The EMBO Journal vol.10 no.13 pp.4219-4229, 1991
A protein domain conserved between yeast MCM 1 and human SRF directs ternary complex formation
Chris G.F.Mueller and Alfred Nordheim Institute for Molecular Biology, Hannover Medical School, PO Box 610180, D-3000 Hannover 61, FRG Communicated by A.Nordheim
MCM1 and SRF bind to the same DNA sequence and form ternary complexes with STE12 and p62TCF, respectively. We show that in gel retardation assays, MCM1 recruits both ternary complex factors whereas SRF interacts only with p62ICF. A protein domain of 90 amino acids, shared by MCM1 and SRF, was found to be sufficient for ternary complex formation. The domain is also required for dimerization and DNA binding. Similar regions are found in other proteins, such as ARG80, Deficiens and Agamous. ARG80 and Agamous exhibit similar DNA binding specificities but do not interact with either STE12 or p62TCF. By exchanging three residues of ARG80 with those of corresponding positions in SRF (residues 198, 200 and 203), the ARG80 protein acquires the ability to recruit p62TCF into a ternary complex. Likewise, the substitution of four SRF amino acids by MCM1-derived residues (amino acids 73, 75, 77 and 78) confers on SRF the ability to interact with STE12. Thus, we have identified specific amino acids in MCM1 and SRF that are critical for ternary complex formation and which map to equivalent positions within the shared domains. Therefore, the structural basis for specific protein- protein interaction appears to be conserved in evolution between a class of transcription factors. Key words: c-fosIp62TCF/STE 12/transcriptional regulation/ yeast mating type control
Introduction It has become a widely recognized phenomenon that transcription of many genes is controlled by multicomponent protein complexes that are in specific contact with DNA (for review see Shaw, 1990). The herpes simplex virus capsid protein gene VP16, for instance, controls the expression of a number of host genes by interacting with the DNA binding protein Oct-I (Gerster and Roeder, 1988; O'Hare and Goding, 1988; O'Hare et al., 1988; Stern et al., 1989). A comparable ternary complex, i.e. a protein -protein complex stabilized by a DNA-bound protein component, was shown to be involved in the regulation of the mammalian c-fos gene (Shaw et al., 1989a; Schroter et al., 1990). Recently, cyclin A and the retinoblastoma susceptibility protein (Rb) were reported to form ternary complexes with transcription factors (Mudryj et al., 1991; Bandara et al., 1991; Chellappan et al., 1991). DNA-specific multicomponent protein complexes are also found in the mating control of the yeast Saccharomyces cerevisiae, where the protein MCM 1 (GRM or PRTF) plays Oxford University Press
a pivotal role (Passmore et al., 1988; Ammerer, 1990). It is a specific DNA binding protein, recognizing the DNA sequence CC(A/T)6GG (termed CArG box) (Hayes et al., 1988; Passmore et al., 1989). MCM1 controls the expression of a number of different genes by recruiting specific transcription regulatory proteins. An MCM1-a2 complex leads to the repression of a-specific genes, whereas the interaction with a l activates another set of genes (aspecific genes) (Strathern et al., 1982; Sprague et al., 1983; Bender and Sprague, 1987; Tan et al., 1988; Keleher et al., 1988, 1989; for recent review see Dolan and Fields, 1991). In addition, MCM1 forms a ternary complex with the protein STE12, which is involved in conferring transcriptional induction by mating pheromones (Errede and Ammerer, 1989). STE12 itself recognizes the DNA sequence ATGAAACT (pheromone response element or PRE), which is found in the upstream regulatory region of several a-, aand haploid-specific genes (Van Arsdell and Thorner, 1987; Kronstad et al., 1987; Fields and Herskowitz, 1985; Dolan et al., 1989; Fields et al., 1988). In the c"ase of the a-specific gene STE2, the PRE is positioned adjacent to an MCM1 binding site and it was found that STE12 binds the PRE of the STE2 gene most efficiently as a ternary complex in cooperation with MCM1 (Errede and Ammerer, 1989). Parallels can be drawn between the yeast pheromone response and the serum response of mammalian cells. The stimulation of quiescent cells by serum is mirrored in the very rapid transient induction of c-fos transcription (Greenberg and Ziff, 1984; Kruijer et al., 1984; Muiller et al., 1984). c-fos promoter dissection studies have identified a DNA sequence (serum response element or SRE) that mediates this process (Treisman, 1985, 1986; Gilman et al., 1986; Shaw et al., 1989a; Rivera et al., 1990; for review see Treisman, 1990). The 30 bp element contains a sequence (DSE) which is recognized by the serum response factor (SRF) (Treisman, 1987; Prywes and Roeder, 1987; Schroter et al., 1987). The DSE resembles the MCM1 binding site in that it comprises a CArG box. It was shown that in addition to SRF, MCM1 can bind to the DSE, contacting the same bases as SRF (Hayes et al., 1988; Passmore et al., 1989). SRF was found to form a ternary complex over the SRE with another protein, the ternary complex factor p62TCF (Shaw et al., 1989a; Schroter et al., 1990). Genomic DMS footprinting in A431 and NIH 3T3 cells has revealed that the ternary complex is present before and after c-fos gene induction by EGF, serum or TPA (Herrera et al., 1989; Konig, 1991). It has been shown that the inability of SRF to recruit p62TCF results in impaired c-fos induction by serum in NIH 3T3 fibroblasts and by TPA in BALB/c 3T3 fibroblasts (Shaw et al., 1989a; Graham and Gilman, 1991). However, the precise function of p62TCF has yet to be determined. MCM1 and SRF have similar DNA sequence binding specificities and recruit ternary complex factors to promote gene expression. The two proteins exhibit strong amino acid
