The SRF and MCM1 transcription factors Richard Treisman and Gustav Ammerer Imperial Cancer Research Fund, London, UK, and Forschungsinstitut for Molekular Pathologie and University of Vienna, Vienna, Austria The mammalian transcription factor SRF (serum-response factor) and the related Saccharomyces cerevisiae transcription factor MCMI are the prototypes of a new class of dimeric DNA-binding proteins. Their function is regulated in part by the interactions of their DNA-binding domains with accessory proteins. Recent work has advanced the functional characterization of the contributions of SRFand MCMI, and their accessory proteins to transcriptional activation. Current Opinion in Genetics and Development 1992, 2:221-226
Introduction The mammalian transcription factor SRF (serum-response factor) and the transcription factor MCM1 (also known as PRTF and GRM) are 70% identical within their DNA-binding and dimerization domains but are otherwise unrelated. Both proteins are apparently involved in the cellular response to extracellular signals and the celltype-specific regulation of transcription. The DNA-binding domains of both factors interact with a number of structurally unrelated accessory proteins that are listed in Table 1. Although these accessory proteins show only weak affinity for DNA, they make sequence-specific DNA contacts at positions adjacent to the SRF or MCM1 DNAbinding sites. SRF-binding sites are found in promoters and enhancers of growth factor-regulated and muscle-specific genes: they form the central sequence of serum-response elements (SREs) (for reviews see [1-3]). At the c-los SRE, DNA-bound SRF directs binding of an accessory protein, p62/TCF, that in some cells appears to be required for signal transduction [4,5]. Over the past year cDNAs encoding a number of SRF accessory proteins have been isolated (M Grueneberg, M Gilman, personal communication) [6°.,7 ' ' ] but the precise contributions of these proteins to SRE function remains unclear. MCM1, the product of the MCMI gene (for minichromosome maintenance) fulfills an unknown function that is essential for cell viability. MCMl-binding sites are found in several types of yeast promoter, particularly those of cell-type-specific genes [8-10] (see [11,12] for reviews). MCM1 recruits at least four different accessory proteins to its binding sites, depending on the sequence context of the particular MCMl-binding site and the cell type. The functions of the different MCMl-accessoryprotein complexes have been well characterized: in Saccharomyces cerevisiae a cells, complexes includ-
ing the MATczl and IVlAT0t2 proteins activate 0t-specific and repress a-specific genes, respectively, while MCM-1-STE12 complexes link the promoter to extracellular signals. A recently identified accessory factor, SFF, appears to be involved in cell-cycle-regulated transcription [13"]. As yet no obvious sequence similarities between the different MCM1 accessory proteins and the DNA sequences that they contact have been observed. In this review we first consider progress in the understanding of the structure of the SRF/MCM-like DNA-binding domain and its interaction with both DNA and the various accessory proteins. We then consider functional studies of SRF and MCM1 with particular reference to the role of the accessory proteins in activation. Finally, we consider to what extent functional comparisons between the two proteins are warranted.
The SRF/MCM-type DNA-binding domain SRF and MCM1 define a gene family An alignment of the SRF and MCM1 DNA-binding domains with other related sequences from },east, mammals and plants is shown in Figure 1. The yeast ARG80 protein is highly related to MCM1 and also appears to function as part of multiprotein complexes [ 14,15]. Several homeotic gene products that are involvecl in flower morphogenesis are members of a large family of plant gene products containing sequences distantly related to the DNA-binding domains of SRF and MCM1 [16-18] (for reviews see [19,20]). A set of equally divergent human gene products, the related to SRF (RSRF) proteins, that contain a common SRF-/MCM-type DNA-binding domains have also been characterized. The binding site of the RSRF proteins occurs in muscle-specifc and growth-factor-regulated genes, suggesting that their function may be mechanistically similar to SRF [21.]. No accessory proteins for
Abbreviations CKIl--casein kinase II; PKC--protein kinase C; RSRF~related to SRF; SRE--serum-response element; SRF~serum-response factor; UAS~upstream activating sequence. (~) Current Biology Lid ISSN 0959-437X
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Gene expression and differentiation
Table 1. Accessory proteins.
Core
Accessory protein
Source of accessory protein
SRF
p621TCF
HeLa
Ets domain los: CAGG
Detected in HeLa nuclear extracts; homologous to Elk-1 (A Rogers, R Treisman, unpublished data)16ee]; function unclear, see text 141°L43"°], can interact with MCM1 [26°'1
SRF
SAP-1
HeLa
Ets domain los: ACAGGAT
Isolated as an SRF-dependent SRE-binding protein by in vivo cDNA screening; function unclear, see text; related to Elk-l; can interact with MCM1 17"'1
SRF
SAP-2
Placenta
Ets domain los: ACAGGAT
Isolated by homology to SAP-l; also related to Elk-l; function is unknown IA Rogers, S Dalton, R Treisman, unpublished data)
SRF
Phoxl
Human
MCM1
MAT0cl
Diploids, c~ cells
? STE3: CTGTCA'FI-G
Positive- and pheromone-regulated transcription at a-specific MCM1 operators, e.g. STE3 (reviewed in [11,12l); response to a factor 149"]; cannot bind MCMI-SRF fusion protein 125"1
MCM1
MAT~z2
Diploids, a cells
Homeodomain STE6: CATGTAAI-i" CGTGTAAAT STE2: CATGTAAAT CATGTACTT
Negative regulation at a-specific MCM1 operators, eg. STE2 and STE6 [11,12]; functions negatively and indepently of MCM1 as part the of al-c~l repressor (reviewed in 111,12]; binds MCMI-SRF fusion protein weakly 12S°'l
MCM1
STE12
All types
Divergent homeodomain? STE2: ATGAAACA
Required for high basal level activity of a-specilic promoters, e.g. STE2; also functions independently of MCMI at promoters, such as FUSI, that contain multiple STE12-binding sites [50,51]; cannot interact with SRF [25"o,26 ° ' ]
MCM1
SFF
All types
? SW15: AAACAATA
Identilied only as a binding activity in yeast cell extracts implicated in cell-cell-dependent activation of the 5W15 promoter 113°°l; can interact with the MCM-SRF fusion protein (D Lydall, unpublished data)
DNA-interaction site and motif
Protein function, and comments
Isolated as human cDNA that activates a PQ box in the promoter of a MAT~I: TRP1 yeast strain; contains a homeobox (M Grueneberg, M Gilman, personal communication)
The different SRF and MCM1 accessory proteins that have thus far been identified are listed. Where known, the type of DNA-binding motif used by the accessory proteins to contact the DNA is shown, together with examples of the sequences contacted by each protein. See text for discussion.
the plant and human SRF-/MCM-related DNA-binding domains have been reported. Sequence homology between the SRF, MCM1 and ARG80 group of proteins, and also between the RSRF proteins, extends throughout their DNA-binding regions (Fig. 1). Sequences conserved between the entire group of proteins are, however, con~ned to the amino-terminal twothirds of the region that presumably represents a conserved structural domain, and which has been called the 'MADS-box' (derived from MCM-ARG80-AgamousDeficiens-SRF [19]). Careful study of the properties of chimeric proteins from clitTerent family members has provided significant insight into the structural and functional subdomains of the SRF/MCM-like DNA-binding domain.
