(Received for publication, August 18, 1995; and in revised form, November 28, 1995)
From the
Basal as well as induced transcription from the human urokinase-type plasminogen activator gene requires an enhancer containing two elements, a combined PEA3/AP-1 and a consensus AP-1 site. The integrity of each of these binding sites as well as their cooperation is required for activating transcription. The two elements are separated by a 74-base pair cooperation mediating (COM) region required for the cooperation between the transactivating sites. The COM region contains binding sites for four different unidentified urokinase-type plasminogen activator enhancer factors (UEF 1 to 4), all four required for correct COM activity. We have purified UEF3 from HeLa nuclear extracts by several chromatographic steps including DNA affinity purification. Purification and UV cross-linking data showed that UEF3 is a complex of three polypeptides (p40, p50, and p64). Amino acid sequence from one peptide of p64 was obtained, which showed no homology to other known proteins. Both crude and purified UEF3 specifically bound to the sequence TGACAG as shown by electrophoretic mobility shifts and methylation interference studies. DNA-binding specificity of purified UEF3 was identical to that of NIP, a non-characterized factor binding and regulating multiple AP-1-regulated promoters like stromelysin and interleukin-3. Thus UEF3 appears to be a general DNA-binding factor involved in modulating the transcriptional response of AP-1 containing promoters.
Urokinase plasminogen activator (uPA) ()is a serine
protease and a key enzyme in the proteolytic cascade activating
plasminogen and thereby leading to degradation of the extracellular
matrix (Blasi and Verde, 1990). The uPA gene is expressed
constitutively in the ectoplacental cone during embryo implantation, in
kidney and lung in the adult (Larsson et al., 1984; Sappino et al., 1989). Expression of the uPA gene is induced in
inflammatory and tumoral pathological conditions, and appears to be
regulated by a very large number of growth factors and cytokines (Blasi
and Verde, 1990). Transcriptional regulation of the human uPA gene
relies upon an enhancer element positioned 2 kilobase pairs upstream of
the transcriptional start site (Cassady et al., 1991; Nerlov et al., 1991; Rørth et al., 1990; Verde et
al., 1988). Two elements within the enhancer have been shown to be
essential for its activity: a combined PEA3/AP-1 site similar to that
of the collagenase gene, and a 74-base pair downstream consensus AP-1
site (Gutman et al., 1990; Nerlov et al., 1991;
Rørth et al., 1990) (see Fig. 1A).
These sites are important for both basal level and induced enhancer
activity (Cassady et al., 1991; Nerlov et al., 1991;
Rørth et al., 1990) and appear to cooperate to activate
transcription.
Figure 1: Structure of the uPA, IL-3, and stromelysin promoters. A, organization of the uPA enhancer, showing the upstream combined PEA3-AP-1 site, the downstream AP-1 site, and the interspacing COM element divided into the functional regions, u-COM and d-COM. Binding positions for the different UEFs within the u-COM and d-COM are indicated. B, comparison of overall structure similarity between the uPA enhancer PEA3-AP-1 and UEF3 region with regions from the interleukin-3 and stromelysin promoters containing an inducible AP-1 site.
A cooperation mediating (COM) element is positioned
between the combined PEA3/AP-1 and the AP-1 site (Nerlov et
al., 1992). The COM element does not appear to have
transactivation-mediating activity by itself, but is important for
synergistic activation of the PEA3/AP-1 and AP-1 sites. DNase
footprinting and site-directed mutagenesis have identified two areas of
the COM region (u-COM and d-COM) important for the function of the
enhancer. Four different so far unidentified nuclear factors, uPA
enhancer factors (UEF) 1 to 4, appear to bind to these regions.
Mutations within the COM element, affecting the binding of the
different UEFs impair both inducibility (Nerlov et al., 1992)
as well as basal level activity ()of the enhancer.
Promoters of the interleukin-3, LD78, and stromelysin genes (Mathey-Prevot et al., 1990; Sirum-Connolly and Brinckerhoff, 1991; Nomiyama et al., 1993) are also regulated via AP-1 sites, and appear to have sequence homologies to the uPA enhancer not only in the transactivating elements, but also in the COM region (Fig. 1B). In particular, a NIP site is present, close to both PEA3 and AP-1 sites in the stromelysin promoter (Sirum-Connolly and Brinckerhoff, 1991) and to AP-1 and Elf-1 sites in the interleukin-3 promoter (Gottschalk et al., 1993; Mathey-Prevot et al., 1990). The activity of the NIP sites in these promoters appears to be that of modifying the efficiency of elements responding to the transcription-inducing signals. In this view, and because of the sequence homologies, one or more UEF factor of the uPA enhancer might correspond to the NIP-binding proteins. In this paper we have purified and preliminarily characterized UEF3 and shown that it recognizes a TG(A/G)CAG sequence, common to both the uPA COM region and the NIP element.