C.G.F.Mueller and A.Nordheim
conservation over a short protein region of only 90 amino acids (Norman et al., 1988). This region is also shared by other proteins, such as ARG80 (or ARG RI), Deficiens (DEF A) and Agamous (AG). ARG80 is involved in arginine metabolism in S. cerevisiae (Dubois et al., 1987). DEF A and AG are prototypical members of large homeotic gene families in plants and are involved in flower development (Schwarz-Sommer et al., 1990; Ma et al., 1991). Recently, the cDNA for Xenopus SRF was cloned, and it was found that human SRF and Xenopus SRF are identical over the conserved domain (Mohun et al., 1991). It has been shown that the SRF region of homology encodes the DNA binding and dimerization properties (Norman et al., 1988). This study aims to investigate the structural basis of ternary complex formation by comparing human SRF, MCM1, ARG80, DEF A and AG with respect to DNA binding and ternary complex formation with p62TCF and STE12. We found that all the proteins except DEFA have similar DNA binding specificities but only MCM1 and SRF could form ternary complexes. The conserved protein domains of MCM1 and SRF were sufficient to recruit STE12 and p62TCF, respectively. Furthermore, specific amino acids in MCM 1 and SRF were identified to be of critical importance for ternary complex formation. They are positioned at equivalent locations within the region of homology, which suggests that the underlying mechanisms for ternary complex formation of MCM 1 with STE12, and SRF with p62 F, are very similar.
Results We have chosen the gel retardation assay (Fried and Crothers, 1981; Garner and Revzin, 1981) to study human SRF, MCM1 and the related proteins ARG80, DEF A and AG in terms of DNA binding and formation of a ternary complex with p62TCF or STE12. All proteins were expressed using an in vitro reticulocyte lysate translation system or by using recombinant vaccinia virus, with the exception of p62T F, which was partially purified from HeLa cells. Typically, two main protein -DNA complexes were observed in the gel retardation assay: a higher mobility complex (complex I) which arises from SRF, MCM 1 or the related protein binding to the DNA probe alone, and a lower mobility complex (complex II) that corresponds to complex I assembled in a ternary complex with p62TCF or STE12. This is illustrated schematically in Figure 1A and Figure 2A. The corresponding complexes in a gel retardation assay with vaccinia-produced SRF, MCM1 and STE12 and HeLa cell partially purified p62TCF are shown in Figure 1C and Figure 2C (lanes 5 and 6). Vaccinia mock-infected cell extract did not give rise to probe retardation in the absence or presence of p62TCF or STE 12 (lanes 1 and 2). The core domains of SRF and MCM 1 form ternary complexes with p62TCF SRF binds as a homodimer to the dyad symmetry element (DSE) that comprises the CArG box (Treisman, 1986) (Figure iB). Two guanine bases at the 5' boundary of the DSE are necessary to attract p62TCF into the ternary complex (Shaw et al., 1989a) (Figure iB). The DNA binding and dimerization properties of SRF have been delineated to an internal region, spanning amino acids 133 -222, which is conserved between vertebrates, yeast and
plants (Norman et al., 1988) (Figure 7). We refer to this 90 amino acid protein region as the core domain. An SRF derivative spanning amino acids 93-222 has previously been reported to interact with the SRE and P62TCF (Schroter et al., 1990). We have now further truncated SRF to residues 132-222 (SRF core), and as shown in Figure IC, lanes 7 and 8, SRF core binds the SRE and forms a ternary complex with p62TCF. Unprogrammed reticulocyte lysate does not give rise to a complex over the SRE probe in the absence or presence of p62TCF (lanes 3 and 4). Consequently, we tested whether full-length MCM 1 and a core version of MCM1 (residues 1-98) behaved similarly to SRF with respect to DNA binding and ternary complex formation with p62TCF. High affinity binding of full-length MCM 1 to the DSE has been reported previously (Hayes et al., 1988 and Passmore et al., 1989). Indeed, the vaccinia-expressed full-length MCM1 bound the SRE and could assemble a p62TCF-specific ternary complex (lanes 9 and 10). This, together with the SRF core result, predicted that MCM1 core can also interact with both the SRE and p62TCF. This was in fact observed (lanes 11 and 12). In order to ascertain that complex II is not produced by a different protein present in the HeLa cell extract, gel purified p62TCF was tested for ternary complex formation on MCM1 core as well as SRF core and full-length SRF. All three proteins generated complex II with gel purified p62TCF The complex showed the same gel mobility as complex II formed with partially purified p62TCF (data not shown). The near identical mobility of MCM 1 core and SRF core complex I suggests that MCM1 core binds the probe as a dimer. Indeed, by co-translating MCM1 core and full-length SRF in a reticulocyte extract, MCM 1- SRF heterodimers were formed that bind to the SRE in gel retardation assays (C.G.F.Mueller, N.Kahlin and A.Nordheim, unpublished observations). Thus, for SRF and MCM1, three properties are co-localized within the core domain, namely dimerization, DNA binding and ternary complex formation with p62TCF.