DNA-binding specificity The DNA-binding domains of SRF and MCM1 are 70% identical, and both proteins can bind the c-fos-SRE, which can function as an upstream activating sequence (UAS) in yeast [9,22]. Detailed analysis of the binding specificities of the two proteins shows that they are overlapping rather than identical; sites selected in vitro by MCM1 have the consensus (notC)CCYAAWNNGG (W = Aor T ) (J Wynne, S Dalton, R Treisman, unpublished data), contrasting with the SRF consensus CCWWWWWWGG
(W = Aor T ) (see [9,23] for other studies). The MCM1 sites in ~-specific promoters appear to represent weak binding sites compared with those in a-specific promoters [24]. The consensus binding site for the RSRF proteins, YTAWWWWTAR (Y = C or T, R = A or G, W = A or T), is not detectably bound by SRF in vitro [21-]. Studies on the binding specificities of chimeric RSRF-SRF and MCM1-SRF proteins show that the sequences in the amino-terminal, basic region of the DNA-binding domain determine sequence specificity (J Wynne, S Dalton, R Treisman, unpublished data) (Fig. 1) [21,,25"o]. Although the specificities of the plant proteins and ARG80 have not been thoroughly characterized, in vitro binding studies suggest that the), may be related ~o those of SRF and MCM1 [26".].
Accessory factor specificity Differences in the ability of the various domains to interact with accessory proteins are suggested by the observations that SRF and MCM1, but not ARG80, can interact with HeLa p62/TCF [26 ,o] and that MCM1, but not SRF, can interact with MAT(zl, MAT~2 and STE12 [25o°,26o.]. Experiments with chimeric proteins show that residues crucial for the specificity of accessory protein recruitment are located in the divergent region carboxy-terminal to the 'MADS' homology (Fig. 1). Replacement of the MCM1
The SRF and MCM1 transcription factors Treisman, Ammerer
(a) MCM1 ARGS0
;PprsOpttdOr235 132sgakpgkkTrglv IkmeFidNKll ! i F~ k{t )iMl~ti TGTOYLLLYaSETGhVYTFaTrKRIPmITsetGKaLIOtCLNsP[ TGTQVLLLVvSETGLVYTFsTPKEPiVTqqEGrnLIQACLNAP[ leeedeeedgdd II0 7 gtptnngqqke leIkF}ENKTI F~ (H I N I I ~ :PdatDtspa(le 172 69 tkdssdtsTvt ,qp!r) IENKTI F$ rH I N H ~ V i rGantLLLIlanSGLYYTFtTPKINPvVredEGKsLIrACiNAs[
AG
41 ggesspl sg] iielkr enttn q~ f(
SRF
I
DEF
rn
144
cdaevlal vfssrgrlyeysnnsvkgti erykkaysensntgsv
1
I¢ iiq|kr enqtn qvl Ys *n i f l l
cdakvslilltlsstqkl heyisptt atkqlfdqyqkavgvdlwssl
M~~ IOls~[IOqRNOV{FTI ( F l ; i ~ M~ ~ ,OIt~!mOeRNQVIFT! (F
RSRFR,?. RSRFC4
Co) Conserved sequence motif
L
kkl n 95
CDCEIALIIFNSaNrLFQYASTDMDrVLLKYTEYsEPHESRTNt~tLkrrglgl g5 CDCEIALIIFNSsNkLFQYASTDM DkVLLKYTEYnEPHESRTN~ ~aLnkkehrg95
Bgl .........
-I-ill ..... | .....
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!-- l**i P - I ~ * * i i i - I
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"MADS-Box" DNA binding
(c)
b •
Dimerization
ii
Sequence specificity •
Accessory proteins ..............
,IP
Fig. 1. (a) The SRF-like DNA-binding domains from SRF, MCM1 and ARG80, the plant homeotic gene products Agamous (AG) and Deficiens (DEF), and the human RSRF proteins are shown. Identities between the different proteins are shown in dark grey shading, and conservative substitutions by light grey shading and asterisks. The regions of homology between the SRF, MCM1, ARG80 and the RSRF subgroups are boxed. (b) The conserved 'MADS box' motif (derived from MCM-ARG80-Agamous-Deficiens-SRF), is shown. (c) Functional regions of the DNA-binding domain are indicated below the figure. These include regions that determine DNA binding [3], dimerization [3], sequence specificity [21",25"L and accessory protein interaction [25",26"]. The dotted line indicates that sequences in this region effect the affinity, but not the specificity, of accessory factor interaction.
dimerization domain with the corresponding sequences from SRF prevents interaction of the resulting protein with the MCM1 accessory proteins M_ATcxl, M_ATcz2and STE12 [25--]. Substitution of the SRF amino-acid residues 196-203 with the corresponding residues from MCM1 generates a chimera that can recruit STE12; likewise, substitution of the corresponding residues in ARG80 with SRF residues 196-203 generates a chimera that can recruit p62/TCF [26..]. It is possible, however, that sequences outside this small region may also contribute to the affinity of the accessory factor interaction [26-.]. Thus the observed failure of MCM1 to substitute in vivo for ARG80, and vice versa, may reflect differences in the specificity of both DNA binding and accessory factor recognition [15,27]. Finally, the divergence between the RSRF proteins and SRF/MCM in this region suggests that any accessory proteins recognized by the RSRF proteins may be distinct from those recognized by SRF.
overexpression of SRF expression vectors transactivates a cotransfected SRE, but only in certain cell types (S John, R Treisman, upublished data) [30]. Expression of a LexA-SRF fusion protein is sufficient to render a cotransfected LexA operator weakly serum-responsive and this suggests that SRF itself may be capable of responding to extracellular signals (S John, R Treisman, unpublished data). It will be interesting to apply this approach to the study of muscle-specific promoters that are apparently dependent on SRF for constitutive activity [31]. Microinjection studies indicate that SRF and the SRE also fulfill an unknown function during the G1 stage of the cell cycle [32"]. Such 'late' functions might require newly synthesized SRF, consistent with the activation of SRF mRNA and protein synthesis following growth factor stimulation [3,33"]. In stimulated cells the newly synthesized protein is extensively modified after synthesis, apparently by phosphorylation [33"]. The protein is localized to the nucleus in both quiescent and growing cells, and remains chromatin associated throughout the cell cycle [32",33"].
