°C in 10% piperidine for 30 min. Subsequently, the samples were lyophilized in a vacuum evaporator until dry. Addition of 30 µl of water, freezing, and lyophilizing were repeated twice. Positions of the cleavages were determined by running through the 7% polyacrylamide/8 M urea gel in TBE running buffer (89 mM Tris base, 89 mM boric acid, and 2 mM EDTA). The sample of the G1A reaction of the MaxamGilbert sequencing technique (60) was also run simultaneously as the marker. The gel was dried and exposed to film. Immunohistochemistry of transgenic mice. A second reporter minigene consisting of 22500/11222 bp of the FSP1 promoter, which is the same as pF-2500.IntL shown in Fig. 1, driving the herpes simplex virus thymidine kinase (Hsv TK) cDNA, was assembled (pFSP1.tk) for injection. Blastocysts were injected with pFSP1.tk, and subsequently two lines were established and bred against SJL mice. Adult mice were killed, and their organs were fixed in 4% paraformaldehyde. Immunohistochemistry on 4-µm tissue sections was carried Fig. 2. Position and orientation effects of the 58 fragment from 2187/288 bp on the activity of native minimal 58 promoter (287/167 bp) plus 1st intron in fibroblasts and tubular epithelium. Sequence between 2187 and 288 bp increased the transcriptional activity in fibroblasts more than in epithelium when it was located in its native orientation (pF-87.IntL vs. pF-87.IntL; P # 0.001), less so in its reverse orientation [pF(2187/288)R-87.IntL vs. pF-87.IntL; P # 0.05], and not at all when the 2187/288 bp fragment was located downstream of the 1st intron, but in native orientation [pF-87.IntL(2187/288) vs. pF-87.IntL P 5 NS]. Activities of each construct in MCT tubular epithelium were similar (P 5 NS). Luciferase activity of each reporter in all experiments was normalized for transfection efficiency using b-galactosidase activity and then expressed as relative percentage of control pGL2c activity. Downloaded from http://ajprenal.physiology.org/ by 10.220.32.247 on August 3, 2017 letters represent restriction sites to facilitate cloning. The amplicon was cloned into the vector pCRII (Invitrogen) and later digested with appropriate restriction enzymes leaving 58 overhangs which were dephosphorylated with calf intestinal alkaline phosphatase. This fragment was end-labeled with [g-32P]ATP using T4 kinase, generating a probe for gel shift assays (1). In addition, short fragments of 50 bp and 20–25 bp within region 100–58, with or without mutations, were synthesized for use as competitive oligomers. A quantity of 104 cpm of probe was incubated with 10 µg of nuclear extract in the presence of poly-d(I-C) and competitor oligomers as indicated, 12% glycerol, 10 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 300 µg/ml BSA, and 0.1% Triton X-100 in a 25-µl volume for 30 min at 4°C. Reaction mixtures were electrophoresed through a 5% polyacrylamide gel in low ionic running buffer [6.7 mM Tris (pH 7.5), 3.3 mM sodium acetate, and 1 mM EDTA]. Dried gels were exposed to X-ray film at 270°C with an intensifying screen. Methylation interference assay. Methylation interference assay was minimally modified from Hendrickson and Schleif (35). The 100–58 was excised with Mlu I and Sal I from the pCRII plasmid and end-labeled with [32P]dCTP and [32P]dGTP, or [32P]dCTP and [32P]TTP, respectively, using Klenow fragment, yielding 1/2 strand probes. Purines were methylated by adding 1 µl of dimethyl sulfate to the DNA probe in 200 µl of a solution of 50 mM sodium cacodylate (pH 8.0), 10 mM MgCl2, and 0.1 mM EDTA and incubating for 5 min at room temperature. This reaction was stopped by adding 50 µl of a solution of 1 M Tris · HCl (pH 7.5), 1 M 2-mercaptoethanol, 1.5 F309 A REGULATORY ELEMENT IN THE FSP1 GENE out using polyclonal anti-FSP1 antibodies (65) and antithymidine kinase antibodies (provided by W. C. Summers, Yale University) developed by the ABC-peroxidase