MCM1 core but not SRF core forms a ternary complex with STE12 An MCM1 -STE12 ternary complex cannot be assembled over the SRE (data not shown). Therefore, a new DNA probe was designed, termed STE2*, which essentially represents the STE2 upstream regulatory sequence (Miller et al., 1985; Errede and Ammerer, 1989) with its CArG box replaced by that of the c-fos SRE (Figure 2B). The STE2* probe was tested for complex I and II formation with MCM1 and STE12. As shown in Figure 2C, lanes 5 and 6, the STE2* probe is bound by MCM1 and allows the assembly of a ternary complex with MCM1 and STE12. We then examined MCM1 core and found that it also interacts with STE12 (lanes 7 and 8). Unprogrammed reticulocyte lysate does not generate a complex over the STE2* probe in the absence or presence of STE12 (lanes 3 and 4). The identity of STE12 in the ternary complexes formed with fulllength MCM1 and the core derivative of MCM1 was confirmed by including an anti-STE12 antibody to the binding reaction. It resulted in the loss of complex II (data not shown). Thus, the MCM1 amino acids sufficient for ternary complex formation with p62TCF as well as STE 12 map within MCM 1 core. In contrast, neither full-length SRF
MCM 1 and SRF ternary complex formation
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Fig. 1. A. Schematic illustration of complexes I and II formed by SRF and SRF-p62TCF, respectively, over the SRE probe. B. Sequence of the synthetic oligonucleotide SRE probe. Inverted arrows locate the palindromic DSE. Bases forming the CArG box are boxed. The guanine bases of the CArG box that, when methylated, interfere with SRF binding are indicated by arrowheads (Treisman, 1986; Shaw et al., 1989a; Ryan et al., 1989). The two marked guanine bases at the 5' boundary of the DSE are contacted by p62TCF, as mapped by methylation interference and protection (Shaw et al., 1989a; Herrera et al., 1989; Konig, 1991). C. p62TCF interacts with SRF and MCM1. Gel retardation with the SRE probe and vacciniaexpressed full-length SRF (lanes 5 and 6), vaccinia-expressed full-length MCM1 (lanes 9 and 10), in vitro synthesized SRF core (residues 132-222) (lanes 7 and 8) and in vitro synthesized MCM 1 core (residues 1-98) (lanes 11 and 12). SRF and MCM1 have apparent molecular weights of 67 kDa and 45 kDa respectively (Norman et al., 1988; Ammerer, 1990) and the core derivatives are 13 kDa in size. Vaccinia mock-infected cell extract (lanes 1 and 2) and unprogrammed reticulocyte lysate (URL) (lanes 3 and 4) served as negative controls. A plus sign above lanes indicates the addition of HeLa cell purified p62TCF (62 kDa) to the binding reaction. Arrowheads mark complexes I and II formed with full-length SRF and SRF-p62TCF, respectively, as seen in lanes 5 and 6.
led us to construct a series of MCM1/SRF swap mutants. They should all bind the SRE or STE2* probes and recruit p62TCF. However, their ability to assemble a ternary complex with STE 12 would depend on the presence
Mapping of MCM1 core amino acids that are critical for ternary complex formation with STE12 Amino-terminal or carboxy-terminal truncations of MCM 1 core or SRF core disrupt their ability to bind DNA (Norman et al., 1988; Jarvis et al., 1989; C.G.F.Mueller, unpublished observations). Therefore, this approach is not viable for mapping within the core domain the amino acids critical for recruiting the ternary complex factors. However, the inability of SRF to interact with STEl 2, combined with the ability of MCM 1 and SRF to bind to the same DNA
of the critical MCM 1 amino acids. Figure 3A schematically illustrates the MCM1/SRF chimeras. They could all bind the SRE and generate a p62TCF_specific ternary complex (data not shown). All chimeras also bound the STE2* probe, but only a subset could interact with STE12 (panels B and C). The identity of STE12 in the complex II shifts was confirmed using the anti-STE12 antibody (data not shown). The chimeric protein MSM1 carries the DNA binding region of SRF and the dimerization region of MCM1 (Norman et al., 1988) and forms a ternary complex with STE 12 (lanes 5 and 6). Swap
SRF core interacted with STE 12 over the STE2* probe (lanes 9-12). In conclusion, MCM1 and its core derivative can recruit both p62TCF and STE12, but SRF is specific for nor
C.G.F.Mueller and A.Nordheim
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Fig. 2. A. Schematic illustration of complexes I and II assembled over the STE2* probe with MCMl and MCMl-STEl2. B. Nucleotide sequence of the synthetic STE2* probe. Boxed regions outline the SRE CArG box and the PRE. Methylation interference data with MCM1 and STE12 bound to the related STE2 UAS sequence is published (Errede and Ammerer, 1989). C. STE12 interacts with MCM1 but not with SRF. Gel retardation with the STE2* probe and vaccinia-expressed MCM1 (lanes 5 and 6), in vitro synthesized MCM1 core (residues 1-98) (lanes 7 and 8), vacciniaexpressed SRF (lanes 9 and 10) and in vitro synthesized SRF core (residues 132-222) (lanes 11 and 12). Vaccinia mock-infected cell extract (lanes 1 and 2) and unprogrammed reticulocyte lysate (URL) (lanes 3 and 4) served as negative controls. A plus sign above lanes indicates the addition of vaccinia-expressed STE12 (85 kDa). Arrowheads mark complexes I and II formed with full-length MCM1 and MCMl-STE12, respectively, as seen in lanes 5 and 6.