Functional studies SRF function and expression
Regulation of SRF DNA-binding
Two experimental approaches suggest that SRF is directly involved in signal transduction and transcriptional activation. In a system where c-los expression requires SRE-binding proteins, depletion of SRF by the microinjection of specific antibodies is sufficient to block induction, strongly suggesting that SRF interacts directly with the SRE in vivo to promote growth-factor-regulated transcription [28,29" ]. In transfection experiments,
In the recent past, studies of SRF in crude extracts suggested that phosphorylation is absolutely required for DNA-binding [34-36] but since then studies with purified protein suggest that this is not the case [19,37",38"]. SRF contains an evolutionarily conserved site for phosphorylation by casein kinase II (CKII) [3,39], and the recombinant protein can be phosphorylated by this kinase in vitro [37%38",40]. Partial proteolysis studies suggest that
223
224
Gene expression and differentiation CKII phosphorylation causes a conformational change in the protein [40], which dramatically increases the in vitro SRF-DNA exchange rate rather than an alteration in DNAbinding affinity [38"]. Although CKII activity is increased by growth factor stimulation, and microinjection of the enzyme is sufficient to induce c-fos transcription in an SRF-dependent manner [29"'], the significance of CKII phosphorylation in the regulation of SRF function in vivo remains unclear. SRF accessory proteins Recent studies have led to the identification of a number of Ets-domain proteins that have binding properties similar to those of p62/TCF (see Table 1; [6"',7.']). In addition a homeodomain protein, Phoxl, appears to potentiate the binding activity of SRF at the c-fos SRE (M Grueneberg, M Gilman, personal communication). The role of p62/TCF-type proteins in SRE function remains unclear. For example, in Balb/C 3T3 cells, mutations that prevent p62/TCF binding in vitro were shown to block protein kinase C (PKC)-dependent signalling to the SRE but to leave PKC-independent signalling pathways unaffected [41.,]. In contrast, other reports suggest that binding of such factors is apparendy required for both PKC-dependent and independent signalling [42 o-] or that it potentiates but is not essential for signalling [43"]. In certain cell lines inducible binding of p62/TCF-like factors to the c-fos SRE has been reported, suggesting a direct link between these factors and signalling pathways [42.-]. The situation is further complicated by the identification of multiple TCF-like proteins and by the observation that other proteins, including Ets-domain proteins, can bind to the DNA contacted by p62/TCF independen@ of SRF [30, 44]. Moreover, it is not clear whether all SRE sequences can bind p62/TCF-type proteins. Obviously, we need to know much more about the types and structures of SRF accessory proteins before we can draw firm conclusions about their role in SRE function. MCM1 and its accessory proteins Using standard techniques of mutagenesis, functional studies of MCM1 have concentrated on attempts to delineate regions of the molecule required for cell viability and mating phenotype. These studies have shown that an 80 amino acid region of the protein, which contains the minimal DNA-binding domain, is sufficient to restore cell viability, minichromosome stability and mating. From this observation it would be expected that the carboxy-terminal region of the protein might serve a modulatory role: indeed, the acidic region neighbouring the DNA-binding domain is required for full activation of c~-cell-specific genes and the glutamine.rich carboxy-terminal region appears to modulate the degree of transcriptional activation [25..,27,45]. What is the role of the accessory proteins in MCM1 function? Three basic models, which are not mutually exclusive, have been proposed and are based, for historical reasons, on the mechanism of co-specific gene expression. The first proposes differential MCM1 binding. Although the protein is an activator, intracellular levels of the protein are insufficient to allow site saturation at weak sites
in c~-specific promoters in the absence of cooperatively binding MATcxl protein [24]. In the second, a conforrnational model, promoters bind MCM1, but in a conformation incompatible with transcriptional activation. Interaction with accessory proteins such as MATcxl brings about a conforrnational change that converts bound MCM1 to an activating form [46]. Finally, in the adaptor model, • accessory factors such as MAT0tl provide the activation function and linkage to signalling pathways, while MCM1 itself is only a weak activator. Although MCM1 can activate from a palindromic site in any cell type, consistent with the first model [47], recent studies also provide support for the second and third models. For example, genomic footprint analysis of two MCM1 sites at the Mf~l promoter showed that one of the two sites is not occupied by MCM1 in the absence of MATcxl, consistent with model one or three [25"'], while the other is bound by MCM1 but fails to activate transcription, consistent with models two or three (H Winkler, G Ammerer, unpublished data). Further evidence consistent with models two or three comes from the study of the SWI5 UAS: here, mutants that fail to bind the SFF factor are defective in activation even though MCM1 binding is not affected [13"]. Similar results are obtained with an a-cell-specific promoter fragment containing MCM1 and STE12 binding sites: here MCM1 can bind even in the absence of STE12, but only weak activation is observed (M Primig, G Ammerer, unpublished data) [48]. Direct evidence for model three comes from experiments in which either MCM1 or MATotl are fused to the LexA DNA-binding domain. This showed that MATotl has, if anything, greater activator activity than MCM1 itself; moreover, only the LexA-MAT0tl fusion protein will render a LexA-operator pheromone inducible by a factor [49"']. A reasonable proposal is thus that the principal role of MCM1 is as a functionally neutral DNA adaptor whose transcriptional competence and specificity is established by its accessory proteins. In particular, it appears that it is the accessory proteins that provide the target for signalling pathways. How similar are SRF and M C M I ? The similarities in the structure and functions of MCM1 and SRF has led to the suggestion that the two proteins are functional homologues. Strictly interpreted, this would imply that the two proteins might be functionally interchangeable, but this appears not to be the case: the results we have reviewed suggest that both the DNAbinding specificity and the accessory factor interactions of SRF and MCM1 are different. Indeed, when MCM1 is replaced by a chimeric protein comprising the MCM1 basic region (determining sequence specificity) and the SRF dimerization domain, cell viability is maintained but the cells do not mate owing to defects in the ability of the chimeric protein to interact with MCMI's accessory proteins [25"]. A simple view of this result is that although MCM1 and SRF may have similar functions, substantial divergence between the systems has occurred at the the level of the accessory proteins. The fact that the SRF accessory proteins p62/TCF and SAP-1 can interact with both SRF and MCM1 does, however, raise the possibility
The SRF and MCM1 transcription factors Treisman, Ammerer that at least some aspects of accessory protein structure have been evolutionarily conserved [6",7"•,26"']. Thus, it is tempting to speculate that there exist as yet uncharacterized accessory proteins that are structurally conserved between the two systems.
Conclusion Here we have outlined the close similarities between MCM1 and SRF. It is likely that their relationship extends at least in part at the functional level, but as yet no accessory protein from one system has been shown to have a structurally related counterpart in the other. The existence of closely related combinatorial systems of gene regulatory proteins in yeast and mammalian cells should provide valuable tools for their dissection at the molecular level.
s p o n s e Factor, is Encoded by t h e MCM1 Gene. Genes Dev 1989, 3:936-945. 11.
DOLANjW, FIELDSS: Cell-type-specific Transcription in Yeast. Biocbim Biophys Acta 1991, 1088:155-169.
12.
SPRAGUEGF: Combinatorial Associations of Regulatory Proteins and the Control of Cell Type in Yeast. Adv Genet 1990, 27:33--62.
LYDALLD, AMMERERG, N,'6MYI'H K: A New Role for MCMI in Yeast: Cell Cycle Regulation of SWI5 Transcription. Genes Dev 1991, 5:2405-2419. Demonstrates a new role for MCM1 in the regulation of cell.cycle-dependent transcription. Identifies a novel accessory protein, SWI5 factor (SFF), that is distinct from other MCM1 accessory proteins, and which is implicated in transcriptional regulation. 13. ••
14.
MESSENGUY F, DUBOIS E, BOONCHIRD C: Determination of the DNA-binding Sequences of ARGR Proteins to Arginine Anabolic and Catabolic Promoters. Mol Cell Biol 1991, 11:2852-2863.
15.
DUBOISE, ME~,ENGUY F: In Vitro Studies of t h e Binding of the ARGR Proteins to the ARG5,6 Promoter. Mol Cell Biol 1991, 11:2162-2168.
16.
SOMMERH, BELTRANJP, HUUSERP, PAPE H, LONNIGWE, SAEDLER H, SCHWARZ.SZ: Deficiens, a Homeotic Gene Involved in the Control of Flower Morphogenesis in Antirrhinum Majus: the Protein Shows Homology to Transcription Factors. Embo J 1990, 9:605-613.
17.
YANOFSV,~'MF, IvlA H, BOWMANJL, DREWS GN, FELDMANN KA, MEYEROXXq'VZEM: T h e Protein Encoded by the Arabidopsis Homeotic Gene Agamous Resembles Transcription Factors. Nature 1990, 346:35-39.
18.
NORMANC, RUNSWlCKM, POLLOCKR, TREISMANR: Isolation and Properties of cDNA Clones Encoding SRF, a Tanscription Factor that Binds to the c-los Serum Response Element. Cell 1988, 55:989-1003.
MA H, YANOI"SKYMF, MEYEROWrI'Z EM: AGL1-AGL6, an Arabidopsis Gene Family with Similarity to Floral Homeotic and Transcription Factor Genes. Genes Dev 1991, 5:484-495.
19.
SCHROTERH, MUELLER CG, MEESE K, NORDHEIM A: Synergism in Ternary Complex Formation Between the Dimeric Glycoprotein p67SRF, Polypeptide p62TCF and the c-fos Serum Response Element. E m b o j 1990, 9:1123-1130.
