The EMBO Journal vol.9 no.4 pp. 1 123 - 1130, 1990

Synergism in ternary complex formation between the dimeric glycoprotein p67SRF, polypeptide p62TCF and the c-fos serum response element Hennrik Schroter, Chris G.F.Mueller, Klaus Meese and Alfred Nordheim Zentrum fur Molekulare Biologie (ZMBH), Universitat Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg, FRG Communicated by H.Schaller

Transcriptional regulation of the c-fos proto-oncogene requires the serum response element (SRE) which is complexed by a multi-protein assembly observed both in vitro and in vivo. Two protein factors, p67SRF and p62TcF (previously called p62), are required to interact with the SRE for efficient induction of c-fos by serum. By quantitative band shift electrophoresis we measure at least a 50-fold increase in SRE affinity for p67SRF/p62TCF over p67SRF alone. Stoichiometrically we determine that the ternary complex with p62TCF involves 67SRF is a in dimeric form. We demonstrate that p675 glycosylated nuclear transcription factor carrying terminal

N-acetylglucosamine (GlcNAc) as a post-translational modification. A proteolytic limit digestion product, -13 kd in size, was generated from the p67SRF_ SRE complex. This p67SRF-core domain binds SRE, can dimerize with p67SRF and is still able to form a ternary complex with p62TCF. Therefore, three functional activities can be ascribed to this small p67SRF-core domain: specific DNA binding, dimerization and interaction with p62TF. We demonstrate that these functions map within the p67SRF core fragment containing the region between amino acids 93 and 222. Key words: synergy/complex formation/p67SRFp62TcF/ c-foslserum response element

recently able to identify a new protein, xgolypeptide p62, which specifically binds to the SRE-p67 DNA -protein complex (called cI) and demonstrated that the ability to form the resulting ternary complex (called cH) was a requirement for efficient gene induction by serum (Shaw et al., 1989b). To distinguish polypeptide p62 from other SRE-binding proteins of comparable size (Ryan et al., 1989) we shall in future refer to this protein as Ternary Complex Factor p62 (or p62TCF) Our genomic footprinting analysis suggested that in vivo p62TCF contacts the SRE structure as part of a multi-protein complex (Herrera et al., 1989). The latter study also revealed that during induction, elongation and subsequent repression of c-fos transcription no changes in the pattern of SRE protein occupancy are discernable. Since this finding may suggest important regulatory roles for protein -protein interactions between DNA bound and free proteins a better understanding of these interactions is required. Toward this aim, we present here further structural detail on p67SRF and its interaction with p62TCF in forming the cII ternary complex with the SRE. We demonstrate that p67SRF exists as a glycoprotein and in that way is similar to other eukaryotic transcription factors (Jackson and Tjian, 1988, 1989; Lichtsteiner and Schibler, 1989). Our experiments also show that stoichiometrically the SRE ternary complex involves p62TCF and a dimer of p67SRF. Three functionalities of p67S , namely specific SRE recognition, dimerization and interaction with p62TCF, are all found to be located within a small p67SRF proteolytic core domain of 13 kd in size. More specifically, in vitro synthesized fragments of p67SRF locate these functions within an internal segment spanning amino acids 93-222. -

Results Introduction The cellular c-fos proto-oncogene is rapidly induced to high transcriptional activity upon treatment of quiescent cells with serum growth factors (Greenberg and Ziff, 1984; Kruijer et al., 1984; Muller et al., 1984; for review: Curran, 1988; Shaw et al., 1989a). It represents the best studied member of the class of 'rapid response' genes (Almendral et al., 1988 and references therein) which are subject to stringent transcriptional control. The essential cis-acting 5' element for c-fos induction by growth factors and other extracellular stimuli is the serum response element (SRE) (Treisman, 1985, 1986; Gilman et al., 1985; Greenberg et al., 1987). The human c-fos SRE is recognized specifically by the serum response factor (SRF or p67SRF) (Treisman, 1986), a nuclear protein with an apparent molecular weight of 67 kd which has been purified to homogeneity (Prywes and Roeder, 1987; Schroter et al., 1987; Treisman, 1987). The structure of the protein-bound SRE, however, is likely to be quite complex. We were

Polypeptide P67sRF purified from chloroquine extracts of HeLa nuclei is glycosylated Chloroquine-released proteins from HeLa nuclei contain polypeptides p67SRF and p62TCF which together form the specific ternary complex cdI over c-fos SRE sequences (Shaw et al., 1989b; Figure IA, lanes 1, 2 and 9). Passage of a nuclear chloroquine extract over an agarose column carrying the lectin wheat germ agglutinin (WGA) leads to quantitative retention of p67 RF. The protein can be released by 0.1 M N-acetylglucosamine (GlcNAc) thereby recovering binding activity for formation of complex cI, the SRE-p675 complex (Figure IA, lanes 4, 5, 8). No such activity is found in the WGA column flow through (lane 3). However, this flow through contains p62TCF activity since it is able to form complex cIl when mixed with either the WGA-retained p67SRF (lane 6) or purified p67SRF (lane 7). Thus, employing WGA - agarose permits complete separation of polypeptides p6751w and p2 . To obtain further evidence for glycosylation of p67 F we subjected this protein to in vitro labelling by [3H]galactose using the enzyme galactosyl 1 123

H.Schroter et al.

