Vol. 10, No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1990, p. 2349-2358

0270-7306/90/052349-10$02.00/0 Copyright © 1990, American Society for Microbiology

A Negative Element Involved in Vimentin Gene Expression FRANCIS X. FARRELL,1 CHRISTINA M. SAX,2 AND ZENDRA E. ZEHNERl* Department of Biochemistry and Molecular Biophysics and The Massey Cancer Center, Medical College of VirginialVirginia Commonwealth University, Richmond, Virginia 23298,1 and Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland 208922 Received 17 August 1989/Accepted 9 February 1990

Vimentin is one member of the intermediate ifiament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.

Current models of eucaryotic gene regulation invoke multiple DNA-binding sites interacting with trans-acting factors. These DNA-protein interactions can exert an effect on gene regulation by several mechanisms (42, 44). Some act in a constitutive manner (10), while others are tissue specific (18, 19, 22, 32). Yet others modulate transcriptional activity in response to environmental signals such as metal ions (13), serum (41), mitogens (5, 45), and phorbol esters (1). Although much is known about cis sequences which interact with trans factors to stimulate transcription, i.e., enhancer elements, it is also possible for these interactions to repress transcription (2, 6, 14, 38). Recent evidence has shown that these negative regulatory elements act much like enhancer elements in that they are position and orientation independent (24, 46). Furthermore, two DNA-binding proteins which repress transcription have been cloned and characterized. A Drosophila homeodomain protein (eve) has recently been shown to repress transcription of the Ultrabithorax (Ubx) gene in a binding site-dependent manner in the Ubx promoter (4). Secondly, a protein that binds to G+C-rich sequences has been found to repress transcription in the promoters of several genes, including the epidermal growth factor receptor, beta-actin, and calcium-dependent protease (21). This result indicates that negative factors may repress transcription by binding to the same or overlapping DNA sequences found to activate transcription, since G+C-rich sequences are the site of the well-characterized transcriptional activator Spl. Intermediate filament proteins are a prominent component of the cytoskeleton and karyoskeleton of most eucaryotic cell types (for a review, see references 25 and 51). Vimentin is the intermediate filament expressed in mesenchymal cells and represents one of the approximate 40 members of the *

Corresponding author.

intermediate filament multigene family (37, 50). Most intermediate filament proteins adhere to a strict tissue-specific distribution (37, 51). Vimentin is the only member which can be coexpressed with another intermediate filament protein in certain cell types and in tissue culture. Expression of intermediate filament genes is thought to be regulated at the transcriptional and/or posttranscriptional levels (51). This suggests that the vimentin gene must contain multiple modes of regulation, which could include both positive and negative DNA elements, given its complex yet strict tissue-specific expression pattern in vivo. Vimentin is encoded by a single-copy gene in the chicken (56), the hamster (36), and man (28, 35, 39). The expression of vimentin is modulated by several mechanisms, including cell cycle (11, 40), serum (41), culture conditions (12), and phorbolesters (47). Recent data suggests that two AP-1/ jun-binding sites may be responsible for both the serum and phorbolesters induction of the human vimentin gene (41). We have previously characterized sequence elements that regulate chicken vimentin expression (47). The 5'-flanking region contains a variant (CATAAGAG) of the TATAAATA consensus sequence, an inversely oriented CAAT box, and 5 GC boxes. We have identified both a major and minor start site of transcription. The proximal GC boxes have been shown by DNase I footprinting to bind Spl and are responsible for the basal expression of vimentin in many tissues (47). We have localized an enhancer element to a region -321 to -161 from the start site of transcription. This region is also responsible in part for the down regulation of vimentin expression during myogenesis (48). In this report, we show by gel retardation assays, DNase I footprinting, and Southwestern (DNA-protein) blot analysis that a negative regulatory element located approximately 568 base pairs (bp) from the start site of transcription binds a protein factor. This region shares DNA sequence similarity with other characterized silencer elements, including the 2349

2350

FARRELL ET AL.

human immunodeficiency virus type 1 long terminal repeat (43), rat growth hormone (24), and the chicken lysozyme gene (2). Interestingly, our 40-bp region contains some sequence similarity to the octamer-binding sequence (3) which has been observed in a negative element within the human c-myc gene (53). This study suggests that in addition to the multiple positive cis-acting elements, a negative element and factor are involved in the complex regulatory circuit of vimentin gene expression.