SCHWARZ-SOMMER, Z, HUIJSER, P, NACKEN, W, SAEDLER, H, SOMMER, H: Genetic Control of Flower Development by Homeotic Genes in A n t i r r h i n u m Majus. Science 1990, 250:931-936.
20.
COEN ES, MEYEROW1TZE: T h e War of the Whorls: Genetic Interactions Controlling Flower Development. Nature 1991, 353:31-37.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
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2.
RIVERAVM, GREENBERG ME: Growth Factor-induced Gene Expression: the Ups and D o w n s of c-fos Regulation. N Biol 1990, 2:751-758.
3.
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SHAW PE, SCHROTERH, NORDHEIM A: T h e Ability of a Ternary Complex to Form Over the Serum Response Element Correlates with Serum Inducibility of the H u m a n c-fos Promoter. Cell 1989, 56:563-572.
6. ••
HIPSKIND RA, RAO VN, MUELLER CGF, REDDY EP, NORDHEIM A: Ets-related Protein EIk-I is Homologous to the c-fos Regulatory Factor p62TCF. Nature 1991, 354:531-534. This paper provides DNA-binding, protein-protein interaction and immunological evidence that I-leLa p62/TCF is homologous to Elk-l, an Ets-domain protein of previously unknown function. 7.
21.
POLLOCK RiM, TREISMAN RH: H u m a n SRF-related Proteins: DNA-binding Properties and Potential Regulatory Targets. Genes Det, 1991, 5:2327-2341. Characterization of cDNAs encoding mammalian SRF-related proteins of distinct sequence specificity, and the identification of potential target genes regulated by these proteins. Uses chimeric proteins to show that the basic region of the DNA-binding domain determines sequence specificity. •
22.
HAYES "rE, SENGUPTA P, COCHRAN BH: The H u m a n c-fos Serum Response Factor and the Yeast Factors GRM/PRTF have Related DNA-binding Specificities. Genes Dev 1988, 2:1713-1722.
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POLLOCKR, TREISMAN R: A Sensitive Method for the Determination of Protein-DNA-binding Specificities. Nucleic Acids Res 1990, 18:6197-6204.
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BENDER A, SPRAGUE GJ: MAT Alpha 1 Protein, a Yeast Transcription Activator, Binds Synergistically with a Second Protein to a Set of Cell-type-specific Genes. Cell 1987, 50:681-691.
DALTONS, TREISMANR: Characterisation of SAP-l, a Protein
••
Recruited by Serum Response Factor to the c-fos Serum Response Element. Cell 1992, 68:597-612. This paper uses a novel genetic screening method to identify a HeLA eDNA encoding the SRF accessory protein SAP-l, a protein related to Elk-1 and that has identical DNA-binding properties to the HeLa p62/TCF protein. These findings together with those in [6 • ' ] demonstrate that SRF has multiple accessory proteins. 8.
AMMERERG: Identification, Purification, and Cloning of a
Polypeptide (PRTF/GRM) that Binds to Mating-specific Prom o t e r Elements in Yeast. Genes Dev 1990, 4:299-312. 9.
PASSMORE S, ELBLE R, TYE BK: A Protein Involved in Minichromosome Maintenance in Yeast Binds a Transcriptional Enhancer Conserved in Eukaryotes. Genes Dev 1989, 3:921-935.
10.
JARVlSEE, CLARK KL, SPRAGUE GJ: T h e Yeast Transcription Activator PRTF, a Homolog of t h e Mammalian Serum Re-
25. ..
PRIMIG M, WINKLER H, AMMERER G: T h e DNA-binding and Oligomerisation Domain of MCMI is Sufficient for its Interaction with O t h e r Regulatory Protein. I~IBO J 1991, 10:4209-4218. Demonstration that the major functions of MCM1 require only its DNAbinding domain, and that a hybrid protein in which the MCM1 dimerization region is replaced by that of SRF will maintain cell viability but cannot interact with STE12, MAT0d and MATa2.
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Gene expression and differentiation MUELLERCGF, NORDHEIMA: A Protein Domain Conserved Between Yeast MCM1 and H u m a n SRF Directs Ternary Complex Formation. EMBOJ 1991, 10:4219--4229. Use of chimeric proteins to identify residues in the SRF/MCM1 DNAbinding domain that mediate interactions with accessory proteins. 26. ..
27.
CHRIST C, TYE BK: Functional Domains of the Yeast Transcription/replication Factor MCM1. Genes Dev 1991, 5:751-763.
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GAUTHIER,RC, FERNANDEZ,A, L~tB, NJ: Ras-induced c-fos Expression and Proliferation in Living Rat Fibroblasts Involves C-kinase Activation and the s e r u m Response Element Pathway. E)dBO J 1990, 9:171-180.
29. •.
GAUT~IER-ROUVIEREC, BASSET M, Bt.ANCHARDJ-M, CAVADOREJC, FERNANDEZA, LAMB NJC: Casein Kinase II Induces c-fos Expression Via the Serum Response Element Pathway and p67/SRF Phophorylation in Living Fibroblasts. EMBOJ 1991, 10:2921-2930. Describes the elegant use of microinjection to study the regulation of c-fo~ Shows that immunodepletion of SRF from cell nuclei by antibody microiniection prevents SRE-dependent induction of the c-fos gene by serum or microinjected CKII. 30.
GUTMANA, W&SYLYKC, W&S~XYKB: Cell-specific Regulation of Oncogene-responsive Sequences of the c-fos Promoter. Mol Cell Biol 1991, 11:5381-5387.
31.
SARTOREI.LIV, WEBSTER KA, KEDES I- Muscle-specific Expression of the Cardiac Alpha-actin Gene Requires MyoD1, CArG-Box Binding Factor, and Spl. Genes Dev 1990, 4:1811-1822.
32. •
39.
MOHUNTJ, CHAMBERSAE, TOWERS N, TAYLORM'V: Expression of Genes Encoding the Transcription Factor SRF During Early Development of X e n o p u s Laevis: Identification of a CArG Box.binding Activity as SRF. EMBOJ 1991, 10:933-940.
40.
MANAKJR, PRYWES R: Mutation of Serum Response Factor Phosphorylation Sites and the Mechanism by w h i c h its DNA-binding Activity is Increased by Casein Kinase II. Mol Cell Biol 1991, 11:3652-3659.
41. •.
GRAHam!R, GIL~IAN M: Distinct Protein Targets for Signals Acting at the c-fos Serum Response Element. Science 1991, 251:189-192. This paper shows that in Balb/C 3T3 cells, mutations that block p62/TCF binding reduce the response of the human c-fosgene to PKCdependent signalling pathways but have no effect on PKC-independent signal transduction. 42. ,•
MA~aKRK, ROE MXV, BLACKSHEARPJ: Epidermal Growth Factor and Other Mitogens Induce Binding of a Protein C o m p l e x to the c-los s e r u m Response Element in H u m a n Astrocytoma and O t h e r Cells. J Biol Cbem 1991, 266:8576-8582. E~fidence that in several cell t3q3es binding of p62/TCF or a similar protein to the c-fos SRE is regulated by growth-factor stimulation operating by both PKC-dependent and PKC-independent pathways.
43. t.
KONIGH: Cell-type Specific Multiprotein C o m p l e x Formation Over the c-fos Serum Response Element in Viv~ Ternary C o m p l e x Formation is not Required for the Induction of c-fos. Nucleic Acids Res 1991, 19:3607-3611. A genomic footprinting analysis of the c f o s SRE is given. Shows that the p62/TCF genomic footprint is not always observed at this SRE, even in cells in which the gene is inducible by serum stimulation.