mutant MSM2 also gives rise to complex II, which localizes the amino acids critical for the MCMl -STE12 interaction further towards the carboxy-terminus of core, that is between residues 71 and 98 (lanes 7 and 8). However, further substitution of MCM1 core amino acids by SRF-derived amino acids (MSM3 and MSM4) abolishes ternary complex formation (lanes 9-12). Inspection of the protein sequences (see Figure 7, boxed residues) reveals that MSM3 differs from MSM2 by only four residues (Ser73, Pro75, Phe77 and Glu78). To address the question of whether these four amino acids are alone able to confer STE 12-specific ternary complex formation on SRF core, chimera MSM6 was constructed. It represents SRF core with these four MCM 1-derived residues at their corresponding positions within the core domain. Panel C, lanes 3 and 4, shows that MSM6 can assemble STE12 into a ternary complex, albeit with reduced efficiency as compared with MSM2. Further replacement of SRF amino acids by MCM 1-derived residues
(MSM5) enhances the interaction with STE12 marginally (lanes 5 and 6). In conclusion, MCM1 residues 73, 75, 77 and 78 are involved in mediating a specific ternary complex with STE12.
ARG8O is a sequence-specific DNA binding protein but cannot form a ternary complex with p62TCF or STE12 A similar mapping approach to that used for MCM1 and STE12 could be adopted to map the interaction of p62TCF with SRF, provided an SRE binding protein with no affinity for p62TCF could be found. These criteria are fulfilled by the ARG80 protein, which displays strong amino acid conservation with SRF over the core domain (Figure 7). It can interact specifically with the SRE but cannot generate a ternary complex with p62TCF (Figure 4, lanes 3 and 4). For comparison, an SRF derivative, spanning residues 70-244 (Schroter et al., 1990), was included (lanes 4 and
MCM 1 and SRF ternary complex formation
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Fig. 3. Mapping of the MCMl core amino acids critical for ternary complex formation with STE12. A. Schematic illustration of the MCM1/SRF swap mutants. SRF is represented as a striped box and MCM1 as a plain box. Numbers above the boxes refer to the protein residues; SRF residues are printed in bold and MCM1 residues in plain type. Plus and minus signs next to the boxes indicate the efficiency in ternary complex formation with STE12. The constructs were tested for complex formation over the STE2* probe as shown in the next two panels. B. Gel retardation assay with unprogrammed reticulocyte lysate (lanes I and 2), MCM1 core (lanes 3 and 4), swap mutants MSM1 (lanes 5 and 6), MSM2 (lanes 7 and 8), MSM3 (lanes 9 and 10) and MSM4 (lanes 11 and 12), and SRF core (lanes 13 and 14). C. Gel retardation assay with SRF core (lanes 1 and 2), swap mutants MSM6 (lanes 3 and 4), MSM5 (lanes 5 and 6) and MSM2 (lanes 7 and 8) and unprogrammed reticulocyte lysate (lanes 9 and 10). All proteins were synthesized in vitro and incubated with the STE2* probe in the absence (-) or presence (+) of vaccinia-expressed STE12. For panel C a different batch of STE12 was used that interacts weakly with the probe alone, shifting it to the position marked by an asterisk. 4223
C.G.F.Mueller and A.Nordheim
5). This protein has a similar apparent molecular weight to ARG80 but forms both complexes. ARG80 was also tested for assembling a complex with STE12 over the STE2* probe and, although ARG80 binds to the STE2* probe, no ternary complex with STE12 was generated (data not shown). The ARG80 complex I migrates with almost identical mobility to the SRF 70-244 complex I, and by co-translating full-length ARG80 with either SRF core or MCM1 core, SRF -ARG80 and MCM 1 -ARG80 heterodimers were formed that bound to the SRE in gel retardation assays (C.G.F.Mueller, N.Kahlin and A.Nordheim, unpublished observations). Together, these observations strongly suggest that ARG80 binds the DSE as a homodimer. Interestingly, an ARG80 core derivative (residues 71-177) binds neither the SRE nor the STE2* probe (data not shown). Mapping of SRF core amino acids that are critical for ternary complex formation with p62TcF The fact that ARG80 interacts with the SRE but not with p62TCF makes it an ideal candidate for swap mutations with SRF core to map the SRF amino acids critical for recruiting p62TCF. The chimeric core proteins, SSM1 and SSM2 (Figure 5A) can form complexes I and II efficiently (Figure SB, lanes 5-8). Therefore, the amino acids critical for the SRF-p62TCF interaction map towards the carboxyterminus of SRF core, i.e. between residues 196 and 222. This observation complements the MCM1 -STE12 mapping data (Figure 3). Further SRF/ARG80 chimeras, comparable to MSM3 and MSM4, could not be tested for complex II formation as they no longer bound the SRE sequence (data not shown). However, on the basis of the MCM1 -STE 12 swap experiments, chimeras SSM3 and SSM4 were constructed (Figure SA), which comprise the full-length ARG80 sequence with internal SRF sequence substitutions, comparable to MSM5 and MSM6 (Figure 3A). As shown in panel C, lanes 3 and 4, SSM4 binds the SRE and in addition assembles a p62TCF_specific ternary complex. p62TCF is sequestered more effectively when additional SRF-derived amino acids are present (SSM3, lanes 5 and 6). SSM4 also assembles gel purified p62TCF into complex II (data not shown). No interaction of SSM3 or SSM4 with STE12 was observed (data not shown). SSM4 differs from ARG80 by only three residues (Figure 7, boxed region). Therefore, by changing the three ARG80 residues (Thrl25, Pro 127 and Glu130) to the SRFderived residues (Alal98, Arg200 and Gln2O2), ARG80 acquires the ability to recruit p62TCF specifically. The ARG80 and SRF amino acids are located at a position within the core domain, which was also identified above as being critical for the MCM1 -STE12 interaction. Deficiens and Agamous bind the SRE but cannot assemble a ternary complex with either p62TCF or STE12 A number of plant genes have recently been identified which encode proteins with sequence similarity to SRF, MCM1 and ARG80 over the core domain (Schwarz-Sommer et al., 1990; Ma et al., 1991). Figure 7 depicts the sequence comparison with Deficiens (DEF A) and Agamous (AG) which are prototypical members of gene families in Antirrhinum (snapdragon) and Arabidopsis (common wall cress), respectively (Sommer et al., 1990; Yanofsky et al., 1990). It is notable that strong amino acid conservation exists throughout the DNA binding region; however, the homology 4224
Fig. 4. ARG80 binds the SRE probe but cannot recruit p62TCF. Fulllength ARG80 (lanes 3 and 4) and SRF derivative (residues 70-244) (Schroter et al., 1990) (lanes 4 and 5) were expressd in vitro and tested for complex formation with p62TCF (+ lanes) over the SRE. Lanes 1 and 2 contain unprogrammed reticulocyte lysate. The SRF derivative has approximately the same apparent molecular weight as ARG80 (25 kDa).