Active fractions from two sequential heparin-Sepharose columns were pooled, dialyzed twice against 2500 ml of H2K150 for a total of 12 h, then cleared by centrifugation. The extract was passed through a 6-ml mutant O-1 Sepharose column at 15 ml/h. Column flow-through with an absorbance higher than 0.025 absorbancy units was collected. The column was washed with 18 ml of H2K150 and step eluted with H2K1000 (same buffer but with 1000 mM KCl). Fractions of 1 ml were collected. The flow-through from the mutant column was supplemented with 25 mg of poly(dI-dC) and cleared by centrifugation. The load was applied to a 3-ml O-1 Sepharose column at a flow rate of 15 ml/min, after which the column was washed with 20 ml of H2K150 and 40 ml of H2K200 and eluted with an 18 ml of H2K200 to 1000 gradient, collecting fractions of 1 ml. Fractions were screened both by EMSA and SDS-PAGE. By both criteria, fractions were selected for the final step. Fractions from the O-1 Sepharose chromatography were pooled and dialyzed against 500 ml of H1N100 (H1 buffer with 100 mM NaCl) for 4 h. The dialysate was spun and loaded onto a 1.6/5 Mono-S column (Pharmacia SMART system) at 100 µl/min. The column was washed with 2.5 ml of H1K100 at 100 µl/min and eluted with 1 ml of H1K100 to 1000 gradient at a flow rate of 25 µl/min. Fractions of 50 µl were collected.
EMSA was performed with purified
UEF3 and the partially methylated oligonucleotides as probe. The wet
gel was blotted to DE-81 paper as described for UV cross-linking and
bands corresponding to UC, LC, and free probe were cut out and eluted
with 1.5 M NaCl and recovered by ethanol precipitation. The
oligonucleotides were resuspended, extracted twice with
phenol/chloroform, precipitated, washed, and dried followed by cleavage
with piperidine. The samples were dried and resuspended in formamide
loading buffer. 10 cpm of each sample were loaded on a 20%
acrylamide gel containing 8 M urea and 0.5
TBE. The
gel was run at 40 W (1 W/cm) for 90 min, dried, and exposed as
described previously.
Figure 2: UEF3 binds to identical sequences contained within the O-1 and O-17 oligonucleotides. Electrophoretic mobility shift assay with HeLa nuclear extracts and radiolabeled O-1 (lanes 1-9) or O-17 (lanes 10-18) probes. Unlabeled competitor oligonucleotides were used at 50- and 500-fold excess, as indicated. The two bands corresponding to UEF3, UC, and LC, are indicated together with UEF-1 and -4. Binding of the previously identified UEF2 was not observed with the extract used in this study.
Figure 3: Initial fractionation of HeLa nuclear extracts. A, S-Sepharose chromatography. Lower panel, optical density profile showing the column flow-through (0-300 ml) and elution (300-600 ml) together with the theoretical salt gradient and collected fractions. AU, absorbance units at 280 nm. Top panel, EMSA assay on 1 µl of each fraction, in the presence of 2 µg of poly(dI-dC) and labeled O-1 oligonucleotide. NE, nuclear extract; FT, flow-through; numbers indicate fraction number. B, heparin-Sepharose fractionation profile. Lower panel, absorbance profile of the column flow-through (0-340 ml) and step elution (340-450 ml); AU, absorbance units at 280 nm. Top panel, EMSA analysis, as above. L, load; FT, flow-through; numbers indicate fraction numbers. C, SDS-PAGE analysis of fraction from the initial fractionation steps after mutant DNA Sepharose. 0.5 µl of each fraction was used. NE, nuclear extracts; SS L, S-Sepharose load; HS L, heparin-Sepharose load; 0-1 mut L, load of the O-1mut chromatography; FT, flow-through of the same; f4, fraction 4 of the same. 8 fractions of 1 ml were collected from the elution of the O-1mut Sepharose. Fraction 4 was the peak fraction containing the majority of the proteins. Molecular weight markers are indicated. The gel was silver stained.