method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Statistics. In some experiments statistics were performed using Student’s t-test. RESULTS 32P-labeled 100-58 100-bp (2187/288) probe 58 CACTCACTACTTGATTGTGCCTGCTGGGGAGGGAGCAGGAAGCCTGGTTCCCAGACTGGGCTGGTCGAGGGTGCTATG ACATTTACTACATCAACCAACA 38 Competitor 50-bp oligomers C1/2(50) 58 CACTCACTACTTGATTGTGCCTGCTGGGGAGGGAGCAGGAAGCCTGGTTC 38 C2/3(50) 58 GGGGAGGGAGCAGGAAGCCTGGTTCCCAGACTGGGCTGGTCGAGGGTGCTA 38 C3/4(50) 58 CGAGACTGGGCTGGTCGAGGGTGCTATGACATTTACTACATCAACAACA 38 Competitor 25-bp oligomers C1(25) C2(25) C3(25) C4(25) 58 58 58 58 CACTCACTACTTGATTGTGCCTGCT 38 GGGGAGGGAGCAGGAAGCCTGGTTC 38 CCAGACTGGGCTGGTCGAGGGTGCTA 38 TGACATTTACTACATCAACCAACA 38 M1-1 M1-2 M1-3 M1-4 M1-5 58 58 58 58 58 TGTCTACTACTTGATTGTGCCTGCT CACTCGTCGTTTGATTGTGCCTGCT CACTCACTACCCAGCTGTGCCTGCT CACTCACTACTTGATCACATCTGCT CACTCACTACTTGATTGTGCTCATC Mutated competitor 25-bp oligomers 38 38 38 38 38 32P-Labeled probe 100-58 was incubated with nuclear extract alone or with 200-fold molar excess of an unlabeled, double-stranded oligonucleotides as a competitor. Competitors [C1/2(50), C2/3(50), C3/4(50), C1(25), C2(25), C3(25), and C4(25)] consist of partial sequences of the native 100 bp of 100-58, and mutated competitors (M1-1, -2, -3, -4, and -5) have serial 5 nucleotide substitutes (shown in boldface letters) within the native 25 bps of C1(25). were resequenced for comparison with the reported sequence (71). Only one discrepancy was found; 2142 to 2140 bp is GGT instead of AGA. All sequences, competitors, and mutants used for the shift assays are listed in Table 1. Nuclear extracts prepared from 3T3 fibroblasts and MCT epithelium were compared in shifts (Fig. 3) using a 32P-labeled 100-bp probe spanning 2187 to 288 bp (100–58). The minor band marked by the solid arrow in Fig. 3 was consistently present in fibroblasts, and all the bands observed in this gel shift assay were completely quenched in the presence of a 200-fold molar excess of unlabeled probe (Fig. 4A). Other gel shifts using the same 100–58 probe and nuclear extracts from TFB fibroblasts and PYS-2 endoderm demonstrated the same set of shifted bands only with fibroblasts (data not shown). To localize the protein DNA-binding site, synthesized oligomers within 100–58 were employed as competitors (Table 1) in gel shifts under similar conditions as reported above. Among the 50-bp competitive oligomers employed in this study, only the 58 fragment C1/2(50) could prevent probe retardation, suggesting that the 58 end of the 100-bp probe is important for binding (data not shown). Subsequently, a 25-bp oligomer from 2187 to 2162 bp, C1(25), competed out the shifted band created by 100–58 probe with fibroblast extract, whereas an adjacent 25-bp oligomer could not (Fig. 4A). By repeating the competition with a series of mutant oligomers made from C1(25), we observed that mutant M1–3 failed to abolish the shifted band, indicating that Downloaded from http://ajprenal.physiology.org/ by 10.220.32.247 on August 3, 2017 Functional characterization of cis-acting regulatory elements in the flanking regions of the murine FSP1 promoter. Since murine transcripts encoding FSP1 are predominantly seen in fibroblasts (65), we began looking for the cis-acting elements responsible for restricting transcription to these cells. Figure 1 shows a restriction map of the murine FSP1 gene. In the first set of constructs (Fig. 1A), a series of luciferase reporters (L) were assembled using a 58 EcoR I-Nhe I fragment (around 22500/167 bp) and its subfragments all ending at 167 bp. Transfection of these constructs demonstrated significantly greater expression in 3T3 fibroblasts than in MCT epithelium. This finding is consistent with transfection results using other tissue fibroblasts and nonfibroblast cells (65); data not shown. A second set of luciferase constructs were assembled by adding the 1st intron with the splice donor