is weak over the dimerization region of SRF, MCM 1 and ARG80. Therefore DEF A and AG represent two natural variants that can be tested with regard to SRE and STE2* binding, dimerization and ternary complex formation. On the basis of the results above, we predicted that DEF A core and AG core should bind to the SRE and STE2* sequences, presumably as homodimers, but that neither should form ternary complexes since the carboxy-terminal 27 amino acids of their core domains are not conserved with SRF or MCM 1 (Figure 7). Figure 6 shows that AG core indeed binds strongly to the SRE but that DEF A core binds only very weakly; neither DEF A core nor AG core can generate a p62TC -specific shift (lanes 1-4). Similarly, AG core binds to the STE2* but cannot generate a ternary complex with STE12 (data not shown). It has not been possible to assess a DEF A-STE12 interaction since DEF A does not bind to the STE2* probe (data not shown). In summary, AG core behaved as predicted for formation of complexes I and II. A higher affinity of DEF A core for the SRE was expected; however, like ARG80, full-length DEF A may bind more strongly than DEF A core.
Discussion We have analysed in vitro homologous protein domains for their ability to bind to the c-fos SRE and the STE2* DNA sequences (Figures lB and 2B) and to assemble a ternary
MCM 1 and SRF ternary complex formation
SSM 3 SSM 4
Fig. 5. Mapping of the SRF core amino acids critical for ternary complex formation with p62TCF. A. Schematic illustration of the SRF/ARG80 swap mutants. SRF is represented as a diagonally striped box and ARG80 as a longitudinally striped box. Numbers above the boxes refer to the protein residues; SRF residues are printed in bold and ARG80 residues in plain type. Plus and minus signs next to the boxes indicate the efficiency in ternary complex formation with p62TCF. The constructs were tested for complex formation over the SRE probe as shown in the next two panels. B. Gel retardation assay with unprogrammed reticulocyte lysate (lanes 1 and 2), SRF core (lanes 3 and 4), and swap mutants SSM1 (lanes 5 and 6) and SSM2 (lanes 7 and 8). C. Gel retardation assay with full-length ARG80 (lanes 1 and 2), and swap mutants SSM4 (lanes 3 and 4) and SSM5 (lanes 5 and 6), and SRF derivative 70-244 (Schroter et al., 1990) (lanes 7 and 8). All proteins were expressed in vitro and incubated with the SRE probe in the absence (-) or presence (+) of HeLa cell partially purified p62TCF.