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Fig. 1. (A) p67SRF but not p62TCF is bound by the lectin WGA. In gel retardation experiments a nuclear chloroquine extract can form complex cII with an SRE-containing oligonucleotide probe in the presence (lane 1) or absence (lane 2) of added p67 RS. Chloroquine extract was applied to a WGA-agarose matrix and unbound proteins were washed off with 200 mM KCI-buffer A (flow through) and analysed in lane 3. After a further wash with 1 M KCI-buffer A the matrix was equilibrated to 100 mM KCI-buffer A and was subsequently eluted with this buffer containing 0.1 M GlNAc. The eluate was bound to the oligonucleotide directly (lanes 4, 5 and 8) or in a mixture with flow through proteins (lane 6). Flow through proteins were also recombined with affinity purified p67SRF (lane 7). Lane 9 contains affinity purified p67SRF and p62TCF as control for complex clI. The reactions of lanes 1-7 contained poly(dI-dC) DNA carrier in amounts of 80, 80, 80, 40, 8, 80 and 80 ng/4l, respectively. Lanes 8 and 9 had no carrier. (B) 3H-galactosylation of p67SRF. 3Hgalactosylated p67SRF was analysed by SDS-PAGE followed by fluorography. Prior to galactosylation the protein was purified either by lectin affinity chromatography followed by a single SRE-affinity chromatography step (lane 2) or by three consecutive passages over the SRE-affinity matrix (lane 1). Molecular weights of marker proteins are indicated by arrows. (C) Galactosylated p67pRF binds to the SRE. p67SRF (lane 1) and 3H-galactosylated p67SRF (lanes 2-5) were analysed for SRE binding by gel retardation and fluorography. The proteins were bound to unlabelled or radioactively labelled DNA probes A according to the tabulated scheme below. Proteins used in lanes 2-4 and lane 5 are displayed in (B), lanes 2 and 1, respectively. All reactions were done in the presence of 0.5 jig of

poly(dI-dC). Lane

1

p67SRF [3Hlgal_p67SRF

x

[32P]DNA probe A Unlabelled DNA probe A

x x

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3

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polypeptide p62TCF to the lectin column was not observed. Synergism in formation of the ternary complex cil In our original identification of p62TCF (Shaw et al., 1989b) we provided evidence that this protein alone cannot bind to DNA sequence-specifically, although general DNA binding activity can be observed by DNA-cellulose chromatography (unpublished). Instead, p62TCF specifically recognizes the SRE -p67SIF DNA -protein complex to form the ternary complex cII. We characterized this interaction further by quantitative DNA-binding studies (Figure 2, A -C) using both gel purified renatured and partially purified native proteins. p67wRF and p62TCF were bound to SRE-containing oligonucleotides either individually or mixed in varying ratios. Gel purified and renatured p67SRF specifically binds the DNA probe to form complex cI (Figure 2A, lane 1). In contrast, no specific complex is formed by similarly purified p62TCF (lane 3). However, if these same amounts of protein are mixed, a cdI band is generated with an intensity which exceeds that of cI formation several-fold (lane 2). This reveals synergistic interaction between SRE, p67S'F and p62TCF leading to higher structural stability of the p62TCFmediated complex cdI versus cI. Equally, this demonstrates how p62TCF, which alone is not able to bind DNA sequencespecifically, is attracted to this specific DNA site at which

p67SRF is positioned.

This observation was investigated further in titration experiments using native, partially purified protein preparations. Within two different ranges of concentration, a fixed amount of p62TCF was titrated with increasing amounts of p67SRF (Figure 2A, lanes 4-7; Figure 2B, lanes 5-10; Figure 2C, lower panel, dotted curve). Complex cIl is formed very efficiently and formation of cI is observed only when all p62TCF is bound, i.e. when cII has reached a plateau level. The rapid, non-linear ascent to the plateau level of cdI is apparent in the quantitation shown in Figure 2C (lowerpanel, dotted line). In contrast, titrations of a mixture of p67sF plus p62TCF (Figure 2B, lanes 4-1) or p67SRF alone (Figure 2B, lanes 11 - 15) display linear increases in formation of complexes cdI and cI, respectively (for quantitation, see Figure 2C, top panel and bottom panel/ connected triangles respectively). We proceeded to determine the equilibrium dissociation constants for the formation of complexes cI and cII, respectively. The reaction is formulated as: k,

transferase. As shown in Figure 1B, specific labelling of a 67 kd protein was obtained with p67l which had been purified either with SRE DNA affinity chromatography (Figure 1B, lane 1) or by a WGA column (lane 2). The labelleda roteins shown in Figure lB displayed the activity of p67S by SRE DNA binding assays (Figure IC). The 3H-labelled proteins generated complex cI with either 32plabelled (Figure IC, lane 4) or unlabelled (lane 2) SREcontaining DNA probes in the same way as did purified, untreated p67sRF (lane 1). No cI band was generated in the absence of SRE probe (lane 3). Our data provide evidence that chloroquine-extracted p67SRF is a nuclear glycoprotein and also demonstrate that [3H]galactose labelling of this protein does not inhibit its DNA-binding characteristics. The analysis did not reveal any non-glycosylated subfractions of this protein. Binding of 1124