GAOTHIER-ROU',qEREC, CAVADOREJ-C, BL~d'~CHARDJ-M, L~IB NJC, FERNANDEZA: p67SRF is a Constitutive Nuclear Protein Implicated in the Modulation of Genes Required Throughout the G1 Period. Cell Regul 1991, 2:575-588. Demostrate.s the use of SRF-specific antibodies, immunofluorescence and microinjection to study the function and intracellular localization of SRF.
44.
GUT~N A, W&SYL','K B: The Collagenase Gene Promoter Contains a TPA and Oncogene-responsive Unit Encompassing the PEA3 and AP-1 Binding Sites. F_A,IBOJ 1990, 9:2241-2246.
45.
Eu3LE R, TYE B-K: Both Activation and Repression of aMating-type-specific Genes in Yeast Require Transcription Factor M c m l . Proc Nail Acad Sci USA 1991, 88:10966-10970.
33. •
46.
TAN S, RICHMOND TJ: DNA-binding-induced Conformational Change of the Yeast Transcriptional Activator PRTF. Cell 1990, 62:367-377.
47.
JAR\qSEE, PL.~.GEN DC, SPRAGUE GJ: Identification of a DNA s e g m e n t that is Necessary and Sufficient for Alpha-specific Gene Control in Saccharomyces Cerevisiae: Implications for Regulation of Alpha.specific and a-specific Genes. Mol Cell Biol 1988, 8:309-320.
48.
ERREDEB, AMMERER G: STE12, a Protein Involved in Celltype-specific Transcription and Signal Transduction in Yeast, is Part of Protein-DNA Complexes. Genes Dev 1989, 3:1349-1361.
MISRARP, RWERAV/vl, \Vth'qGJM, FAN PD, GREENBERG ME: The Serum Response Factor is Extensively Modified by Phosphorylation Following its Synthesis in Serum-stimulated Fibroblasts. Mol Cell Biol 1991, 11:4545-4554. This work identifies the gene encoding SRF as an immediate early gene, and demonstrates that the protein encoded is extensively modified following sTnthesis. 34.
35.
36.
BOXER LM, MIWA T, GUSTAFSON TA, KEDES lz Identification and Characterization of a Factor that Binds to Two Human Sarcomeric Actin Promoters. J Biol Cbem 1989, 264:1284-1292. PRYWESR, DLrYI'AA, CROMHSHJA, ROEDER RG: Phosphorylation of Serum Response Factor, a Factor that Binds to the Serum Response Element of the c-FOS Enhancer. Proc Nail Acad Sci USA 1988, 85:7206-7210. SCHAL&~AG, DOPPLER C: Inhibition of c-fos Transcription and Phosphorylation of the Serum Response Factor by an Inhibitor of Phospholipase C-type Reactions. Mol Cell Biol 1990, 10:5558-5561.
37. •
MANAKJR, DE BN, KRIS RM, PRYWES R: Casein Kinase It Enhances the DNA-binding Activity of Serum Response Factor. Genes Det, 1990, 4:955-967. Demonstration that CKII phosphorylation results in a conformational change in the SRF DNA-binding domain. MARAISRM, HSUAN JJ, McGuIGAN CM, WYNNE J, TREISMAN R: Casein Kinase II Phosphorylation Increases the Rate of Serum Response Factor-binding Site Exchange. EMBO J 1992, 11:97-105. The use of recombinant SRF produced in insect cells to show that the effect of CKII phosphorylation is to accelerate the rate rather than the extent of SRF DNA binding.
49. SENGUPTAP, COCHRAN BH: MATed can Mediate Gene Acti•, vation by a Mating Factor. Genes Dev 1991, 5:1924-1934. Use of Lex.A gene fusions to demonstrate that MAT~xl but not MCM1 can mediate a response to mating pheromone, and that MATcxl contains a powerful actix~tion domain. 50.
YOAN YO, FIELDS S: Properties of the DNA.binding Domain of t h e S. cerevisiae STE12 Protein. Mol Cell Biol 1991, 11:5910-5918.
51.
HAGEN DC, MCCAFFREY G, SPRAGUE GJ: P h e r o m o n e Response Elements are Necessary and Sufficient for Basal and P h e r u m o n e - i n d u c e d Transcription of the FUS1 Gene of Saccharomyces cerevisiae. Mol Cell Biol 1991, 11:2952-2961.
38. •
R Treisman, Imperial Cancer Research Fund, PO Box 123, Lincolns Inn Fields, London WC2A 3PX, UK. G Ammerer, Forschunginstitut fur Molekular Pathologie (IMP), A-1090 Vienna, and lnstitut fUr Algemeine Biochemie and Ludwig Boltzmann Forschungstelle, University of Vienna, A-1090 Vienna, Austria.
Introduction The mammalian transcription factor SRF (serum-response factor) and the transcription factor MCM1 (also known as PRTF and GRM) are 70% identical within their DNA-binding and dimerization domains but are otherwise unrelated. Both proteins are apparently involved in the cellular response to extracellular signals and the celltype-specific regulation of transcription. The DNA-binding domains of both factors interact with a number of structurally unrelated accessory proteins that are listed in Table 1. Although these accessory proteins show only weak affinity for DNA, they make sequence-specific DNA contacts at positions adjacent to the SRF or MCM1 DNAbinding sites. SRF-binding sites are found in promoters and enhancers of growth factor-regulated and muscle-specific genes: they form the central sequence of serum-response elements (SREs) (for reviews see [1-3]). At the c-los SRE, DNA-bound SRF directs binding of an accessory protein, p62/TCF, that in some cells appears to be required for signal transduction [4,5]. Over the past year cDNAs encoding a number of SRF accessory proteins have been isolated (M Grueneberg, M Gilman, personal communication) [6°.,7 ' ' ] but the precise contributions of these proteins to SRE function remains unclear. MCM1, the product of the MCMI gene (for minichromosome maintenance) fulfills an unknown function that is essential for cell viability. MCMl-binding sites are found in several types of yeast promoter, particularly those of cell-type-specific genes [8-10] (see [11,12] for reviews). MCM1 recruits at least four different accessory proteins to its binding sites, depending on the sequence context of the particular MCMl-binding site and the cell type. The functions of the different MCMl-accessoryprotein complexes have been well characterized: in Saccharomyces cerevisiae a cells, complexes includ-
ing the MATczl and IVlAT0t2 proteins activate 0t-specific and repress a-specific genes, respectively, while MCM-1-STE12 complexes link the promoter to extracellular signals. A recently identified accessory factor, SFF, appears to be involved in cell-cycle-regulated transcription [13"]. As yet no obvious sequence similarities between the different MCM1 accessory proteins and the DNA sequences that they contact have been observed. In this review we first consider progress in the understanding of the structure of the SRF/MCM-like DNA-binding domain and its interaction with both DNA and the various accessory proteins. We then consider functional studies of SRF and MCM1 with particular reference to the role of the accessory proteins in activation. Finally, we consider to what extent functional comparisons between the two proteins are warranted.
The SRF/MCM-type DNA-binding domain SRF and MCM1 define a gene family An alignment of the SRF and MCM1 DNA-binding domains with other related sequences from },east, mammals and plants is shown in Figure 1. The yeast ARG80 protein is highly related to MCM1 and also appears to function as part of multiprotein complexes [ 14,15]. Several homeotic gene products that are involvecl in flower morphogenesis are members of a large family of plant gene products containing sequences distantly related to the DNA-binding domains of SRF and MCM1 [16-18] (for reviews see [19,20]). A set of equally divergent human gene products, the related to SRF (RSRF) proteins, that contain a common SRF-/MCM-type DNA-binding domains have also been characterized. The binding site of the RSRF proteins occurs in muscle-specifc and growth-factor-regulated genes, suggesting that their function may be mechanistically similar to SRF [21.]. No accessory proteins for
Abbreviations CKIl--casein kinase II; PKC--protein kinase C; RSRF~related to SRF; SRE--serum-response element; SRF~serum-response factor; UAS~upstream activating sequence. (~) Current Biology Lid ISSN 0959-437X
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Gene expression and differentiation
Table 1. Accessory proteins.