MATERIALS AND METHODS Plasmid constructions. The expression vector (p8CAT) is a derivative of pEMBL8 (8) in which the Fl origin of replication has been removed and the bacterial CAT gene inserted. Various 5'-flanking sequences were cloned into the multicloning site of p8CAT as previously described (47). pcV-767, pcV-608, pcV-321, and pcV-161 were generated from the restriction enzyme HindIll, BstNI, AluI, and AvaIl sites found 767, 608, 321, and 161 bp upstream of the transcriptional initiation site (+ 1), respectively. pcV-568 was con-

structed by BAL 31 digestion of pcV-608. Synthetic oligonucleotides of the region -608 to -568 synthesized with BamHI restriction sites at their ends were annealed and subcloned into pUC18 for gel retardation and footprint studies or into a unique BamHI site located upstream of the herpes simplex virus (HSV) thymidine kinase (tk) promoter in the expression vector ptkCAT (30). Plasmids containing concatamers of this 40-bp region were generated by ligation of the region alone for 6 h, followed by the addition of

BamHI-digested ptkCAT. The number of copies in each construct was determined by DNA sequencing (29). Cell culture, DNA transfections, and CAT assays. Mouse L

or HeLa cells were plated at a density of 5 x 105 cells per 100-mm dish 24 h prior to transfection. Cultures were transfected via calcium phosphate precipitation (17) with 10 ,g of chimeric plasmid and 1 ,ug of pMSV-beta-galactosidase (34) to serve as an internal control for standardization between different transfections. Cultures were harvested 48 h posttransfection, and cell lysates were obtained by repeated freeze-thawing in 250 mM Tris, pH 7.8. CAT assays were performed by the method of Gorman et al. (16). Experiments were quantitated by excising the radioactive spots from silica plates and determining their '4C content by liquid scintillation counting. Beta-galactosidase activity was assayed in all cultures as described by Nielsen et al. (34). CAT enzyme activity was expressed as picomoles of chloramphenicol acetylated per minute per microgram of protein divided by units of beta-galactosidase activity. All values reported for mouse L cells are the average of at least three separate transfections. Preparation of nuclear extracts from HeLa cells. HeLa cells were grown to a density of 5 x 105 cells per ml in spinner flasks. Nuclear extracts were prepared typically from 5 liters of cells by the method of Dignam et al. (9), with slight modifications. Extracts were ammonium sulfate precipitated to 50% saturation. The pellet obtained was desalted on a G-75 (Sigma Chemical Co.) column equilibrated in buffer D. A280 fractions were collected and stored at -70°C for up to 6 months. Further fractionation was performed on some preparations. Extracts were dialyzed in buffer D minus KCl and applied to a 10-ml phosphocellulose (Whatman Inc.) column equilibrated in buffer D minus KCI. The column was washed with 3 column volumes of buffer D minus KCI. Proteins were eluted from the column via a step gradient of buffer D containing 0.1 M, 0.2 M, 0.4 M, and 1.0 M KCl, respectively.

MOL. CELL. BIOL.