SRE +

(p67SRI)2

+

p62TCF =

cI +

k2 p62TCF = clI

(1)

By quantitative band shift electrophoresis (Figure 2D) we obtain the values k, = (5.3 0.01) x 10-10 M and k2 < 10" M (see Materials and methods). The affinity of the SRE-p67SIF complex is therefore increased at least 50-fold upon addition of p62TCF. Generation of p67SRF%core: a DNA binding proteolytic

fragment

As was previously demonstrated with the papillomavirus BPV-l E2 protein (Dostatin et al., 1988) and the actin gene CArG box binding factor (Boxer et al., 1989a), proteolytic treatment of the respective DNA -protein complexes generated a limit digestion product which retained the ability to specifically interact with its DNA-binding site.

Ternary

p67SRF, p62TCF and SRE complexes

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band shift Fig. 2. (A) Synergistic interaction of p67SRF and p62TCF leading to formation of complex cHI. Gel-purified, renatured proteins used in this of constant titration a show 4-7 Lanes alone 3). (lane p62 2), p62 (lane plus 1), (lane alone p67SRF with the SRE-containing oligonucleotide: p67SRF solution, 5 and 4 3, Al p675RF 2, contain 7 and 6 5, 4, (lanes amounts of partially purified p62TCF with increasing amounts of purified p67SRF of respectively). (B) Protein titrations in gel retardation experiments using the SRE-containing oligonucleotide. Lanes 1-5: titration of a mixture 3, 4 1, 2, in lanes 1 and 1 used 2, were Al 4, (6, and S67SRF of mixture p62TCF the purified of partially amounts plus Decreasing p67SRF p62TCF. 5 lane for used as mixtures the To previously RF. protein with F p67 plus of a mixture of p62T titration p67SRF and 5, respectively). Lanes 6-10: In of titration p67SRF Lanes and 9 1-l15: 10, respectively). lanes 8, 6, 7, SRF in increasing amounts of p67SRF were added (1, 2, 3, 4 and 5 IJ p67 The the absence of added p62TCF the same amounts of p67SRF were used as in lanes 6-10. (C) Quantitation of the gel retardation signals of (B). dotted upper panel represents the data of (B), lanes 4, 3, 2 and 1 which assayed a titration of the p67SRF plus p62TCF mixture. The lower panel, of the curves Both -l15. lanes of 1 (B), the quantitation represents triangles connecting curve the lanes 5-1O; in curve, measures complex cII (B), for and cI k2 for panel) (upper complex constants dissociation k, of lower panel represent titrations with p67SRF. (D) Determination equilibrium lines complex cIl (lower panel). Scatchard plots for the binding of p67SRF to the SRE (upper) and p62TCF to complex cI (lower) are shown. The We drawn are the least-square fit to the data points. The insert in the lower panel shows the gel retardation of complex cI titrated with p62TCF. determine the values k, = (5.3 :1 0.01) x 1O-10 M and k2 ' x 10"- M.

We employed the same strategy with p67SF. Figure 3 shows that over a range of proteinase K concentrations a limit digestion product of p67SRF can be obtained (p67SRFI core) which retains the ability to specifically bind the SRE. We then generated p67SRF-core on a preparative scale by binding p67SRFto the SRE-affinity column (Schroter et al., 1987) and incubating it with proteinase K. Preparative SDS -PAGE of such affinity purified p67SRF-core and renaturation of the gel extracted material permitted us to determine its approximate molecular weight as 13 kd (data not shown). This finding contrasts with the estimate of Boxer et al. (1989a) which suggested a molecular mass for SRFcore in the 30 kd range. -

p67SRF-core can dimerize to form a heterodimer with p67SRF

Norman et al. (1988) demonstrated that SRF binds DNA as a dimer. The availability of affinity purified p67OS1-core protein enabled us to perform DNA binding studies with mixtures of p67SRF and p67SRFIcore in order to determine the stoichiometries in their complexes with SRE probes. Figure 4A demonstrates that the complexes formed with native proteins of p67SRf (i.e. p67ww; lane 1) or p67SRFcore (i.e p67cc; lane 2) are the only complexes obtained when a mixture of the two proteins is offered to the DNA