The flow-through from the mutant O-1m column was supplemented with poly(dI-dC), loaded on the O-1 Sepharose column and eluted with a 200 to 680 mM KCl gradient (Fig. 4). The fractions were screened by EMSA (Fig. 4A) and SDS-PAGE analysis (Fig. 4B). UEF3 activity was eluted from 270 to 450 mM KCl, correlating with the observed absorbance peak. Active fractions displayed major bands of 40, 50, and 64 kDa on SDS-PAGE. The enriched 64-, 50-, and 40-kDa bands will from now on be referred to as p64, p50, and p40. Based on EMSA and SDS-PAGE, fractions 5-12 were selected for the last chromatography step. These fractions were pooled, dialyzed, and applied onto the PC 1.6/5 Mono-S column for the SMART system (Fig. 5A). The column was eluted with a 100-1000 mM KCl gradient. Screening of the fractions by EMSA showed that UEF3 eluted as a small peak in fractions 11-14 from 220 to 350 mM KCl. SDS-PAGE analysis, Fig. 5B, showed that fractions 11-13 which had the highest activity also had the highest level of p64, p50, and p40. The less active adjacent peak, fractions 14 and 15, contained other polypeptides including a 116-kDa band. The presence of this protein band correlated with a nonspecific binding activity seen by EMSA (arrow in Fig. 5A) which did not co-migrate with UC and LC.
Figure 4: Fractionation of UEF3 by O-1 DNA-Sepharose chromatography. A, lower panel: O-1-Sepharose absorbance profile showing the column flow-through (0-150 ml), the 200 mM KCl wash (150-225 ml), and the elution (225-250 ml). Inset, blow-up of elution profile from 225 to 245 ml showing the absorbance of the collected 1-ml fractions together with the theoretical salt gradient. AU, absorbance units at 280 nm. Top panel, 1 µl of each fraction were analyzed by EMSA with 0.5 µg of poly(dI-dC) and labeled O-1 oligonucleotide. L, column load; FT, flow-through; numbers indicate fraction number. B, SDS-PAGE analysis of O-1-Sepharose fractions. 1 µl of each fraction was used. L, load; FT, flow-through; numbers, fraction numbers. Molecular weight markers are indicated. The gel was silver stained.
Figure 5: Fractionation of UEF3 by Mono-S column chromatography. A, lower panel: absorbance profile showing the elution of the Mono-S column with the measured salt gradient and the collected 50-µl fractions. AU, absorbance units at 280 nm. Top panel, 0.5 µl of each fraction were analyzed by EMSA with 0.05 µg of poly(dI-dC) and labeled O-1 oligonucleotide. L, column load; FT, flow-through; numbers indicate fraction number. The N arrow indicates the position of the nonspecific band observed. B, SDS-PAGE analysis of O-1-Sepharose fractions. 0.2 µl of each fraction was used. Numbers indicate fraction numbers. Molecular weight markers are indicated. The gel was silver stained.
The result of this last chromatographic step indicates that p64, p50, and p40 all participate in the binding activity of UEF3. Table 2summarizes the purification. This procedure gave a calculated yield of more than 1.7% as measured by UEF3 binding activity in EMSA and a purification of almost 5,000-fold. The major loss of UEF3 activity occurred after fractionation by O-1-Sepharose chromatography. The last Mono-S step gave another 5-fold loss. However, it is our experience that UEF3 loses activity upon purification and as such, the overall yield may be higher than the calculated 1.7%.
Figure 6: UV cross-linking analysis of the binding of UEF3 to O-1 oligonucleotide. A, sequence of the 5-bromodeoxyuridine (BrdUrd) substituted O-1 oligonucleotide showing the positions of BrdUrd on both strands. All three BrdUrd substitutions lie within the TGACAG motif. B, SDS-PAGE analysis of the UV cross-linked adducts. UC and LC were separated by a preparative EMSA with labeled BrdUrd substituted O-1 oligonucleotide, exposed to UV irradiation, and loaded onto an SDS gel. Molecular weight markers are indicated. Cross-linked adducts of 80 and 65 kDa in the UC lane and of 80 and 55 kDa in the LC lane are indicated. Some 65-kDa adduct observed in the LC lane are likely to be a comtamination from the UC band. High molecular weight adducts of 200 and 190 kDa in the UC and LC lane, respectively, are also observed.