Core
Accessory protein
Source of accessory protein
SRF
p621TCF
HeLa
Ets domain los: CAGG
Detected in HeLa nuclear extracts; homologous to Elk-1 (A Rogers, R Treisman, unpublished data)16ee]; function unclear, see text 141°L43"°], can interact with MCM1 [26°'1
SRF
SAP-1
HeLa
Ets domain los: ACAGGAT
Isolated as an SRF-dependent SRE-binding protein by in vivo cDNA screening; function unclear, see text; related to Elk-l; can interact with MCM1 17"'1
SRF
SAP-2
Placenta
Ets domain los: ACAGGAT
Isolated by homology to SAP-l; also related to Elk-l; function is unknown IA Rogers, S Dalton, R Treisman, unpublished data)
SRF
Phoxl
Human
MCM1
MAT0cl
Diploids, c~ cells
? STE3: CTGTCA'FI-G
Positive- and pheromone-regulated transcription at a-specific MCM1 operators, e.g. STE3 (reviewed in [11,12l); response to a factor 149"]; cannot bind MCMI-SRF fusion protein 125"1
MCM1
MAT~z2
Diploids, a cells
Homeodomain STE6: CATGTAAI-i" CGTGTAAAT STE2: CATGTAAAT CATGTACTT
Negative regulation at a-specific MCM1 operators, eg. STE2 and STE6 [11,12]; functions negatively and indepently of MCM1 as part the of al-c~l repressor (reviewed in 111,12]; binds MCMI-SRF fusion protein weakly 12S°'l
MCM1
STE12
All types
Divergent homeodomain? STE2: ATGAAACA
Required for high basal level activity of a-specilic promoters, e.g. STE2; also functions independently of MCMI at promoters, such as FUSI, that contain multiple STE12-binding sites [50,51]; cannot interact with SRF [25"o,26 ° ' ]
MCM1
SFF
All types
? SW15: AAACAATA
Identilied only as a binding activity in yeast cell extracts implicated in cell-cell-dependent activation of the 5W15 promoter 113°°l; can interact with the MCM-SRF fusion protein (D Lydall, unpublished data)
DNA-interaction site and motif
Protein function, and comments
Isolated as human cDNA that activates a PQ box in the promoter of a MAT~I: TRP1 yeast strain; contains a homeobox (M Grueneberg, M Gilman, personal communication)
The different SRF and MCM1 accessory proteins that have thus far been identified are listed. Where known, the type of DNA-binding motif used by the accessory proteins to contact the DNA is shown, together with examples of the sequences contacted by each protein. See text for discussion.
the plant and human SRF-/MCM-related DNA-binding domains have been reported. Sequence homology between the SRF, MCM1 and ARG80 group of proteins, and also between the RSRF proteins, extends throughout their DNA-binding regions (Fig. 1). Sequences conserved between the entire group of proteins are, however, con~ned to the amino-terminal twothirds of the region that presumably represents a conserved structural domain, and which has been called the 'MADS-box' (derived from MCM-ARG80-AgamousDeficiens-SRF [19]). Careful study of the properties of chimeric proteins from clitTerent family members has provided significant insight into the structural and functional subdomains of the SRF/MCM-like DNA-binding domain.
DNA-binding specificity The DNA-binding domains of SRF and MCM1 are 70% identical, and both proteins can bind the c-fos-SRE, which can function as an upstream activating sequence (UAS) in yeast [9,22]. Detailed analysis of the binding specificities of the two proteins shows that they are overlapping rather than identical; sites selected in vitro by MCM1 have the consensus (notC)CCYAAWNNGG (W = Aor T ) (J Wynne, S Dalton, R Treisman, unpublished data), contrasting with the SRF consensus CCWWWWWWGG
(W = Aor T ) (see [9,23] for other studies). The MCM1 sites in ~-specific promoters appear to represent weak binding sites compared with those in a-specific promoters [24]. The consensus binding site for the RSRF proteins, YTAWWWWTAR (Y = C or T, R = A or G, W = A or T), is not detectably bound by SRF in vitro [21-]. Studies on the binding specificities of chimeric RSRF-SRF and MCM1-SRF proteins show that the sequences in the amino-terminal, basic region of the DNA-binding domain determine sequence specificity (J Wynne, S Dalton, R Treisman, unpublished data) (Fig. 1) [21,,25"o]. Although the specificities of the plant proteins and ARG80 have not been thoroughly characterized, in vitro binding studies suggest that the), may be related ~o those of SRF and MCM1 [26".].
Accessory factor specificity Differences in the ability of the various domains to interact with accessory proteins are suggested by the observations that SRF and MCM1, but not ARG80, can interact with HeLa p62/TCF [26 ,o] and that MCM1, but not SRF, can interact with MAT(zl, MAT~2 and STE12 [25o°,26o.]. Experiments with chimeric proteins show that residues crucial for the specificity of accessory protein recruitment are located in the divergent region carboxy-terminal to the 'MADS' homology (Fig. 1). Replacement of the MCM1
The SRF and MCM1 transcription factors Treisman, Ammerer
(a) MCM1 ARGS0
;PprsOpttdOr235 132sgakpgkkTrglv IkmeFidNKll ! i F~ k{t )iMl~ti TGTOYLLLYaSETGhVYTFaTrKRIPmITsetGKaLIOtCLNsP[ TGTQVLLLVvSETGLVYTFsTPKEPiVTqqEGrnLIQACLNAP[ leeedeeedgdd II0 7 gtptnngqqke leIkF}ENKTI F~ (H I N I I ~ :PdatDtspa(le 172 69 tkdssdtsTvt ,qp!r) IENKTI F$ rH I N H ~ V i rGantLLLIlanSGLYYTFtTPKINPvVredEGKsLIrACiNAs[
AG
41 ggesspl sg] iielkr enttn q~ f(
SRF
I
DEF
rn
144
cdaevlal vfssrgrlyeysnnsvkgti erykkaysensntgsv
1
I¢ iiq|kr enqtn qvl Ys *n i f l l
cdakvslilltlsstqkl heyisptt atkqlfdqyqkavgvdlwssl
M~~ IOls~[IOqRNOV{FTI ( F l ; i ~ M~ ~ ,OIt~!mOeRNQVIFT! (F
RSRFR,?. RSRFC4
Co) Conserved sequence motif
L
kkl n 95
CDCEIALIIFNSaNrLFQYASTDMDrVLLKYTEYsEPHESRTNt~tLkrrglgl g5 CDCEIALIIFNSsNkLFQYASTDM DkVLLKYTEYnEPHESRTN~ ~aLnkkehrg95
Bgl .........
-I-ill ..... | .....
.......
!-- l**i P - I ~ * * i i i - I
, ...........
,
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
"MADS-Box" DNA binding
(c)
b •
Dimerization
ii
Sequence specificity •
Accessory proteins ..............