(lane 3). A new complex, p67cw, is found, however, when the proteins are mixed in denatured form and are permitted to co-renature prior to DNA binding (lanes 6, 7, 9 and 12). The yield of complex p67cw is dependent on the relative concentrations of denatured p67S and p67SRF-core at the time of renaturation (lanes 6, 7, 9 and 12). In addition, measurements of gel migration distances of these complexes (Figure 4C) and knowledge of the molecular masses of the involved proteins permits us to assign the p67cw complex as the heterodimeric form of p67SRF and p67SRI-core (Bading, 1988). The migration of p67cc does not unambiguously prove this complex to contain a dimeric form of p67 RFcore, since the deviation from linearity in migration of this complex (Figure 4C) would also be compatible with the presence of the monomeric protein component. However, the apparent high affinity and stability of this complex (Norman et al., 1988) and the associated occupancy of both palindromic half sites of the SRE, as assayed by DNase I footprinting (P.E.Shaw, unpublished) and methylation interference studies (Gustafson et al., 1989), argue for dimeric protein arrangement in p67cc. A schematic representation of complexes p67cc, p67cw and p67ww is shown in Figure 4B, center. This analysis provides evidence that the protein domain of p67SRF-core harbours both the DNA binding and dimerization functions. 1125

H.Schroter et al. 4

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by lane 8, the addition of p62 to the heterodimer p67cw also leads to formation of a slower migrating ternary complex, p67cw/p62 (compare lanes 7 and 8). This complex migrates at the same position as p67ww (see lanes 1, 3 and 13) and its identity as p67cw/p62 is shown in Figure 4E. The finding that ternary complexes with p62 can be formed

I

with both p67cc and p67cw reveals that the p67SRFIcore domain also contains the function for p62 binding, in addition to DNA binding and dimerization.

if;67;;:-_

Locating the p67SRF region that mediates p62TCF binding

1 2 3 4 5 6

Fig. 3. Digestion of complex cI with proteinase K. Complex cI was formed for a gel retardation assay in the presence of 2.5 mg/ml BSA. Lanes 1 and 6: no addition of proteinase K. Lanes 2-5: complex I was digested at room temperature for 10 min with 4, 1, 0.5 or 0.1 g proteinase K, respectively, in a 12 iLl reaction. PMSF was added to 5 mM before electrophoresis.

Protein stoichiometries in ternary complexes of SRE,

p62TcF and p67SRF proteins The availability of three different types of p67SRF dimers in complex with SRE (for schematic see Figure 4B, center) which can be separated electrophoretically (Figure 4A, lane 9) and which all can be bound by p62 (lanes 4, 5 and 8), permits us now to determine whether the ternary complex formed upon addition of p62TCF contains p67StF in monomeric or dimer form. In the former situation one would expect only two types of ternary complex to be generated (p67w/p62 and p67c/p62) whereas the latter scenario would give rise to three types of ternary complex (p67ww/p62, p67cw/p62, p67cc/p62) (see Figure 4B for graphic display of these interpretations). We find that three types of ternary complex are formed (lane 10; compare lanes 10 and 9) and therefore deduce that the ternary complex with the c-fos SRE contains a dimer of p67RF plus p62TCF. In addition, the measured migration distances are consistent with p62TCF being monomeric within the ternary complex (Figure 4C). Our gel system does not resolve the cI complex containing the dimericp67ww from the cdI ternary complex containing p67cw/p62 F (lanes 9 and 10). To unambiguously prove our assignments, we used preparative gel electrophoresis to obtain in pure form the three types of cI complex containing either p67ww, p67cw or p67cc (Figure 4D, lanes 1-3). To each of these gel-embedded complexes (Figure 4E; lanes 3, 5 and 7) p62MF was added and the electrophoretic banding positions of the respectively generated ternary complexes were observed (Figure 4E; lanes 4, 6 and 8).

To map within p67SRF the region which mediates the activities of dimerization, SRE binding and p62TCF interaction various N- and C-terminal truncations of the SRF cDNA were constructed and the corresponding polypeptides synthesized in vitro. Synthetic full length SRF can bind to the SRE (Figure 5, lane 7) and interact with p62TCF to generate the ternary complex (lane 8). Removal of N- and C-terminal protein segments, 1-69 and 223 -508, preserves all three SRE functionalities mentioned above (lanes 9-14). Preliminary results suggest that a further SRF derivative, only encompassing region 93-222, also retains these activities (data not shown).

Discussion

CF

The p67SRF-core domain can specifically interact with polypeptide p62TCF We investigated whether the specific ternary complex between SRE probes, p67SRF and p62TCF (Shaw et al., 1989b; Figure 4A, lane 4) could also be established with p67SRF-core protein replacing the intact p67SRF. As shown in Figure 4A (lane 5) the p67cc complex can indeed by converted into the slower migrating p67cc/p62 ternary complex upon addition of p62. Furthermore, as evidenced