Eluted fractions were adjusted to 0.1 M KCI and stored as described above. Gel mobility shift assays. A radiolabeled fragment (1 to 2 ng) containing the region -608 to -568 cloned into pUC18 was incubated with 1 to 4 RI of crude nuclear extract or column fraction and 1 ,ug of poly(dI-dC) in a final buffer concentration of 25 mM Tris (pH 7.9)-6.25 mM MgCl2-0.5 mM EDTA-50 mM KCl-0.5 mM dithiothreitol-10% glycerol. Reactions were incubated on ice for 15 min followed by 2 min at room temperature. Samples were loaded onto a 4% nondenaturing gel prepared in 0.25x Tris-boric acid-EDTA. Electrophoresis was carried out for 90 min at 10 V/cm in 0.25 x Tris-boric acid-EDTA. Gels were dried and placed on XAR film overnight for visualization. DNase I footprinting. A 20-fmol portion of a fragment containing the 40-bp silencer region was incubated in a final volume of 50 RI as described above. After the 2-min incubation step at room temperature, 50 p1 of a 5 mM CaCl2-10 mM MgCl2 solution was added, followed by 2.5 p1 of a freshly diluted 5 ,ug/ml solution of DNase I (Pharmacia). Digestion was allowed to continue for 1 min. The reactions were terminated by a 100-pI solution containing 200 mM NaCl, 20 mM EDTA, 1% SDS, and 250 pug of tRNA per ml. Reactions were incubated at 65°C for 15 min, extracted with phenolchloroform, and precipitated with 2.5 volumes of ethanol. The pellets were rinsed with 70% ethanol and dried before loading on a 6% acrylamide-8 M urea gel. Southwestern blotting. Southwestern blotting was performed as described by Singh et al. (49). A 100-,ug portion of nuclear extract or column fraction was diluted 1:1 with sample resuspension buffer (2% sodium dodecyl sulfate [SDS], 100 mM Tris [pH 7.5], 280 mM beta-mercaptoethanol, 20% glycerol, 0.002% pyronin Y), boiled for 5 min, and loaded onto a 10% SDS denaturing gel in a buffer of 50 mM Tris-400 mM glycine-0.1% SDS. Electrophoresis was carried out at a constant current of 25 mA. The gel was incubated for 30 min in gel transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol) and then blotted to nitrocellulose. Transfer of proteins was carried out on a Bio-Rad transfer cell at 0.5 A for 2 h in gel transfer buffer. Transfer of proteins was monitored by use of prestained molecular weight standards (Sigma). The nitrocellulose was blocked for 1 h at room temperature in a solution containing 5% Carnation nonfat dry milk, 50 mM Tris (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, and 1 mM dithiothreitol. The nitrocellulose was washed in TNED (10 mM Tris [pH 7.5], 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol) for 2 to 5 min and then placed in a sealed plastic bag containing 20 ml of TNED and 106 cpm/ml of probe as described in the figure legends. Binding was allowed to continue for 1 h at room temperature. The nitrocellulose was washed two times with TNED for 10 min each time. The blot was allowed to dry at room temperature and placed on film for visualization. Synthetic oligonucleotides. All oligonucleotides were synthesized on a BioSearch Cyclone DNA Synthesizer. Oligonucleotides were purified on a 20% denaturing urea gel. Complementary oligonucleotides were annealed in a buffer containing 0.3 M KCl, 10 mM Tris (pH 7.5), and 1 mM EDTA. The silencer fragment -608 to -568 was as follows:

5'-GATCCAGGAGCGCTGTGCCCGAAGCAAAGCGATG CCCCTCCTGCAG-3' and 5'-GATCCTGCAGGAGGGGC ATCGCTTTGCTTCGGGCACAGCGCTCCTG-3'. The octamer binding sequence was generated as follows: 5'-TCGA CATGCAAATG-3' and 5'-TCGACATTTGCATG-3'. Two subregions of the 40-bp region were synthesized as follows: 5'-CTAGACAGGAGCGCTGTGCCCGT-3' and 5'-

A NEGATIVE ELEMENT IN VIMENTIN GENE EXPRESSION

VOL. 10, 1990

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FIG. 1. Transfection of mouse L and HeLa cells with chicken vimentin 5' deletions and silencer-HSV tk constructs. The activity of each CAT fusion is determined as picomoles of ["4C]chloramphenicol (specific activity, 60 mCi/mmol) acetylated per unit of beta-galactosidase activity. Fold denotes the fold induction of CAT activity relative to the promoterless p8CAT vector.