In conclusion, we have found evidence for a direct DNA interaction of the three proteins present in purified UEF3. The 55-kDa cross-linked adduct may correspond to p40, the 65-kDa adduct to p50, and the 80-kDa adduct to p64. Furthermore, the cross-linking data also give information on the composition of UC and LC. In fact, Fig. 6shows that LC contains p40 and p64 while UC contains p50 and p64. As such, UC and LC would be formed by p40 or, respectively, p50 with p64 as a common factor. The suggested composition of UC and LC also seems to be supported by the comparison of EMSA and SDS-PAGE analysis as seen from Fig. 5, A and B. Fraction 11 from the Mono-S column contained mainly UC activity and was mainly composed of p64 and p50. Similarly, LC-enriched fraction 13 displayed mainly p40 and p64.
Figure 7: Methylation interference analysis of UEF3 binding to O1 oligonucleotide. A, methylation interference reaction. Methylated O-1 oligonucleotide labeled on either the top strand or the bottom strand was used with purified UEF3 in a preparative EMSA. The DNA from recovered UC or LC bands was cleaved and separated on a denaturing sequencing-type gel along with unbound (F) probe. The sequence of the oligonucleotide corresponding to the observed bands is shown. Filled arrows show the bases where strong interference was observed; open arrows, weak interference; open arrows with asterisk, weak interference specific for UC only. Band marked X is an artificial band co-migrating with the dye-front. B, summary of the methylation interference on UC and LC. Closed and open triangles indicates strong and weak interference, respectively.
Figure 8: UEF3 binds the NIP element with the same specificity of u-COM. Purified UEF3 was incubated with labeled IL-3 NIP element (sequence in Table 1) and 0.1 µg of poly(dI-dC) and separated by EMSA. Binding of UC and LC to the NIP element is indicated. Competition was carried out with a 50- and 500-fold excess of unlabeled NIP and O-1, and with a 500-fold excess of O-1m oligonucleotides, respectively.
Figure 9: Reverse phase (C4) column chromatography analysis of the p64 band of UEF3. Vydac 218TP54 column (internal diameter 4.6 mm, 250 mm height) was eluted at a flow of 1 ml/min with a linear gradient of solution A (0.1% trifluoroacetic acid) and buffer B (0.1% trifluoroacetic acid in 70% acetonitrile) (see ``Materials and Methods''). The percentage of buffer B is indicated by the straight line. The left ordinate show the optical density at 214 nm (AU, absorbance units), the abscissa the elution time in minutes. The arrow indicates peak 14 which gave the sequence shown in Table 3. The other peaks identified with filled squares show other peptides used for microsequencing (see ``Results'').
A 106-base pair conserved enhancer is located about 2 kilobase pairs upstream of the transcription start site in the human, mouse, and porcine urokinase genes (see Fig. 1A). This enhancer has been shown to mediate both the high basal transcription activity of the human, as well as the induction by phorbol 12-myristate 13-acetate and epidermal growth factor in the mouse and the human uPA genes (Verde et al., 1988; Rørth et al., 1990). The activity of the uPA enhancer is dependent on the integrity of each of two protein binding sites: the upstream combined PEA3-AP-1 site and the downstream consensus AP-1 site (Rørth et al., 1990; Nerlov et al., 1991). These two sites are separated by the 74-base pair COM sequence, made up of two regions endowed with specific protein binding activity which overall bind 4 different factors (UEF 1-4). Three mutations in the COM element affect the inducibility of the uPA gene, and the combined mutations totally destroy enhancer activity (Nerlov et al., 1992). A further characterization of the different UEFs is therefore important to understand the function of the COM element. In this paper we have addressed the nature of UEF3, since this factor seems also to bind similar regulatory sites in other promoters. In three promoters, uPA, stromelysin, and IL-3, the UEF3 binding site maps close to a consensus AP-1 site and to a site binding Ets-family proteins (Fig. 1B) (Gottschalk et al., 1993; Mathey-Prevot et al., 1990; Nerlov et al., 1991; Rørth et al., 1990; Sirum-Connolly and Brinckerhoff, 1991), suggesting similar regulatory features.
In the O-17
oligonucleotide spanning the u-COM element, the UEF3 binding site
overlaps with binding sites for UEF1, -2, and -4. ()We have,
however, identified an oligonucleotide (O-1) in another region of the
uPA promoter (-1500 to -1470), that binds solely UEF3. By
EMSA, UEF3 forms two complexes, UC and LC, which are common to O-1 and
O-17. Competition and mutagenesis studies suggest that both complexes
constitute the same DNA binding activity, UEF3 (Fig. 2).