1126

The human c-fos SRE is bound by the SRF (p67SRF) (Treisman, 1986) to form the specific DNA-protein complex cI. This cI complex is bound by polypeptide p62TCF to generate the ternary complex cII (Shaw et al., 1989b). For a better understanding of the macromolecular interactions that underly c-fos transcriptional control here we present further biochemical characterization of the p67SRF protein itself and its interplay with polypeptide

p6TCF*

Glycosylation of eukaryotic transcrption factors Binding to the lectin WGA and enzymatic galactosylation by galactosyl transferase identify the RNA polymerase II transcription factor p67SRF as a glycoprotein. The binding specificity of WGA, the displacement of p67SRF from the lectin column by 0.1 M N-GlcNAc, and the efficient transfer of [3H]galactose by galactosyl transferase argue for p67SRF being posttranslationally modified by terminal GlcNAc residues. Glycosylation has recently been demonstrated with other transcription factors, including Spl (Jackson and Tjian, 1988), CTF (Jackson and Tjian, 1989) and HNF1 (Lichtsteiner and Schibler, 1989). Agarose matrices carrying covalently bound WGA proved an efficient tool for the selective enrichment of p67SRF. This lectin chromatography step quantitatively separated p67SRF from p62TCF. At present the biological functions regarding glycosylation of p67SRF

remain unclear. Functions due to this modification may include protein transport to the nucleus (Baglia and Maul, 1983), assembly of multimeric protein complexes (Holt et al., 1987; Shaw et al., 1989b; Herrera et al., 1989) or interference with protein phosphorylation (Holt et al., 1989). The latter modification has already been demonstrated to occur on

p67SRF (Prywes et al., 1988; Ryan et al., 1989)

and influence its DNA binding characteristics (Prywes et al.,

1988).

p67SRF, p62TCF and SRE complexes

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Fig. 4. Protein stoichiometries in complexes involving the SRE-containing oligonucleotide. (A) The proteins used for complex formation are indicated for each lane (top part of figure) and are denoted as follows: p67ww (homodimer of p67SRF), p67cw (heterodimer of p67SRF and p67SRF-core), p67cc (homodimer of p67SR -core) and p62TCF. Proteins were used either in partially purified, native form (indicated by filled circles) or in denatured/renatured form (open circles; relative amounts of protein are indicated by two different sizes of circle). (B) Schematic representation of cII ternary complexes (left, right) which can be postulated to occur when p67SRF-SRE cI complexes (center) are bound by p62W. Protein components of the complexes are denoted as in (A), whereby the additional p67w/p62 and p67c/p62 represent p62-bound monomers of p67SRF or p67SRF-core, respectively. (C) Migration of DNA-protein complexes in a native retardation gel. The measured distances of migration of all complexes of (A) are plotted versus the molecular weights of their calculated protein content (logarithmic scale). The dotted line indicates that deviation from a linear migration relationship with respect to p67cw and p67ww is observed if p67cc is here assumed to be the dimeric form of p67SRF-core (see text). (D) Preparative gel retardation to separate SRE-protein complexes containing p67ww, p67cw and p67cc. Lane shows gel retardation with the material used for (A), lane 7, which contained p67cw and p67cc. Lane 2 shows complex cI formed with p67cc alone and lane 3 with p67ww. The wet gel was autoradiographed over night at 4C and gel slices of the bands indicated by arrows were recovered. (E) Formation and identification of cII complexes by incubation of cI complexes with polypeptide p62. Gel slices containing cI complexes, as obtained in (D), were cut into two halves and incubated for h at 4C either in the absence or presence of p62. The gel slices and liquid of the incubation mix were applied directly to the slots of an analytical gel for retardation analysis. Lanes 3, 5 and 7: cI complexes containing p67cc, p67cw or p67ww, respectively, incubated without the addition of p62. Lanes 4, 6 and 8: same complexes as in lanes 3, 5 and 7, but incubated in the presence of p62. For comparison, the SRE complexes with p67cc and p67cw dimers, prepared in solution according to Materials and methods, were incubated in the presence or absence of p62 and run in lanes and 2, respectively. 1127

H.Schroter et al.

with the PRTF protein and DNA control elements of mating type-regulated genes in Saccharomyces cerevisiae (Tan et

+

+

+

+

+

+

+

_=

.4.,

al., 1988).

In attempting to obtain kinetic data for formation and association of complexes cI and clI our experimental system did not permit accurate quantitation of the observed high on-rates. However, half-life measurements of cI (in the presence of up to 1000-fold molar excess of SRE binding sites) or cII (in the presence of equal molar amounts of cI) revealed tl/2 = 1.6 min and tl/2 = -2.5 min, respectively (data not shown). If cII was analysed instead in the presence of excess p62TCF plus excess free SRE a tl/2 = 20-30 min was measured. This extended half-life supports the postulated pathways for a stepwise assembly of cII via cI, as formulated in equation (1). An interaction between the SRE and a heterodimeric (p67SRF/p62TCF) protein entity is made

unlikely.