and acceptor sequences in native configuration (Int) to the constructs used above. Overall the expression of FSP1 promoter (Fig. 1B) in 3T3 fibroblasts was greatly enhanced by the addition of the first intron compared with the intronless promoters (pF-2500.L, pF-263.L, pF-187.L, and pF-87.L) described in Fig. 1A. This enhancement was also seen to proportionally affect epithelial cells and therefore was not cell lineage specific. Subsequent experiments isolated this enhancement to a discrete region in the first intron (1777 to 1964 bp; data not shown). Of special note in Fig. 1B was the strong luciferase activity of pF-187.IntL in fibroblasts, which fell to levels registered in epithelium with the promoter deletion of 2187 to 288 bp (pF187.IntL vs. pF-87.IntL; P # 0.001), suggesting this proximal region spanning 2187 to 288 is important to the fibroblast phenotype. All of the transfection experiments in MCT epithelium were confirmed using several other nonfibroblastic cell lines (data not shown). We next created a series of constructs in which the putative fibroblast-specific promoter-proximal fragment (2187 to 288 bp) was placed either upstream or downstream of pF-87.IntL in the reverse and native orientation, generating pF(2187/288)R-87.IntL and pF-87.IntL(2187/288), respectively. In Fig. 2 this fragment increased the transcriptional activity in 3T3 fibroblasts best when it was located at the upstream, native orientation (pF-187.IntL P # 0.001), somewhat less in the reverse upstream position (pF(2187/288)R87.IntL; P # 0.05), and not at all when located downstream in a forward orientation [pF-87.IntL(2187/ 288); P 5 not significant (NS)]. Mapping a proximal element in the 58-flanking region of the FSP1 promoter. Both strands of the FSP1 promoter region contained within the construct pF-187.L Table 1. Gel shift oligomers F310 A REGULATORY ELEMENT IN THE FSP1 GENE the base pairs changed in this oligomer were critical for the binding of the fibroblast nuclear factors (Fig. 4B). Thus a core binding site was approximately localized to 58 TTGAT 38, from 2177 to 2173 bp in the promoter of Fig. 4. Competition gel shift analysis of nuclear extracts from fibroblasts. A: all shifted bands seen in lane 2 were abolished in lane 3 by cold 100–58 fragment (see Table 1 for description of competitors). Band shifted in fibroblast (indicated by arrow for lane 2) was attenuated by the C1(25) inhibitor (lane 4) but not by other 25-mer competitors (lanes 5–7). B: competition gel shift analysis of nuclear extract from fibroblasts using mutated competitors. Band shifted in fibroblasts (indicated by arrow, in lane 2) was competed out, not only by intact C1(25) (lane 3) but also by mutated C1(25) competitors, M1–1, -2, -4, and -5 (lanes 4, 5, 7, and 8, respectively). In contrast, mutated competitor M1–3 did not attenuate this shifted band (lane 6). Downloaded from http://ajprenal.physiology.org/ by 10.220.32.247 on August 3, 2017 Fig. 3. Gel shift of 3T3 (fibroblast) and MCT (tubular epithelium) nuclear extracts. 100–58 probe (see Table 1 for description of competitors) was incubated with 3T3 (lanes 2–6) or MCT (lanes 7–11) nuclear extracts with varying amounts of poly-d(I-C) ( 0, 1, 3, 6, and 10 µg, respectively). Free probe showed no shifted bands (lane 1). Band seen in fibroblasts but not epithelium, is indicated by the arrow. FSP1. Tandem repeats of fragment 2182 to 2168 were cloned into 58 sites in front of various heterologous promoters. This tandem repeat region as well as cisfragment 2187 to 287 was unable, however, to enhance heterologous promoter function in fibroblasts compared with native promoter, pF-187.IntL (data not shown), suggesting the candidate elements were promoter dependent. A methylation interference assay was then performed using a gel cutout of this specific, shifted band as template (data not shown). The 2177 to 2173 region is AT-rich and only contains one