CTAGACGGGCACAGCGCTCCTGT-3' and 5'-GATGCC CCTCCTGCA-3' and 5'-GGAGGGGCATCTGCA-3'. RESULTS We have shown previously by use of 5'-deleted plasmid constructs transfected into mouse L cells that the chicken vimentin gene contains a proximal promoter region between -161 to +1 and an enhancer region between -321 to -161 (47). The proximal promoter region contains an inverted CAAT box and three GC boxes, which we have shown to bind Spl in vitro in gel retardation and DNase I footprint assays (47). In addition, we showed that these GC boxes were responsible for the low but constitutive level of CAT expression found in several different cell types and Xenopus oocytes (47). On the other hand, the tissue specificity of the enhancer region was demonstrated by its ability to significantly enhance CAT activity in cells of mesenchymal origin, such as mouse L cells or chicken embryo fibroblasts (C. M. Sax, F. X. Farrell, Z. E. Zehner, and J. Piatigorsky, Dev. Biol., in press) and not in cells of nonmesenchymal origin, i.e., rat MH1Cj (ATCC CCL144), which produce no vimentin mRNA via Si or Northern blot analysis. When constructs were transfected into mouse L cells (Fig. 1), we observed evidence for both a distal enhancer and a negative regulatory element. The largest construct, pcV-767, yielded a CAT activity of 42-fold over the promoterless p8CAT. On the other hand, the construct pcV-608 had an activity of only fivefold. This low value suggests that sequences which suppress CAT activity must reside between

2351

-608 and -321. However, a construct deleting 40 bp from pcV-608, pcV-568, yielded a CAT activity of 52-fold. This result represents a 10-fold increase in activity over the construct pcV-608 and indicates that 44% of the CAT activity of pcV-321 is recovered by removing this 40-bp sequence. The construction pcV-321 yields high CAT activity (118-fold) only in cells where vimentin is highly expressed. Since removal of the 40-bp sequence does not restore CAT activity to that of pcV-321, this suggests that other negative acting sequences may be present between -566 to -321. To assess whether the repression due to this 40-bp sequence is found in other cell types, we transfected these same constructs into a nonrelated, i.e., nonfibroblast, cell type. We chose the HeLa cell line for two reasons. First, we know by Northern (RNA) blot analysis that vimentin mRNA is found in HeLa cells grown in suspension or monolayer by using a human vimentin cDNA probe, albeit at a lower level than mouse L cells (data not shown). Secondly, since it is well documented that vimentin is expressed in some cells when grown in culture (12), HeLa is an excellent model system to use, since it is well characterized, can be easily grown in large quantities, and has been used as a source for the purification of several transcription factors (7). HeLa cells exhibit similar trends in CAT activity for each construct as was observed in mouse L cells; however, it is at much lower levels (Fig. 1). The construct pcV-767 yields a CAT activity of 4.5-fold over p8CAT, while pcV-608 has an activity of 1.2-fold more. The construct pcV-568 yields an activity of 2.3-fold, representing a 48% increase in CAT activity due to the removal of the 40-bp sequence. As observed for mouse L cells and all other cell lines assayed to date which express vimentin, pcV-321 yields the highest activity, i.e., 5.0-fold over the promoterless p8CAT. Evidence from others (18, 31, 46) has suggested that negative regulatory elements exert their effect like enhancer elements, in that functionality is orientation and position independent. To determine whether our negative regulatory element is analogous to an enhancer element, i.e., position and orientation independent, we made constructs containing this region fused to a heterologous promoter and transfected them into mouse L cells. This also allowed us to test the silencing effect, if any, on the promoter of a nonrelated gene. The plasmid used for these experiments contained the CAT gene under the control of the HSV tk promoter, ptkCAT (30). The tk promoter was chosen because it is very similar to the chicken vimentin promoter in that it contains an inverted CAAT box flanked by GC boxes (20). The 40-bp DNA sequence was generated synthetically with BamHI linkers and cloned into a unique BamHI site 5' of the tk promoter. The plasmid ptkCAT yields 110-fold CAT activity over p8CAT. When the 40-bp region was inserted 5' to the promoter in the sense direction, CAT activity dropped to 70-fold. This represents a 36% drop in CAT activity. The 40-bp element inserted in the antisense orientation yielded a similar result, a drop which was 76-fold. To test whether the region could suppress CAT activity to a greater extent if present in multiple copies, we constructed concatamers of the element in the BamHI site of ptkCAT. A construct containing two copies was able to suppress CAT activity to 35-fold. This represents an additional 50% decrease in CAT activity as compared with a single copy and an overall decrease of 68%. A construction containing four copies gave a CAT activity value of 10-fold. This result indicates that multiple copies are more effective than a single copy in suppressing CAT activity. Because this region appears to

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FARRELL ET AL.