2 1 2 3 4 5 6 7 8 9 10 t1 12

13 14

Fig. 5. Mapping of the internal p67SRF region which interacts with p62TCF RNAs coding for the entire p67 protein or segments of it spanning amino acid coordinates 1-273, 70-244 or 70-222 were synthesized and translated in vitro. The resulting protein products were bound to the SRE either alone or together with biochemically purified p62TCF (+lanes). In vitro synthesized translation products were: full length p67SRF (lanes 7, 8), region 1-273 (lanes 9, 10), region 70-244 (lanes 11, 12), region 70-222 (lanes 13, 14). The reticulocyte lysate unprogrammed by RNA is shown in lanes 5, 6. p67SRF purified from cells, was used for comparison in intact form (lanes 1, 2) or as proteolytic p67SRF_core (lanes 3, 4). The shaded segment in the graphic display of p67SRF represents the 90 amino acid segment of high homology with the S.cerevisiae PRTF DNA binding protein (Norman et al., 1988).

Stoichiometry of SRE- protein complexes involving

P67SRF and p62TCF

Using SRF protein generated by in vitro transcription/translation Norman et aL (1988) determined that this protein binds the dyad symmetry element (DSE) of the SRE as a dimer. Our analysis with protein material purified from HeLa cell nuclear extracts confirms the dimeric nature of SRE-bound p675RF. Generation in vitro of dimeric forms of p67s'F could only be achieved upon denaturation and co-renaturation of the polypeptides, in line with the study of Norman et al. (1988) which used co-translation to achieve dimerization. This suggests a very high stability of preformed p67SRF dimers. We also show that it is the dimeric form of DNAbound p67SRF which is contacted by polypeptide p62TCF to form the ternary complex cII. In addition, the gel migration behaviour of cII suggests that p62TCF is bound as a monomer. This divergence from structural 2-fold symmetry in formation of cII may be attributed to additional DNA-protein contacts upstream of the DSE (Shaw et al., 1989b). Cooperation of p675RF and p62TCF By determining the equilibrium dissociation constants associated with complexes cI and cII we find that p62TCF, which under no conditions tested is able to recognize SRE sequences by itself, contributes significantly to stabilization of the multicomponent SRE complex. The observed 50-fold smaller dissociation constant of cII over cI compares very well with the MATalI stabilized ternary complexes formed 1128

We observe that under conditions of excess SRE the halflife of cII is short (-2.5 min) in the presence of equal amounts of cI and long (20-30 min) if we provide excess p62TCF protein (not shown). From this we deduce the occurrence of rapid exchange of the p62TCF moiety since the p62TCF protein dissociated from clI appears to be rebound quickly before dissociation of the cI intermediate has occurred. Therefore it seems possible that such exchange of p62TCF can also occur in vivo. This might suggest a potential mechanism for c-fos induction by which active and inactive forms of p62TC are exchanged rapidly in situ. Such an exchange process would be compatible with our previous in vivo foot printing data (Herreva et al., 1989) which did not detect altered DNA -protein interactions at the SRE during induction and subsequent autorepression of

c-fos.

Localization of p67SRF functions within the internal

region 93-222 p67SRF is a ubiquitous nuclear transcription factor which has been implicated to function in both positive (Treisman, 1985, 1986; Greenberg et al., 1987; Mohun et al., 1987; Gilman, 1988; Shaw et al., 1989b; Siegfried and Ziff, 1989; Walsh, 1989) and negative (Leung and Miyamoto, 1989; Konig et al., 1989; Shaw et al., 1989c; Subramaniam et al., 1989) regulation of c-fos expression. Its DNA binding characteristics to SRE sequences have been well characterized in vitro (Gilman et al., 1986; Treisman, 1986; Mohun, 1987; Prywes and Roeder, 1987; Schroter et al., 1987) and in vivo (Herrera et al., 1989). We have shown that in addition to binding DNA p67SRF interacts specifically with polypeptide p62iCF which is essential for efficient c-fos induction by serum (Shaw et al., 1989b). The isolation of cDNA clones encoding SRF (Norman et al., 1988) and their expression in vitro allowed demonstration that this protein, 508 amino acids in length, recognizes the symmetrical SRE sequence as a protein homo-dimer and that efficient DNA

binding and dimerization is retained by a truncated polypepcontaining only SRF amino acids 122 -222 (Norman et al., 1988). The proteolytic p67WRF-core domain, which we demonstrate here to also be able to dimerize with the intact protein and to specifically bind the SRE, is of 13 kd in molecular weight. Polypeptide p675sR-core is additionally able to interact with polypeptide p62TCF. This locates three important functionalities within the relatively small p67SRFcore protein domain: specific DNA binding, dimerization tide