complex with either p62TCF or STE 12. The 90 amino acid domains, termed core, are conserved to different extents between the proteins SRF, MCM 1, ARG80, AG and DEF A, from vertebrates, yeast and plants. The amino-terminal half of the core is strongly conserved between the five proteins, whereas the carboxy-terminal half
is less conserved between SRF, MCM1 and ARG80 and very weakly in AG and DEF A. Figure 7 depicts the sequence comparison of the five proteins along the 90 amino acid core domain. It highlights the SRF amino acids necessary for DNA binding and dimerization (striped and shaded boxes) (Norman et al., 1988). The boxed area localizes the MCM1 4225
C.G.F.Mueller and A.Nordheim
and SRF amino acids identified in this study to be critical for ternary complex formation with STE12 and p62TCF, respectively. DNA binding and dimerization It was previously reported that full-length SRF, SRF core and full-length MCM1 bind to the SRE, and their binding sites were mapped to the CArG box of the DSE (Norman
Fig. 6. Deficiens and Agamous bind the SRE probe but cannot interact with p62TCF. Gel retardation assay with Deficiens core (residues -10 to 82) (lanes 1 and 2), Agamous core (residues 41 -131) (lanes 3 and 4) and SRF core (residues 132-222) (lanes 5 and 6). All proteins were synthesized in vitro. Plus and minus signs refer to the addition of p62TCF
et al., 1988; Hayes et al., 1988; Passmore et al., 1989). We have presented evidence that MCM 1 core, AG core and full-length ARG80 also bind to the SRE. DEF A core interacts with markedly reduced affinity and ARG80 core not at all. The strongly conserved amino-terminal half of the core contains many hydrophilic and basic residues (Figure 7), which is reminiscent of DNA binding domains (reviewed in Struhl, 1989; Busch and Sassone-Corsi, 1990). It is likely that sequence specificity for DNA binding is determined by the amino-terminal half of the core and that amino acids outside the core domain contribute to overall DNA affinity. The observation that ARG80 can interact directly with the SRE or STE2* probes is particularly interesting in the context of an observation by Dubois and Messenguy (1991) showing that ARG80 cannot bind to a promoter element of the ARG5,6 gene unless it is a component of a multi-protein complex. This element does not contain a perfect CArG box as it lacks two nucleotides within the AT-rich spacer sequence (Messenguy et al., 1991). This may explain the inability of ARG80 to bind this promoter sequence in the absence of other proteins. The more divergent carboxy-terminal half of the core is largely hydrophobic (Figure 7). By a dimerization interference assay the SRF dimerization region has been mapped to the carboxy-terminal half of the core (Norman et al., 1988) (Figure 7). These experiments also established that dimerization is necessary for efficient SRE-binding. The observation that MCM1, ARG80, DEF A and AG can interact with the SRE, strongly suggests that they bind the SRE in dimeric form. Consistent with this notion is the fact that SRF, MCM 1 and ARG80 can form heterodimers with one another over the SRE.
Ternary complex formation We have shown that MCM1 recruits p62TCF as well as STE12 but that SRF forms a ternary complex only with p62TCF. ARG80, AG and DEF A have no affinity for either ternary complex factor. We have exploited these differences to map the amino acids critical for ternary complex formation. By exchanging short protein regions between MCM1 and SRF as well as between SRF and ARG80, it could be demonstrated that amino acids within a carboxy-terminal sub-domain of MCM1 core and SRF core (mutants MSM2 222
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Fig. 7. Sequence comparison of MCM1 (yeast), SRF (human and toad), ARG80 (yeast), Agamous (AG) (common wall cress) and Deficiens (DEF A) (snapdragon) over the core domain. Identical amino acids are indicated by a connecting line, residues conserved between all five proteins are in bold type. The SRF protein regions required for DNA binding and dimerization are outlined (Norman et al., 1988). The MCM1 and SRF protein regions which harbour the amino acids identified as critical for specific ternary complex formation with STE12 and p62TCF, respectively, are boxed, as are the ARG80 residues which were mutated to SRF-derived amino acids such that ARG80 acquires the ability to recruit p62TCF specifically. The amino acid sequence for human SRF and the yeast proteins is taken from Norman et al. (1988), Xenopus SRF is from Mohun et al. (1991), and the sequences for Deficiens and Agamous are taken from Sommer et al. (1990) and Yanofsky et al. (1990), respectively.
MCM 1 and SRF ternary complex formation
and SSM2) are involved in mediating efficient ternary complex formation with STE12 and p62TCF, respectively. AG and DEF A core proteins exhibit no amino acid conservation with SRF and MCM 1 over the carboxyterminal subregion of the core domain and do not recruit either p62TCF or STE12 over the tested DNA sequences. More detailed mapping identified four amino acids in the case of MCM (Ser73, Pro75, Phe77 and Glu78) and three amino acids in the case of SRF (Alal98, Arg200 and Gln203) to play a critical role in mediating specific ternary complex formation. When these four MCM1 residues were replaced by the corresponding SRF amino acids (Alal98, Arg200, Leu2O2 and Gln203) (MSM3), ternary complex formation with STE12 was abolished while maintaining the interaction with p62TCF. On the other hand, when introduced into SRF core at their corresponding position (MSM6), these four MCM1 residues conferred STE12specific ternary complex formation to SRF core, while not disrupting the ability of SRF core to interact with p62TCF Likewise, by exchanging ARG80 residues Thrl25, Prol27 and Glu120 for the corresponding SRF amino acids (Alal98, Arg200, Gln203) (SSM4), ARG80 acquired the ability to specifically. recruit p62 Thus, in two complementary sets of experiments, identical residues of SRF were identified as being critical for specific ternary complex formation. These experiments demonstrated that by exchanging not more than four amino acids it was possible to engineer transcription factors such that they either lose or acquire the ability to sequester a specific ternary complex factor. A similar experimental approach has been used to identify the amino acids in the transcription factor Octl that mediate its interaction with the viral protein VP16. The substitution of four amino acids in Octl for the corresponding amino acids of the related protein Oct2 resulted in the disruption of Octl -VP 16 interaction (Stern et al., 1989). TCF
paper (Primig et al., 1991) presents evidence that MCM 1 core itself (residues 1 -98) can maintain cell viability and mating competence. A similar conclusion was reached in an independent study (Christ and Tye, 1991). On the other hand, MCM1 core comprising the SRF dimerizing region renders the cells sterile by not being able to sequester STE 12 or ax1. Primig et al. (1991) also present evidence that MCM1 core is sufficient to recruit a(x and ca2. In addition, it appears that yet another protein, the recently identified SWI5 activation factor (SFF), can interact with MCM1 core (D.Lydall, G.Ammerer and K.Nasmyth, submitted). Therefore, MCM1 core appears to be tailor-made for recruiting ternary complex proteins and may act as a DNA adaptor for transcription regulatory proteins. The similarity between MCM1 and ARG80 is illustrated in vivo by the fact that an ARG80 defect can be partly complemented by over-expressing MCM1 (Bercy et al., 1987). Additionally, two conserved ARG80 point mutations that are detrimental to its function have been mapped to the core domain (Ala109 and Gly148). In agreement, the first 60 amino acids that are not part of core could be deleted without any appreciable loss of function (Qiu et al., 1990). In summary, we have shown that the core domain which is conserved between yeast and multicellular eukaryotes plays a central role for DNA binding and, in the cases of MCM 1 and SRF, also for ternary complex formation. It is tempting to speculate that, in analogy to MCM 1, SRF may be able to recruit additional ternary complex factors. For instance, the repression of c-fos transcription which appears to require the SRF bound to the SRE (Shaw et al., 1989b; Konig et al., 1989) may be mediated by SRF sequestering a repressor. It would likewise be interesting to determine whether proteins exist that interact with ARG80 or with members of the Agamous and Deficiens families.