,IP
Fig. 1. (a) The SRF-like DNA-binding domains from SRF, MCM1 and ARG80, the plant homeotic gene products Agamous (AG) and Deficiens (DEF), and the human RSRF proteins are shown. Identities between the different proteins are shown in dark grey shading, and conservative substitutions by light grey shading and asterisks. The regions of homology between the SRF, MCM1, ARG80 and the RSRF subgroups are boxed. (b) The conserved 'MADS box' motif (derived from MCM-ARG80-Agamous-Deficiens-SRF), is shown. (c) Functional regions of the DNA-binding domain are indicated below the figure. These include regions that determine DNA binding [3], dimerization [3], sequence specificity [21",25"L and accessory protein interaction [25",26"]. The dotted line indicates that sequences in this region effect the affinity, but not the specificity, of accessory factor interaction.
dimerization domain with the corresponding sequences from SRF prevents interaction of the resulting protein with the MCM1 accessory proteins M_ATcxl, M_ATcz2and STE12 [25--]. Substitution of the SRF amino-acid residues 196-203 with the corresponding residues from MCM1 generates a chimera that can recruit STE12; likewise, substitution of the corresponding residues in ARG80 with SRF residues 196-203 generates a chimera that can recruit p62/TCF [26..]. It is possible, however, that sequences outside this small region may also contribute to the affinity of the accessory factor interaction [26-.]. Thus the observed failure of MCM1 to substitute in vivo for ARG80, and vice versa, may reflect differences in the specificity of both DNA binding and accessory factor recognition [15,27]. Finally, the divergence between the RSRF proteins and SRF/MCM in this region suggests that any accessory proteins recognized by the RSRF proteins may be distinct from those recognized by SRF.
overexpression of SRF expression vectors transactivates a cotransfected SRE, but only in certain cell types (S John, R Treisman, upublished data) [30]. Expression of a LexA-SRF fusion protein is sufficient to render a cotransfected LexA operator weakly serum-responsive and this suggests that SRF itself may be capable of responding to extracellular signals (S John, R Treisman, unpublished data). It will be interesting to apply this approach to the study of muscle-specific promoters that are apparently dependent on SRF for constitutive activity [31]. Microinjection studies indicate that SRF and the SRE also fulfill an unknown function during the G1 stage of the cell cycle [32"]. Such 'late' functions might require newly synthesized SRF, consistent with the activation of SRF mRNA and protein synthesis following growth factor stimulation [3,33"]. In stimulated cells the newly synthesized protein is extensively modified after synthesis, apparently by phosphorylation [33"]. The protein is localized to the nucleus in both quiescent and growing cells, and remains chromatin associated throughout the cell cycle [32",33"].
Functional studies SRF function and expression
Regulation of SRF DNA-binding
Two experimental approaches suggest that SRF is directly involved in signal transduction and transcriptional activation. In a system where c-los expression requires SRE-binding proteins, depletion of SRF by the microinjection of specific antibodies is sufficient to block induction, strongly suggesting that SRF interacts directly with the SRE in vivo to promote growth-factor-regulated transcription [28,29" ]. In transfection experiments,
In the recent past, studies of SRF in crude extracts suggested that phosphorylation is absolutely required for DNA-binding [34-36] but since then studies with purified protein suggest that this is not the case [19,37",38"]. SRF contains an evolutionarily conserved site for phosphorylation by casein kinase II (CKII) [3,39], and the recombinant protein can be phosphorylated by this kinase in vitro [37%38",40]. Partial proteolysis studies suggest that
223
224
Gene expression and differentiation CKII phosphorylation causes a conformational change in the protein [40], which dramatically increases the in vitro SRF-DNA exchange rate rather than an alteration in DNAbinding affinity [38"]. Although CKII activity is increased by growth factor stimulation, and microinjection of the enzyme is sufficient to induce c-fos transcription in an SRF-dependent manner [29"'], the significance of CKII phosphorylation in the regulation of SRF function in vivo remains unclear. SRF accessory proteins Recent studies have led to the identification of a number of Ets-domain proteins that have binding properties similar to those of p62/TCF (see Table 1; [6"',7.']). In addition a homeodomain protein, Phoxl, appears to potentiate the binding activity of SRF at the c-fos SRE (M Grueneberg, M Gilman, personal communication). The role of p62/TCF-type proteins in SRE function remains unclear. For example, in Balb/C 3T3 cells, mutations that prevent p62/TCF binding in vitro were shown to block protein kinase C (PKC)-dependent signalling to the SRE but to leave PKC-independent signalling pathways unaffected [41.,]. In contrast, other reports suggest that binding of such factors is apparendy required for both PKC-dependent and independent signalling [42 o-] or that it potentiates but is not essential for signalling [43"]. In certain cell lines inducible binding of p62/TCF-like factors to the c-fos SRE has been reported, suggesting a direct link between these factors and signalling pathways [42.-]. The situation is further complicated by the identification of multiple TCF-like proteins and by the observation that other proteins, including Ets-domain proteins, can bind to the DNA contacted by p62/TCF independen@ of SRF [30, 44]. Moreover, it is not clear whether all SRE sequences can bind p62/TCF-type proteins. Obviously, we need to know much more about the types and structures of SRF accessory proteins before we can draw firm conclusions about their role in SRE function. MCM1 and its accessory proteins Using standard techniques of mutagenesis, functional studies of MCM1 have concentrated on attempts to delineate regions of the molecule required for cell viability and mating phenotype. These studies have shown that an 80 amino acid region of the protein, which contains the minimal DNA-binding domain, is sufficient to restore cell viability, minichromosome stability and mating. From this observation it would be expected that the carboxy-terminal region of the protein might serve a modulatory role: indeed, the acidic region neighbouring the DNA-binding domain is required for full activation of c~-cell-specific genes and the glutamine.rich carboxy-terminal region appears to modulate the degree of transcriptional activation [25..,27,45]. What is the role of the accessory proteins in MCM1 function? Three basic models, which are not mutually exclusive, have been proposed and are based, for historical reasons, on the mechanism of co-specific gene expression. The first proposes differential MCM1 binding. Although the protein is an activator, intracellular levels of the protein are insufficient to allow site saturation at weak sites
in c~-specific promoters in the absence of cooperatively binding MATcxl protein [24]. In the second, a conforrnational model, promoters bind MCM1, but in a conformation incompatible with transcriptional activation. Interaction with accessory proteins such as MATcxl brings about a conforrnational change that converts bound MCM1 to an activating form [46]. Finally, in the adaptor model, • accessory factors such as MAT0tl provide the activation function and linkage to signalling pathways, while MCM1 itself is only a weak activator. Although MCM1 can activate from a palindromic site in any cell type, consistent with the first model [47], recent studies also provide support for the second and third models. For example, genomic footprint analysis of two MCM1 sites at the Mf~l promoter showed that one of the two sites is not occupied by MCM1 in the absence of MATcxl, consistent with model one or three [25"'], while the other is bound by MCM1 but fails to activate transcription, consistent with models two or three (H Winkler, G Ammerer, unpublished data). Further evidence consistent with models two or three comes from the study of the SWI5 UAS: here, mutants that fail to bind the SFF factor are defective in activation even though MCM1 binding is not affected [13"]. Similar results are obtained with an a-cell-specific promoter fragment containing MCM1 and STE12 binding sites: here MCM1 can bind even in the absence of STE12, but only weak activation is observed (M Primig, G Ammerer, unpublished data) [48]. Direct evidence for model three comes from experiments in which either MCM1 or MATotl are fused to the LexA DNA-binding domain. This showed that MATotl has, if anything, greater activator activity than MCM1 itself; moreover, only the LexA-MAT0tl fusion protein will render a LexA-operator pheromone inducible by a factor [49"']. A reasonable proposal is thus that the principal role of MCM1 is as a functionally neutral DNA adaptor whose transcriptional competence and specificity is established by its accessory proteins. In particular, it appears that it is the accessory proteins that provide the target for signalling pathways. How similar are SRF and M C M I ? The similarities in the structure and functions of MCM1 and SRF has led to the suggestion that the two proteins are functional homologues. Strictly interpreted, this would imply that the two proteins might be functionally interchangeable, but this appears not to be the case: the results we have reviewed suggest that both the DNAbinding specificity and the accessory factor interactions of SRF and MCM1 are different. Indeed, when MCM1 is replaced by a chimeric protein comprising the MCM1 basic region (determining sequence specificity) and the SRF dimerization domain, cell viability is maintained but the cells do not mate owing to defects in the ability of the chimeric protein to interact with MCMI's accessory proteins [25"]. A simple view of this result is that although MCM1 and SRF may have similar functions, substantial divergence between the systems has occurred at the the level of the accessory proteins. The fact that the SRF accessory proteins p62/TCF and SAP-1 can interact with both SRF and MCM1 does, however, raise the possibility
The SRF and MCM1 transcription factors Treisman, Ammerer that at least some aspects of accessory protein structure have been evolutionarily conserved [6",7"•,26"']. Thus, it is tempting to speculate that there exist as yet uncharacterized accessory proteins that are structurally conserved between the two systems.