potential interference site. No interference was observed for the G at 2175 and other Gs flanking this core sequence, suggesting they are not important for local protein-DNA binding. Another shifted band from that region containing an Ets-like site 58 TCTGGGAA 38, which was detected in gel shift assays under different conditions but proved not to be functional in fibroblast transfections, produced a positive interference reaction as control. Single base mutations of the G at 2175 did not inhibit competition in the gel shift further, suggesting that the G base was not critical (data not shown). To further address the authenticity of the putative cis-acting element defined by M1–3, an identical mutation was introduced into the luciferase reporter construct (pF-187.M1IntL), and a new set of transient transfections were carried out. In Fig. 5, the fibroblastspecific transcriptional activity of pF-187.M1–3IntL was reduced back to the level of the minimal promoter pF-87.IntL compared with the native construct, pF187.IntL (P # 0.001). A mutation at 2151 bp, which did not compete for gel shift, also did not affect the luciferase activity (pF-187.M2IntL). A REGULATORY ELEMENT IN THE FSP1 GENE F311 A larger pF-2500.M1–3IntL construct containing the M1–3 mutation also reduced transcription by 30% compared with wild-type sequence (data not shown). Putative regulatory elements of the FSP1 gene are active in transgenic mice. The FSP1 gene fragment containing the promoter and intronic elements used in the pF-2500.IntL minigene (Fig. 1; around 22500 to 11222 bp) were next used to drive Hsv TK cDNA (pFSP1.tk) in transgenic mice. The distribution of Hsv TK was concordant with FSP1 expression in all tissue examined (data not shown; unpublished observations). Kidney tissue harvested from transgenic progeny was stained by immunohistochemistry (Fig. 6); interstitial cells staining for Hsv TK were also positive for FSP1 protein using a serial section analysis. Two lines of transgenic mice demonstrated the same result in kidney, although data from only one of the two are shown. Sections from the nontransgenic littermates stained positive for FSP1 protein, but were negative for Hsv TK amplicons, and did not stain for Hsv TK protein (data not shown). DISCUSSION The mechanisms regulating the bidirectional transformation of epithelium and mesenchyme are not fully known, although a number of processes have been proposed (34). It is likely that a combination of morphogenic cues including adhesion, matrix, and paracrine stimulation work in combination to activate genes that alter and then stabilize cell phenotype (34, 77). We have approached this issue by looking at the regulation of the FSP1 gene in fibroblasts (65). The FSP1 gene was isolated by subtractive hybridization between murine renal fibroblasts and isogenic epithelium (65). The expression of the FSP1 gene in nonmalignant cells in Fig. 6. Immunohistochemistry with polyclonal anti FSP1 antibodies, anti-thymidine kinase antibodies, and ABC-peroxidase method on serial sections of the kidney of transgenic mice bearing pFSP1.tk. In these animals, thymidine kinase (TK) is expressed under the control of the FSP1 regulatory cassette. A: arrows indicate FSP1-positive cells in the renal interstitium. B: arrowheads indicate TKpositive cells in the next section to the one shown in A, suggesting that same renal interstitial fibroblasts expressed FSP1 as well as TK in these animal. Magnification, 3150. Counterstained with hematoxylin. Downloaded from http://ajprenal.physiology.org/ by 10.220.32.247 on August 3, 2017 Fig. 5. Effects of discrete mutations in the 2187 to 288 bp proximal regulatory region on the native 58 minimal promoter (287 to 167 bp) plus the 1st intron in fibroblasts and tubular epithelium. Whereas the luciferase minigene reporter with mutations between 2151 to 2146 bp (pF-187M2.IntL) demonstrated activity in fibroblasts comparable