We have purified UEF3 from HeLa cell nuclei to near homogeneity by different standard chromatography steps, including DNA affinity. The purified UEF3 has the same DNA-binding properties of the HeLa nuclear extract as it binds the same sequence, forms two complexes UC and LC, and is affected by the same mutations. The purification shows that UEF3 is formed by three polypeptides: p40, p50, and p64. All three polypeptides are able to interact with DNA, as shown by the UV cross-linking experiment, with p64 giving the weakest binding. Both UV cross-linking data and the Mono-S chromatography elution profile suggest that UC was formed by p64 and p50 subunits, LC by p64 and p40. The two complexes may thus have the p64 subunit in common. This implies that the purified UEF3 should contain p64 in a 2:1:1 ratio with respect to p40 and p50, which is actually seen by silver-stained SDS-PAGE (Fig. 5B).
A 5,000-fold purification and a yield of
1.7% show that UEF3 is a low abundant factor. It is important to note,
however, that all activity and recovery measurements are based on the
EMSA data. In fact, after passage through the mutated DNA column, we
observed an increase in specific activity which indicates that
interfering factors partially masked the true activity of UEF3 (Table 2). In addition, after specific DNA chromatography, the
purified UEF3 rapidly lost its activity. Despite these limits, the
purification procedure presented here gave a purification and yield
comparable to procedures employed to purify NF-B factors (Hansen et al., 1994).
UC and LC can be partly separated by S-Sepharose chromatography, reflecting the differences in the two complexes. However, during the course of purification, we were never able to completely separate UC and LC binding activities, neither using other types of ion exchange and general affinity chromotography nor by specific DNA-affinity matrices different from the one described here. This suggests that the protein composition of UC and LC are very similar both in structure as well as DNA binding. Since the UC and LC contain the same p64 subunit, but differ in the p50 versus p40 subunit, we speculate that p50 and p40 may be products of very similar genes or arise from alternatively spliced mRNA from the same gene. A third possibility is that p40 is a cleaved form of p50. This degradation may, however, be physiological since we always observed an equal presence of UC and LC, both when using freshly prepared as well as older nuclear extracts (not shown).
The purification procedure gave a sufficient yield of purified UEF3 to obtain a 17-amino acid residue sequence by microsequencing of the p64 polypeptide. This sequence was obtained after tryptic digestion and therefore probably represent an internal sequence of p64. After another purification we have tried N-terminal sequencing of all of the three subunits: p40, p50, and p64. In all cases the sequencing failed, probably due to blockage of the N-terminal of these polypeptides. Searches in data bases for homologous peptide sequences revealed none, showing that p64 is a novel protein and that UEF3 as such is a novel factor.
We have mapped the binding site for UEF3 by mutation and methylation interference studies. These demonstrate that the sequence TGACAG is the core binding site for UEF3. Mutation or methylation of 5 out of 6 nucleotides in the TGACAG motif interfered with binding of UEF3. In addition, methylation interference showed that nucleotides flanking the TGACAG motif may also participate in binding. These flanking bases are, however, not conserved in the O-1 and O-17 oligonucleotides. If this weak interference is caused by nonspecific steric hindrance or by methylation of bases important for the interaction of UEF3 with its binding site remains to be determined. Searching the transcription factor data base for known factors able to bind the UEF3 sites did not reveal any, supporting the possibility that UEF3 is a novel factor.
The UEF3 binding site, however, has a five out of six nucleotide homology to the NIP binding site (TGACAG versus TGGCAG). The NIP element binds a so far unknown factor that acts as an AP-1 dependent repressor of transcription in the IL-3 promoter (Mathey-Prevot et al., 1990) and as an AP-1 activity modulator in the stromelysin promoter (Sirum-Connolly and Brinckerhoff, 1991). In addition, the UEF3 binding site is homologous to a negative regulatory element, the ICK-1 element, found in promoters of the human LD-78 (Nomiyama et al., 1993) gene and in its murine counterpart, the MIP-1A gene (Ritter et al., 1995). The ICK-1 element binds four different complexes, of which one is a negative factor co-migrating with the NIP activity in EMSA (Nomiyama et al., 1993). The base change in the NIP element occurs at a position which does contact UEF3 (Fig. 7). However, purified UEF3 specifically binds the NIP element (Fig. 8). Our data therefore show that UEF-3 is one of the NIP-binding factors. We are now proceeding with the molecular cloning of cDNAs encoding the various subunits of UEF3, and thereby investigate the nature of this novel factor and the mechanisms involved in regulating transcriptional activity of various promoters.