suppress CAT activity, we initiated studies to determine whether this sequence was a site for protein binding. Evidence that negative regulatory sequences function by binding a factor has been suggested by others (1, 6, 18, 33). The 40-bp negative regulatory element binds a protein(s). Gel retardation assays were used to detect DNA-protein binding in HeLa nuclear extracts. Beside the fact that HeLa cells are easy to grow in a short period of time, they were used as our source because transfection data suggested that the 40-bp region conferred the same relative repression in mouse L cells and HeLa cells. If this region was a site of protein binding, we postulated that the same protein may be involved in the two cell lines. This conclusion, to be discussed in relation to Fig. 4, is supported by Southwestern (DNA-protein) blot analysis. A radiolabeled probe containing the 40-bp sequence cloned in pUC18 was incubated with HeLa nuclear extracts prepared by the Dignam method (9). Multiple retarded species were obtained by using crude extracts (data not shown). We proceeded to fractionate the extract to determine whether we could localize optimal complex formation to a given fraction or fractions and reduce the number of retarded species. As a first step, the extract was adjusted to 50% ammonium sulfate and the precipitated proteins were desalted on a G-75 column. At this step, the extract yielded a major band in the gel retardation assay (data not shown). A minor band of slower mobility was sometimes observed and was sensitive to poly(dI-dC) concentration. Further fractionation was performed on a phosphocellulose column equilibrated with buffer D minus KCl. Proteins were eluted off the column by buffer D containing increasing KCl concentration and were assayed as described previously. We observed retarded bands in the 0.1 to 0.2 M KCl fraction identical to those obtained after ammonium sulfate precipitation (Fig. 2A). Retarded species were not observed in the fractions containing higher KCl concentrations, even after dialyzing against 0.1 M KCl (data not shown). We concluded that the binding activity elutes off the column at low-salt concentrations and that column fractionation is not imperative for optimal complex formation. To ascertain that the observed retarded band in Fig. 2A was due to specific interaction between protein and the 40-bp

region, competition experiments were performed. Assays were carried out as before, except that unlabeled fragments were added to each reaction. The first competitor fragment used was the unlabeled 40-bp sequence (Fig. 2B). As expected, we observed a disappearance of the retarded species at low-molar fragment-probe excess (Fig. 2B). The band was virtually eliminated at a 50-fold molar excess. The second competitor DNA fragment used was a perfect octamerbinding site, ATGCAAAT (3, 52). This competitor was selected because our negative regulatory region contains a partial similarity to an octamer-binding site (see Fig. 7). We did not observe a significant disappearance of the retarded band at a 100 molar fragment-probe excess (Fig. 2C). We did, however, observe partial disappearance of the retarded species at high-molar fragment-probe excess, >500 molar excess (data not shown). Yet a third competitor fragment was a 46-bp fragment isolated from an AluI digestion of pUC18. This nonspecific competitor also did not abolish the retarded species (Fig. 2D). In view of these findings, we proceeded to localize the protein-binding site(s) within this region. Localization of protein-binding site(s) in the vimentin negative regulatory region by DNase I footprinting. The same fragment used for gel retardation studies was subjected to

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FIG. 2. Gel mobility shift assays of a nuclear protein binding to the 40-bp silencer region of chicken vimentin. A 1-ng portion of a radiolabeled fragment, approximately 5,000 cpm, was incubated with 1, 2, 5, or 10 p.g (panel A) or 5 ,ug (panels B, C, and D) of a 0.1 to 0.2 M KCI HeLa nuclear fraction. Zero denotes the fragment incubated in the absence of protein. The radiolabeled fragment was generated by end labeling an EcoRI site of pUC18 containing the 40-bp region cloned into the BamHI site, followed by digestion with HindIII. Competitor fragments were incubated in the reactions in molar excess as indicated and were generated as follows: (B) BamHI digestion of pUC18 containing the 40-bp region; (C) Sall digestion of pUC18 containing a synthetic octamer binding site cloned into the Sall site; (D) 46-bp fragment generated from an AluI digestion of pUC18. B and F at the left of each panel denote bound and free,