-

Ternary

and interaction with p62TCF. Our use of in vitro synthesized truncated p67SRF derivatives maps all three activities within an internal region encompassing at least amino acids 93-222. This includes the 90 amino acid long region of homology to the yeast transcription factor PRTF (Norman et al., 1988; Passmore et al., 1988). It has already been shown that the two proteins have overlapping DNA binding specificities (Hayes et al., 1988) and that they interact with additional protein components when themselves bound to DNA (Bender and Sprague, 1987; Keleher et al., 1988; Tan et al., 1988; Herrera et al., 1989; Shaw et al., 1989b). DNA -protein interactions involving the c-fos SRE An important segment of the c-fos SRE is the DSE, essential components of which are also found in the promoters of muscle and non-muscle actin genes (Minty and Kedes, 1986; Mohun et al., 1987; Walsh and Schimmel, 1987), cellular 'rapid-response' genes (Chavrier et al., 1988) and S. cerevisiae cell type-specific genes (Johnson and Herskowitz, 1985; Bender and Sprague, 1987). Binding proteins that directly contact these sequences have been identified in each case (Bender and Sprague, 1987; Miwa et al., 1987; Mohun et al., 1987; Walsh and Schimmel, 1987; Tan et al., 1988) and relationships between those factors have been investigated (Norman et al., 1988; Boxer et al., 1989b; Ryan et al., 1989; Walsh, 1989). The c-fos DSE can be directly contacted by at least two proteins: p67SRF (Treisman, 1986, 1987; Gilman et al., 1986; Greenberg et al., 1987; Prywes and Roeder, 1987; Schroter et al., 1987) and another protein which seems to contact only one half-site of the DSE (Ryan et al., 1989; Walsh, 1989). The latter protein has an approximate molecular weight of 62 kd (Ryan et al., 1989). Although apparently of the same molecular mass this protein is not identical with polypeptide p62TCF (Shaw et al., 1989b). The present study confirms this conclusion by providing further biochemical detail on the interaction of p62TCF with SRE-bound p67SRF. Functionally we have already shown that this interaction correlates with efficient induction of c-fos by serum (Shaw et al., 1989b). Our in vivo genomic footprinting analysis in human A431 cells revealed an SRE methylation protection pattern which was indistinguishable from the one formed in vitro by the clI ternary complex with p67SRF and p62TCF (Herrera et al., 1989). This is consistent with p62TC being part of the active SRE regulatory complex in vivo. It therefore appears that p62TCF is functionally involved in gene regulation during c-fos induction, albeit with as yet unknown mechanisms. Work is now in progress to understand in functional and structural terms precisely how p67SRF, p62TCF and additional proteins bound immediately downstream of the SRE cooperate in forming the multi-protein complex (Herrera et al., 1989) which confers the tight control of c-fos gene expression.

Materials and methods Chromatography of p67SRF on SRE-affinity and WGA - agarose matrices

SRE-affinity chromatography (Schroter et al., 1987), gel retardation using the SRE-containing oligonucleotide probe A, and renaturation of or p62TCF from gels have been described before (Schroter et al., 1987; Shaw et al., 1989b). For lectin affinity chromatography of p67SRF a chloroquine extract from 3 x 109 HeLa cell nuclei was generated, concentrated

p67SRf

p67SRF, p62TCF and SRE complexes

1 ml and dialysed according to Schroter et al. (1987). This material was to phosphocellulose chromatography. brought to 40 mM KCI and subjected was eluted with 1 M KCI in buffer A (20 mM HEPES, The bound dithiothreitol, 0.1 mM PMSF pH 7.9, 0.1% NP-40, 0.2 mM EDTA, to1 mM and 20% glycerol) and applied directly a WGA-agarose matrix (0.3 ml volume; Sigma). Washing of this column with buffer A packed column equilibration with buffer A (containing (containing 1 M KCI) was followed by of buffer 0.1 M KCI). Bound proteins were eluted with 3 column volumes A (containing 0.1 M KCI, 0.5 M GlcNAc). The proteins obtained were in the presence of 50 applied to SRE-affinitywithchromatography buffer A (containing 1 M KCI). For subsequent and eluted to Jackson and Tjian (1988), buffers were [3H]galactosylation, according G-25 spin-column gel filtration. Sephadex exchanged by

to

p67SRF

dg/ml

poly(dl-dC)

Quantitative band shift analysis of dissociation constants were performed Binding reactions for determination retardation (Schroter et al., 1987), except in 80 mM KCI and analyzed by gel that no unspecific poly(dI-dC) DNA carrier was used. The dissociation constant k, was determined by titrating a constant amount of p67SRF with labelled SRE binding sites. k2 was measured by titration of constant amounts of labelled cI with p62TCF in the presence of a 10-fold molar excess of labelled SRE. Quantitation of complexes was achieved by direct radiation measurements of excised and dried gel segments containing the relevant material. For calculating k2 values the data obtained for cIand cdI within were always normalized over the averaged total complex formation the series of binding reactions. Both dissociation constants were determined by Scatchard blot analysis and calculation of the least-square fit line of the primary data. The error given is the standard deviation of the experimental data about the least square fit line. In determining k2±we obtained in1 two independent sets of experiments 1 the values of (0.52 0.31) x 10- M and (-0.3 1.3) x 10 M. M. 10-11 of statement the values These k2 ' permit

Preparation of p67SRF-core was enriched by WGA chromatography and applied to an SREp67SRF matrix (see above). After a wash with 10 column volumes of buffer affinity A (adjusted to 50 mM KCI, no PMSF), the SRE-bound p67SRF was of 0.1 mg/mil proteinase K (Boehringer, digested in 2 column volumes 2.5 of in the mg/ml bovine serum albumin (BSA) for Mannheim) presence

10 min at room temperature. The matrix was washed with 20 volumes ice cold buffer A (containing 50 mM KCI, 0.5 mM PMSF) including a protease leupeptin (0.5 inhibitor cocktail containing aprotinin (2 A wash with 10 and pepstatin (0.5 (1 a2-macroglobulin followed was M 0.3 by elution A KCI) buffer of column volumes (containing of p67SRF-core with 3 volumes of buffer A (containing 1 M KCI).