Materials and methods Structural similarity of the core domains The core domains of MCM 1, SRF and ARG80 appear to be structurally very similar as they all bind the same DNA sequence and form heterodimers with one another. Moreover, the domain swap mutants described here all dimerize and bind the SRE or the STE2* probes apart from certain SRF/ARG80 swaps. Additionally, the amino acid changes that were critical for ternary complex formation with p62 CF or STE12 all fall within equivalent regions of the MCM 1, SRF and ARG80 core domains. This suggests that the ternary complex factors p62TCF and STE12 recognize a similar core protein structure and that the mechanism by which MCM 1 and SRF form a ternary complex with STE 12 and p62TCF, respectively are related. AG core exhibits similar DNA binding specificity. However, it cannot recruit either p62TCF or STE 12, and AG core does not heterodimerize with SRF (C.G.F.Mueller, N.Kahlin and A.Nordheim, unpublished observations). DEF A core has a low affinity for the SRE sequence and does not interact with p62TCF. This indicates that only over the DNA binding regions may AG and DEF A share structural homology with MCM1, SRF and ARG80. Implications
for function in vivo
Jarvis et al. (1989) have shown that an MCM 1 derivative spanning residues 1 - 157 is sufficient to activate ce-specific genes to wild type levels. Furthermore, the accompanying
Plasmid constructions All in vitro expression constructs are based on pT3G (Schroter et al., 1990). PCR-generated expression clones were sequenced by the chain termination method (Sanger et al., 1977).
MCMJ/SRF swap mutants. pT3G-MCM1 1/98, termed MCM1 core, comprises the EcoRl -PstI restriction fragment of pGA1798, kindly provided by G.Ammerer. pT3G-SRF 132/222, termed SRF core, was constructed as follows. Two kinased oligonucleotides (5'-ACCATGAGCGGGGCCAAGCCGGGT and 5'-TGGCGAGTTGAGGCAGGTCTGAATCAG) and full-length SRF (Norman et al., 1988), which served as template, were used for a polymerase chain reaction (PCR). The PCR protocol remained unchanged from a earlier publication (Schroter et al., 1990). The product was cloned in the correct orientation into SnaI-cleaved pT3G. To make the chimeric gene MSM 1, a silent Sacl site was first introduced into pT3GSRF 132/222 at nucleotide position 883 using PCR with pT3G-SRF 132/222 as template and two primers (5'-ATGAAGAAGGCCTATGAGCTCTCCACG and 5'-TATATGGATCCTATGGCGAGTTGAGGCAGG). The product was inserted as a StuI -BamHI fragment into pT3G-SRF 132/222 to generate pT3G-SRF 132/222(SacI). For pT3G-MSM 1, the oligonucleotide (5'-GCGT-lTGAGCTCTCTGTTCTAACG) and the T7 primer (Stratagene) were used to generate a PCR product with pT3G-MCM 1 1/98 as template. The amplified DNA was cut at its Sacl ends and subcloned into pT3G-SRF 132/222(SacI) in its correct orientation. For pT3G-MSM2, MSM3 and MSM4 the PCR template was pT3G-MCM 1 1/98, and for pT3G-MSM6 and pT3G-MSM5 it was pT3G-SRF 132/222 and pT3G-MSM6, respectively. The T7 primer served as the carboxy-terminal primer and the following oligonucleotides served as amino-terminal primers: MSM2: 5'-ACAGGTTTGGTGTATACTTTCAGC; MSM3: 5'-ACAGGTTTG-
GTGTATACTTTCGCCACGCGGAAGTTGCAACCTATAGTCACG; MSM4: 5'-AGCACGCCGAAGCTGCAGCCTATGATCACGTCGGAGACAGGTAGAAACCTGATC; MSM5: first PCR using 5'-CCAA4227
C.G.F.Mueller and A.Nordheim
AATTCGAGCCCATCGTCACCCAACAGGAGGGCAAGGCACTG, following by second PCR using 5'GGCCATGTGTATACCTTTAGTACTCCAAAATTCGAGCCCATCGTCACC and the previous PCR product as template; MSM6: 5'-GGCCATGTGTATACCTTTAGTACTCCAAAATTCGAGCCCATGATCACC. The PCR products were cloned into prepared vectors: MSM2: pT3GSRF 132/222(SacI) (AccI-BamHI); MSM3: pT3G-SRF 132/222 (AccI-BamHI); MSM4: pT3G-SRF 132/222 (PstI partial, SacI); MSM5: pT3G-SRF 132/222 (AccI, SacI): MSM6: pT3G-SRF 132/222 (AccI, Sacl). SRF/ARG80 swap mutants. pT3G-ARG80 was derived from pSL-3 (a gift from G.Ammerer) by subcloning an EcoRI-XhoI fragment (nucleotides 1-813). For the construction of chimeras SSM1 and SSM2 the ARG80 coding sequence was amino-terminally truncated to give pT3G-ARG80 71/177. For this the T7 primer, an oligonucleotide (5'-GCGCGCAAGCTTCCACCATGGACAGCAGCGACACAAGTACG) and pT3G-ARG80 as template were used for PCR. The product was cloned into pT3G between the HindHI and Sacl sites. For pT3G-SSMl the carboxy-terminal half of ARG80 71/177 was replaced by that of SRF using the Sacl restriction fragment of pT3G-SRF 132/222(SacI). PCR was again used to generate pT3G-SSM2 with the T3 primer and an oligonucleotide (5'-CGTTGTGAAAGTATACACCAGGCC) as primers and pT3G-ARG80 71/177 as template. The HindlIl and AccI ends of the product were cut and inserted into pT3G-SRF 132/222. pT3G-SSM3 and pT3G-SSM4 were constructed with T7 primer and pT3G-ARG80. Additionally, SSM3 and SSM4 required the following primers, respectively: 5'-ACAACGCCTAAGCTGCAGCCGGTGGTACGGGAA and 5'-ATATATATCTGCAGCCCATGATC-