respectively. DNase I footprint analysis. The fragment (32P-labeled on the coding strand) was incubated with the 50% ammonium sulfate-precipitated fraction on ice prior to DNase I addition. Another aliquot was subjected to the A+G sequence reaction of Maxam and Gilbert (29) to define the boundaries of protein binding. We observed protection from DNase I cleavage at position -579 extending through position -608 from the start site of transcription (Fig. 3). The sequence between -591 and -608 in this region is imperative for binding protein by Southwestern blot analysis (see Fig. 5). Taken together, protein binding is probably confined to the first 29 bases (-608 to -579) of the 40-bp region. We obtained the footprint with the ammonium sulfate-precipitated fraction but were not able to observe evidence of protection by using crude nuclear extract preparations. The chicken vimentin negative regulatory element binds a protein of 95 kilodaltons. Because we have shown by gel retardation assays and DNase I footprinting that the 40-bp silencer region binds a protein, we attempted to determine

A NEGATIVE ELEMENT IN VIMENTIN GENE EXPRESSION

VOL. 10, 1990

its molecular weight by using the DNA-binding site as a probe against proteins immobilized to nitrocellulose filters (Southwestern blotting). Proteins from the 0.1 to 0.2 M KCl fraction were separated on a 10o SDS-denaturing polyacrylamide gel. The size-separated proteins were blotted to nitrocellulose and probed with a 32P-labeled fragment or annealed oligonucleotides containing the 40-bp sequence. Our 40-bp region binds to a protein of approximately 95 kilodaltons (kDa), as judged by its comparison to molecular mass standards (Fig. 4A). A predominant band is visible at 95 kDa when crude HeLa nuclear extracts (data not shown) are used, but we did observe some nonspecific binding at higher molecular masses. Because this molecular mass is close to the previously described molecular mass of the transcription factor Spl, we probed our 0.1 to 0.2 M KCI fraction with an annealed 32P-labeled GC box. We did not detect binding to this DNA, as expected, since Spl is known to elute at a higher salt concentration on heparin-agarose (7; Fig. 4B). To confirm the fact that we were observing the result of a protein-DNA interaction, we subjected our fractions to several enzymatic treatments before electrophoresis. Binding was not affected by incubation at 37°C with DNase, RNase, or DNase plus RNase but was abolished by treatment with proteinase K (Fig. 4C). These results confirm that the binding is due to a DNA-protein interaction and not due to a spurious DNA-nucleic acid interaction. To confirm that our sequence did not bind to any given protein extract, we analyzed a whole-cell extract of bacteria, in which we would not expect binding, and an extract from yeast, one that may give binding, with our probe. As shown for bacteria (Fig. 4C) and yeast (data not shown), we did not observe binding in either case. The result in Fig. 4C indicates that the 95-kDa protein is not found in bacteria or yeast; however, we did observe binding in several cell lines assayed in the laboratory,

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FIG. 4. Southwestern blots of HeLa and mouse L-cell nuclear extracts probed with radiolabeled DNA. A 100-pLg portion of a 0.1 to 0.2 M KCl HeLa nuclear fraction (A, B, and C) was separated on a 10o SDS denaturing polyacrylamide gel and transferred to nitrocellulose filters as indicated in Materials and Methods. All filters were probed with a 32P-labeled annealed synthetic 40-bp (-608 to -568) region, except the GC box (panel B), which was probed with an annealed synthetic GC box (14-mers). Enzymatic treatments in panel C were carried out at 37°C for 20 min before loading onto the gel and are indicated. The lane labeled Bacteria was prepared from a whole-cell extract of Escherichia coli. A 100-p.g portion of a nuclear extract made from HeLa or mouse L cells, as indicated (panel D), was probed with the 40-bp

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2354

FARRELL ET AL. A.

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FIG. 5. (A) Replica Southwestern blots probed with the 40-bp silencer region and a perfect octamer-binding site. Parallel lanes of 100 ,ug of HeLa nuclear extract were separated as described in the legend to Fig. 4. The nitrocellulose filter containing identical loadings of extract was cut in half and probed separately with a 32P-labeled annealed synthetic 40-bp (-608 to -568) region or with an annealed synthetic octamer-binding site. Equal counts per minute (5 x 106) were added to each bag and processed in the same fashion. The identity of the probe used for each blot is indicated. (B) Replica blots are as described for panel A, except that probes used for binding are as indicated in the