to that of the native construct pF-187.IntL, a mutation in the putative consensus sequence (2177 to 2173 bp) in pF-187.M1–3IntL dropped the luciferase activity in 3T3 cells to the level of pF-87.IntL (P # 0.001). Activities of each construct in MCT tubular epithelium were similar (P 5 NS). Luciferase activity of each reporter in all experiments was normalized for transfection efficiency using b-galactosidase activity and then expressed as relative percentage of control pGL2c activity. F312 A REGULATORY ELEMENT IN THE FSP1 GENE perhaps the parallel processes in mature cells from adult tissues (32, 34, 44, 53, 76). The future identification of trans-acting factors that bind to elements like FTS-1 should bring us even closer to understanding the plasticity of cell transformation, the modular control of mesenchymal phenotypes, and the gating necessary to selectively engage tissue fibroblasts during organ fibrosis. FSP1 is a critical part of this fibrogenic program, and its role in renal fibrosis is gradually unfolding (52). This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-07006, DK-30280, DK-41110, DK-02334, and DK-45191 and by administrative/educational funds from the DCI RED Fund. H. Okada was a recipient of a fellowship from Eli-Lilly Japan and received financial support from Takeda Science Foundation. F. Strutz was supported by Deutsche Forschungsgemeinschaft Str 388/1–1. A. Fischer was a recipient of a grant from the Swiss National Foundation for Scientific Research and received support from Roche Research Foundation, Ciba-Geigy Jubilaeumsstiftung, and Janggen-Poehn Foundation. Address for reprint requests: E. G. Neilson, C. Mahlon Kline Professor of Medicine, Penn Center for Molecular Studies of Kidney Diseases, 700 Clinical Research Bldg., Univ. of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104-6144. Received 21 July 1997; accepted in final form 30 April 1998. REFERENCES 1. Albert, S. E., F. Strutz, K. Shelton, T. Haverty, M. J. Sun, S. Li, A. Denham, R. A. Maki, and E. G. Neilson. Characterization of a cis-acting regulatory element which silences expression of the class II-A beta gene in epithelium. J. Exp. Med. 180: 233–240, 1994. 2. Alvarez, R. J., M. J. Sun, T. P. Haverty, R. V. Iozzo, J. C. Meyers, and E. G. Neilson. Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines. Kidney Int. 41: 14–23, 1992. 3. Barraclough, R., R. Kimbell, and P. S. Rudland. Increased abundance of a normal cell mRNA sequence accompanies the conversion of rat mammary cuboidal epithelial cells to elongated myoepithelial-like cells in culture. Nucleic Acids Res. 21: 8097– 8114, 1984. 4. Birchmeier, C., and W. Birchmeier. Molecular aspects of mesenchymal-epithelial interactions. Annu. Rev. Cell Biol. 9: 511–540, 1993. 5. Bohinski, R. J., J. A. Huffman, J. A. Whitsett, and D. L. Lattier. Cis-active elements controlling lung cell-specific expression of human pulmonary surfactant protein B gene. J. Biol. Chem. 268: 11160–11166, 1993. 6. Calabretta, B., R. Battini, L. Kaczmarek, J. K. de Riel, and R. Baserga. Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calciumbinding protein. J. Biol. Chem. 261: 12628–12632, 1986. 7. Davies, B. R., M. P. A. Davies, F. E. M. Gibbs, R. Barraclough, and P. S. Rudland. Induction of the metastatic phenotype by transfection of a benign rat mammary epithelial cell line with the gene for p9Ka, a rat calcium-binding protein, but not with the oncogene EJ-ras-1. Oncogene 8: 999–1008, 1993. 8. Davies, M., S. Harris, P. Rudland, and R. Barraclough. Expression of the rat, S-100-related, calcium-binding protein gene, p9Ka, in transgenic mice demonstrates different patterns of expression between these two species. DNA Cell Biol. 14: 825–832, 1995. 9. De, Leon, M., L. J. Van Eldik, and E. M. Shooter. Differential regulation of S100 beta and mRNAs coding for S100-like proteins (42A and 42C) during development and after lesion in rat sciatic nerve. J. Neurosci. Res. 29: 155–162, 1991. 