ACTAGTGAGACCGGTAAGAGCCTCATCAGGGCATGCATCAATG. The SSM4 PCR product was first cloned between the PstI and Sacl sites of PT3G-SRF 132/222, then inserted as a AccI-SacI fragment into pT3GSSM2 and finally into Sacl cleaved pT3G-ARG80 replacing the ARG80 carboxy terminus. The construction of pT3G-SSM3 also required some subcloning of the PCR product: it was inserted into XbaI/PstI cut pT3GSSM2 and then subcloned into pT3G-ARG80 in the correct orientation as a
The cDNA for Deficiens
was kindly provided by Z.Schwarz-Sommer pcDEFIB (Sommer et al., 1990). pCIT15 16, containing the Agamous cDNA, was a gift from E.M.Meyerowitz (Yanofsky et al., 1990). The DEF A protein was originally described as a 227 residue polypeptide with the region of homology at its extreme amino terminus (Sommer et al., 1990).
The cDNA, however, contained no stop codons 5' to the first methionine, which left some uncertainty about the start of its open reading frame. Therefore, the amino terminus was extended by ten residues, with the result that two conserved serines would be included in the core protein. To make pT3G-DEF A - 10/82, a PCR product was generated using pcDEFlB as template and the following primers: 5'-CGCGCAAGCTTCACCATGGAAAACAAGAGCAGTAGT and 5'-ATATATGGATCCTATGAACTCCATAGATCAAC. The product was cloned as a HindLH-BamHI fragment into pT3G. The template pCIT15 16 and two primers (5'-CGCGCAAGCTTCACCATGGGAGATTCCTCTCCCTTGAGG and 5'-ATATATGGATCCTACACCGATCCGGTGTTAGA) were used to generate pT3G-AG 41/131. The PCR product was cloned between the HindIII and BamHI sites of pT3G.
Protein preparation for DNA binding experiments The vaccinia expression vectors for MCM 1 and STE 12 were a gift from H.Stunnenberg. The construction of the vaccinia SRF expression plasmid and the details on protein preparation will be published elsewhere
(Hipskind,R.A., Schmidt,J., Stunnenberg,H. and Nordheim,A., manuscript in preparation). The purification of p62T F from HeLa cells and the in vitro expression system using rabbit reticulocyte lysate have been described before (Schroter et al., 1990). For run-off transcription all expression vectors were linearized at the BamHI site except for those encoding the full-length ARG80 carboxy-terminal sequence, which were cut with XbaI. No protein purification after in vitro expression, or of the vaccinia cell extract, was necessary for the gel retardation experiments which were performed as described earlier (Schroter et al., 1987).
G.Ammerer for providing plasmid clones encoding (residues 1-98), ARG80 and STE12 and for communication of results prior to publication. The support by H.Stunnenberg and J.Schmidt for vaccinia virus-directed expression of MCM1, SRF and STE12 is gratefully acknowledged. Protein preparations were kindly provided by H.Schroter (p62TCF) and R.A.Hipskind (vaccinia-expressed SRF). We are
also wish to thank S.Hall and P.Wagner for making available to us an antiSTE12 antiserum, E.M.Meyerowitz for the Agamous cDNA clone, Z.Schwarz-Sommer for the Deficiens cDNA clone and Marianne Petry and Vera Pingoud for oligonucleotide. synthesis. We appreciate critical review of the manuscript by M.Cahill, R.A.Hipskind and B.Luscher. During part of the work C.G.F.M. was the recipient of a training grant in cancer research (MR4-0162-D), awarded by the Commission of the European Communities. Financial support was provided by the Deutsche Forschungsgemeinschaft, the Bundesministerium fur Forschung und Technologie and the Fonds der Chemischen Industrie.
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Received on August 13, 1991; revised on September 17, 1991