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including mouse L cells. To compare the relative amount of the 95-kDa protein found in HeLa to mouse L cells, we prepared extracts from equivalent amounts of cells and subjected equal amounts of protein from each to analysis. Both extracts exhibited protein binding in the 95-kDa range, yet much more binding was apparent in HeLa cells (Fig. 4D). By use of scanning densitometry, a 12-fold increase in signal intensity was observed in HeLa cells, indicating that the protein is more abundant in HeLa cells. To further rule out the possibility that we were binding to the octamer binding protein in our Southwestern blots, we analyzed replica blots with 32P-labeled probes of the 40-bp region and an octamer-binding site. We observed binding to a 95-kDa protein with the 40-bp probe and an absence of binding with the octamer fragment (Fig. 5A). Analysis of the footprint in Fig. 3, showing that the entire 40-bp region is not protected from DNase I cleavage, suggests that only a part of the region is necessary for protein binding in the Southwestern blots. To address this point, we generated oligonucleotide fragments to the sequences flanking the possible octamer-binding site and assayed for their ability to bind to the 95-kDa protein, as described above. Annealed oligonucleotide fragments containing the sequence -568 to -581 exhibited little or no binding, while the annealed oligonucleotide fragments containing the sequence -608 or -591 bound to an appreciable amount (Fig. 5B). The 95-kDa protein may be developmentally regulated in chicken embryos. When nuclear extracts from tissues of chicken embryos were probed with the 40-bp region, the 95-kDa protein was found in several tissues, especially muscle, where the drop in vimentin expression during myogenesis is well characterized (36, 48, 56). We attempted to observe whether there was any correlation between silencer protein level and vimentin gene activity. We incubated chicken eggs from fertilization through 9 days of age posthatch. Tissues were collected from gizzard and skeletal muscle starting with day 11 of the embryonic period. Nuclear extracts and RNA were prepared at this time point and at subsequent time points thereafter, as indicated in the legend to Fig. 6. The extracts were subjected to Southwest-

ern blot analysis with our 40-bp sequence as described previously, and RNA was quantitated by Northern blot analysis by using a chicken vimentin cDNA probe (pE8) (55). Skeletal muscle and gizzard both exhibit the wellcharacterized drop in vimentin mRNA production during chicken embryogenesis (Fig. 6, N). The appearance of two bands on a Northern blot are due to the utilization of multiple poly(A) sites 3' to the gene (56). For skeletal muscle, we observed strong hybridization at the 11-day embryo stage and almost no signal by day 1 posthatch (Fig. 6). Gizzard, although containing less vimentin mRNA, shows similar down regulation, with little or no signal by 6 days of age. Parallel with these observations was an increase in binding intensity on the Southwestern blot during embryogenesis. We observed a weak signal at the 11-day embryo stage in both tissues that intensified and remained strong through hatching (Fig. 6). This observation would suggest that the protein is produced early in embryogenesis and is present throughout early stages of chicken development. The actual time of appearance of this protein cannot be accurately measured by the Southwestern blot method, since obtaining discrete tissue from chicken embryos before 11 days is not feasible.

DISCUSSION In this study, we have localized in the 5'-flanking region of the chicken vimentin gene a negative regulatory region at -568 to -608 from the start site of transcription. Previously, we characterized a proximal promoter region extending from -161 to +1 and a proximal enhancer element between -321 and -161 (47). When increasing lengths of the chicken vimentin 5'-flanking region were fused to CAT and transfected into cells, a more complex regulatory pattern developed. A construct containing all of the 5'-flanking region sequenced to date, extending to 767 base pairs from the start site of transcription, yielded activity that was approximately double the activity of a construction containing only the proximal promoter, pcV-161. Recent data from transfections in which constructs containing fragments upstream of -767

A NEGATIVE ELEMENT IN VIMENTIN GENE EXPRESSION

VOL. 10, 1990

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FIG. 6. Developmental expression of vimentin and the 95-kDa protein during chicken embryogenesis. Gizzard and skeletal muscle mRNA and nuclear extracts were isolated and prepared as indicated. Results from Northern blot hybridization with a chicken vimentin cDNA probe (E8) is indicated by N, and Southwestern blot hybridization with the radiolabeled 40-bp element is indicated by SW.

were used give CAT activity analogous to that of pcV-321 (unpublished observations). This result suggests that the chicken vimentin gene contains a distal enhancer region. Similar results have been found for the human vimentin gene

(39).