10. Donato, R., F. Battaglia, and D. Cocchia. Characteristics of the effect of S-100 proteins on the assembly-disassembly of brain microtubule proteins at alkaline pH in vitro. Cell Calcium 8: 299–313, 1986. 11. Dorin, J. R., E. Emslie, and V. van Heyningen. Related calcium-binding proteins map to the same subregion of chromo- Downloaded from http://ajprenal.physiology.org/ by 10.220.32.247 on August 3, 2017 mice is exclusively observed in fibroblasts (26, 37, 65). However, the expression of FSP1 homologs in other species or in malignant cells may have a different distribution (8, 12, 15, 23, 28, 48, 74). Although it has recently been suggested that the 58-flanking fragments of the FSP1 gene play no part in its expression in tumor cells (69, 71), their control in cultured fibroblasts has not been explored. In the current study using several murine fibroblasts and nonfibroblast cells, we observed that the 58 cis-acting element 58 TTGAT 38 between 2177 and 2173 bp is critical in fibroblast-specific transcription of the FSP1 gene. Consequently, we refer to this position and promoter-dependent proximal element as FTS-1 (fibroblast transcription site-1). FTS-1 activity is greatly augmented by universal activity located in the first intron (between 1777 and 1964 bp). Our findings are consistent with the observation that promoter sequences in other members of S100 superfamily contain their own cell-specific regulatory elements (38). There seems to be a difference in the pattern of expression of the FSP1 gene in two closely related species, mouse and rat (8). Although 58-flanking sequence of the rat FSP1 gene is not reported, we surmise that differences in the controlling regions may explain why they are differentially expressed in the two rodent species. Finally, a reporter minigene containing the native FTS-1 element as well as other control regions of the FSP1 gene coexpressed only in FSP11 tissue fibroblasts in transgenic mice. A further search of genomic databases with the novel FTS-1 sequence identified identical sites in the early promoter regions of other fibroblastrelevant genes such as a1 and a2(I), and a1(III) procollagens, as well as the aSM-actin gene. These latter genes are typically engaged with some exclusivity by activated fibroblasts (27, 56, 64). Although the presence of FTS-1 sequence in the regulatory regions of these genes only promotes speculation, it is of interest that two FTS-1 sites are present at positions 21707 and 2954 bp in the cis-acting cassette required for cell-specific transcription of a1(I) procollagen in skin and tendon fibroblasts in transgenic mice (36, 59). Similar findings were observed in transgenic mice expressing a2(I) procollagen minigenes, where the promoter region between 22000 and 2350 bp was required for expression in most type I collagen-containing cells (51); an FTS-1 site is also present in that gene at position 2752 bp. Finally, an FTS-1 site was not observed in the a1(I) procollagen promoter region (21656 to 21540 bp) that confers high level, specific expression in osteoblasts (58). These findings are all consistent with the special effect of FTS-1 sites on the definition of a fibroblast. Phenotypic conversions between epithelium and mesenchyme follow a bidirectional pathway during early pattern formation as well as later during the specialization and development of organ tissue (4, 13, 14, 32–34). The dedifferentiation of somatic cells during oncogenesis (19, 44, 59) or the mesenchymalization of epithelium during episodes of fibrogenesis (65) following wounding (51, 64) or inflammation (27, 53, 56) are A REGULATORY ELEMENT IN THE FSP1 GENE 12. 13. 14. 15. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Haverty, T. P., C. J. Kelly, W. H. Hines, P. S. Amenta, M. Watanabe, R. A. Harper, N. A. Kefalides, and E. G. Neilson. 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