1tg/mn), pLg/ml).

unit/mi)

g/mli),

p67SRF-core

In vitro dimerization of p67sRF and fractions were denatured in Laemmli sample buffer and treatment for 15 min at 56°C. The proteins were (Laemmli, 1970) by heat then precipitated with acetone and renatured as described (Wang et al., 1987).

p67SRF

Partial

p67SRF_core

purification

of polypeptide

p62TCF

We find the majority of p62 in the flow through of the phosphocellulose separation of chloroquine extracted proteins (see above). Subsequently,

DNA-cellulose chromatography followed by affinity chromatography on in a p67SRF-containing SRE matrix was performed and will be described

detail elsewhere

(Schroter et al., manuscript in preparation).

Construction of SRF expression

plasmids

All plasmid DNA manipulations and preparations were according to standard kindly provided by R.Treisman (London). techniques. The SRF cDNA waswere based on Bluescript KS (Stratagene). The SRF expression vectors with the rabbit ,B-globin 5' untranslated region (provided by A.Annweiler and R.A.Hipskind) inserted between polylinker KpnI and Hindlll sites

(pT3G). The SRF coding region was excised by Hael (nucleotide 353) and HindII coli expression (nucleotide 1968) cuts. It was subcloned into Escherichiadownstream of a vector pUHE 25-2 (Bujard, manuscript in preparation) T3 ribosomal binding site (25 bp long) preserving the SRF ATG and a Kozak consensus (Kozak, 1986). For the construction of pT3G-SRF the SRF coding region and the T3 RBS were then inserted into a pT3G between polylinker RI and SmaI sites. For expression of the SRF region 70-244 a NarI-Bgll fragment (nucleotide 564-1090) was subcloned into E.coli expression vector T3 ribosomal binding site. pUHE22 downstream of theframe with polylinker ATG (ATG AGG GAT The SRF fragment was in CCG TCG TCG ACG

...

SRF). This SRF construct with the T3 ribosomal

1129

H.Schroter et al. binding site was then cloned into EcoRI-SmaI sites of pT3G

(pT3G-SRF70/244).

Polymerase chain reaction (PCR) DNA fragments coding for SRF segments 1-273 and 7-244 were obtained by the PCR method. For SRF amino acid 1-273 PCR was performed on pT3G-SRF using T3 primer (Stratagene) and primer A (5'-GGATCCCGGCAGGTTGGTGACTGTGA-3'). The SRF amino acid 70-222 fragment was generated with pT3G-SRF70/244 as template, using T3 primer and primer B

(5'-TGGCGAGTTGAGGCAGGTCTGAATCAG-3').

PCR was carried out with 1 ng linearized template DNA, 20 mM Tris-HCI, pH 8.0, 3 mM MgCl2, 0.05% Tween 20, 0.0005% NP-40, 200 jiM dXTP, 20 pmol of each primer and 2.5 U of Taq polymerase (AGS, Heidelberg) in a lOl 100 final reaction volume. The DNA was amplified on a thermal cycler (Techne, PHC-II) with 25 cycles denaturation (92°C, 20 s) annealing (45°C, 20 s) and extension (72°C, 1 min).

In vitro transcription/translation Linearized SRF expression vectors (1 /g) and all of the SRF PCR product was transcribed. The PCR-amplified DNA was purified over a gel prior to transcription. The transcription reaction (Melton et al., 1984) employed T3 RNA polymerase. Approximately 1 /g of cRNA was translated in rabbit reticulocyte lysate (Promega) according to manufacturer's description. The synthesis of SRF protein was followed by [35S]methionine incorporation and subsequent SDS-PAGE. Aliquots of the translation products were used directly for gel retardation assays in the presence of 100 ng of salmon sperm carrier DNA.

Acknowledgements We appreciate the provision of SRF cDNA by Richard Treisman (London). We thank R.A.Hipskind and A.Annweiler for support in establishing the in vitro transcription/translation methodology. During the course of this work many helpful suggestions were made by P.E.Shaw and R.A.Hipskind. We appreciate comments on the manuscript by R.E.Herrera and H.Schaller. Expert secretarial assistance by H.Demuth is gratefully acknowledged. This work was funded by the Bundesministerium fur Forschung und Technologie through grant BCT-0381-5 and the Fonds der Chemischen Industrie.

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Received on

August 10, 1989; revised on December 22, 1989