When pcV-608 was transfected into mouse L cells, we observed a tremendous drop in CAT activity from pcV-767. The lowest activity obtained was 95% less than that of pcV-321 and only fivefold greater than that of p8CAT. pcV-321 yields an activity of over 118-fold greater than that of p8CAT. When a 40-bp sequence was removed from pcV-608, generating pcV-568, we obtained an activity of 52-fold. This resulting increase in CAT activity by the simple removal of 40 bp suggests that this sequence contains an element that suppresses CAT activity. This result is also observed in HeLa cells. We were not surprised to find similar, yet lower, CAT activities in our HeLa transfections, for several reasons. Firstly, vimentin is the only intermediate filament that can be coexpressed with another intermediate filament in a given cell type, despite its embryological origin, i.e., HeLa is derived from cervical epithelium tissue and expresses both the expected intermediate filament cytokeratin and vimentin. For this reason, vimentin is often permissive in many cell types where one would expect to find only the tissue-specific expressed intermediate filament (12). Secondly, vimentin gene expression is often turned on or activated when cells are grown in culture (12). Yet the most convincing evidence for HeLa cells yielding results similar to those of mouse L cells is the fact that HeLa cells grown in suspension or monolayer express vimentin, albeit at different levels, as evidenced by Northern blot analysis with a human vimentin cDNA probe (data not shown). Not surprisingly, vimentin mRNA is more abundant in L cells as compared with HeLa. This would support the conclusion that the relative CAT activities of the transfections are an accurate reflection of what is occurring in the given cell type. Since removal of the 40-bp sequence does not restore CAT activity to pcV-321, this suggests that other negative-acting sequences may be present between -568 and -321. This conclusion is further supported by the fact that some sequence similarity to the 40-bp sequence is found between -568 and -321. A 17- out of 22-bp match to the negative

element sequence (-605 to -583) is found between -484 and -460 from the start site of transcription. This is the region of the negative element that binds protein by DNase I footprinting experiments (Fig. 3). It is possible that another auxiliary negative element may reside in this region. Preliminary results indicate that fragments containing sequences -484 to -460 bind a protein of 95 kDa. Furthermore, constructs containing this homologous region are currently being generated in order to assess their ability to repress transcription. In addition to the ability of the 40-bp region to suppress CAT activity in the context of the chicken vimentin 5' region, it can also suppress CAT activity driven by a heterologous promoter. When one copy of the region is inserted upstream of the HSV tk promoter, CAT activity is decreased 36%, whereas two copies decrease activity 68%, and concatamers of the region decrease activity 90%. It appears this region is able to function in a position-independent manner, since it is able to suppress activity out of the context of the chicken vimentin gene and is orientationindependent, since it can function in a sense or antisense orientation in ptkCAT. Evidence that the 40-bp region is a site of protein binding is shown by gel retardation, DNase I protection, and Southwestern blots. Competition assays indicate that the retarded species is readily competed with the unlabeled 40-bp element but not with nonspecific DNA such as the octamer-binding sequence or plasmid DNA. In some cases, a minor slowermobility band is observed in our gel mobility assays. This band is more readily competed with nonspecific DNA, and, therefore, it is not known whether it is from binding the same factor or some additional factor. DNase I digestion exhibits a protection pattern of approximately 30 bp which is in a very A+G-rich region. Southwestern blot analysis indicating that this region binds specifically to a protein approximately 95 kDa in size, which is easily detected in a variety of cells, leads us to believe that the protein may be a general negative-acting factor found in many cell types and that it probably exerts its effect on several genes. Interestingly, we were not able to detect protein binding to our 40-bp vimentin fragment in extracts made from Swiss 3T3 cells. This conclusion may be explained by the finding by Northern blot

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FARRELL ET AL.

liii III GAAAGC -476 CAGCO3CITI7 -464 1I II 11111 1111 11 11 11 kGCAAGOSIGOOCIM7 -608 aGu III 11111 II 1111111 HE=n Vimetin

A negative element involved in vimentin gene expression.

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo,...
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