Conclusion Here we have outlined the close similarities between MCM1 and SRF. It is likely that their relationship extends at least in part at the functional level, but as yet no accessory protein from one system has been shown to have a structurally related counterpart in the other. The existence of closely related combinatorial systems of gene regulatory proteins in yeast and mammalian cells should provide valuable tools for their dissection at the molecular level.
s p o n s e Factor, is Encoded by t h e MCM1 Gene. Genes Dev 1989, 3:936-945. 11.
DOLANjW, FIELDSS: Cell-type-specific Transcription in Yeast. Biocbim Biophys Acta 1991, 1088:155-169.
12.
SPRAGUEGF: Combinatorial Associations of Regulatory Proteins and the Control of Cell Type in Yeast. Adv Genet 1990, 27:33--62.
LYDALLD, AMMERERG, N,'6MYI'H K: A New Role for MCMI in Yeast: Cell Cycle Regulation of SWI5 Transcription. Genes Dev 1991, 5:2405-2419. Demonstrates a new role for MCM1 in the regulation of cell.cycle-dependent transcription. Identifies a novel accessory protein, SWI5 factor (SFF), that is distinct from other MCM1 accessory proteins, and which is implicated in transcriptional regulation. 13. ••
14.
MESSENGUY F, DUBOIS E, BOONCHIRD C: Determination of the DNA-binding Sequences of ARGR Proteins to Arginine Anabolic and Catabolic Promoters. Mol Cell Biol 1991, 11:2852-2863.
15.
DUBOISE, ME~,ENGUY F: In Vitro Studies of t h e Binding of the ARGR Proteins to the ARG5,6 Promoter. Mol Cell Biol 1991, 11:2162-2168.
16.
SOMMERH, BELTRANJP, HUUSERP, PAPE H, LONNIGWE, SAEDLER H, SCHWARZ.SZ: Deficiens, a Homeotic Gene Involved in the Control of Flower Morphogenesis in Antirrhinum Majus: the Protein Shows Homology to Transcription Factors. Embo J 1990, 9:605-613.
17.
YANOFSV,~'MF, IvlA H, BOWMANJL, DREWS GN, FELDMANN KA, MEYEROXXq'VZEM: T h e Protein Encoded by the Arabidopsis Homeotic Gene Agamous Resembles Transcription Factors. Nature 1990, 346:35-39.
18.
NORMANC, RUNSWlCKM, POLLOCKR, TREISMANR: Isolation and Properties of cDNA Clones Encoding SRF, a Tanscription Factor that Binds to the c-los Serum Response Element. Cell 1988, 55:989-1003.
MA H, YANOI"SKYMF, MEYEROWrI'Z EM: AGL1-AGL6, an Arabidopsis Gene Family with Similarity to Floral Homeotic and Transcription Factor Genes. Genes Dev 1991, 5:484-495.
19.
SCHROTERH, MUELLER CG, MEESE K, NORDHEIM A: Synergism in Ternary Complex Formation Between the Dimeric Glycoprotein p67SRF, Polypeptide p62TCF and the c-fos Serum Response Element. E m b o j 1990, 9:1123-1130.
SCHWARZ-SOMMER, Z, HUIJSER, P, NACKEN, W, SAEDLER, H, SOMMER, H: Genetic Control of Flower Development by Homeotic Genes in A n t i r r h i n u m Majus. Science 1990, 250:931-936.
20.
COEN ES, MEYEROW1TZE: T h e War of the Whorls: Genetic Interactions Controlling Flower Development. Nature 1991, 353:31-37.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
TREISMANR: The SRE: A Growth-factor Responsive Transcriptional Regulator. Semin Cancer Biol 1990, 1:47-58.
2.
RIVERAVM, GREENBERG ME: Growth Factor-induced Gene Expression: the Ups and D o w n s of c-fos Regulation. N Biol 1990, 2:751-758.
3.
4.
5.
SHAW PE, SCHROTERH, NORDHEIM A: T h e Ability of a Ternary Complex to Form Over the Serum Response Element Correlates with Serum Inducibility of the H u m a n c-fos Promoter. Cell 1989, 56:563-572.
6. ••
HIPSKIND RA, RAO VN, MUELLER CGF, REDDY EP, NORDHEIM A: Ets-related Protein EIk-I is Homologous to the c-fos Regulatory Factor p62TCF. Nature 1991, 354:531-534. This paper provides DNA-binding, protein-protein interaction and immunological evidence that I-leLa p62/TCF is homologous to Elk-l, an Ets-domain protein of previously unknown function. 7.
21.
POLLOCK RiM, TREISMAN RH: H u m a n SRF-related Proteins: DNA-binding Properties and Potential Regulatory Targets. Genes Det, 1991, 5:2327-2341. Characterization of cDNAs encoding mammalian SRF-related proteins of distinct sequence specificity, and the identification of potential target genes regulated by these proteins. Uses chimeric proteins to show that the basic region of the DNA-binding domain determines sequence specificity. •
22.
HAYES "rE, SENGUPTA P, COCHRAN BH: The H u m a n c-fos Serum Response Factor and the Yeast Factors GRM/PRTF have Related DNA-binding Specificities. Genes Dev 1988, 2:1713-1722.
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POLLOCKR, TREISMAN R: A Sensitive Method for the Determination of Protein-DNA-binding Specificities. Nucleic Acids Res 1990, 18:6197-6204.
24.
BENDER A, SPRAGUE GJ: MAT Alpha 1 Protein, a Yeast Transcription Activator, Binds Synergistically with a Second Protein to a Set of Cell-type-specific Genes. Cell 1987, 50:681-691.
DALTONS, TREISMANR: Characterisation of SAP-l, a Protein
••
Recruited by Serum Response Factor to the c-fos Serum Response Element. Cell 1992, 68:597-612. This paper uses a novel genetic screening method to identify a HeLA eDNA encoding the SRF accessory protein SAP-l, a protein related to Elk-1 and that has identical DNA-binding properties to the HeLa p62/TCF protein. These findings together with those in [6 • ' ] demonstrate that SRF has multiple accessory proteins. 8.
AMMERERG: Identification, Purification, and Cloning of a
Polypeptide (PRTF/GRM) that Binds to Mating-specific Prom o t e r Elements in Yeast. Genes Dev 1990, 4:299-312. 9.
PASSMORE S, ELBLE R, TYE BK: A Protein Involved in Minichromosome Maintenance in Yeast Binds a Transcriptional Enhancer Conserved in Eukaryotes. Genes Dev 1989, 3:921-935.
10.
JARVlSEE, CLARK KL, SPRAGUE GJ: T h e Yeast Transcription Activator PRTF, a Homolog of t h e Mammalian Serum Re-
25. ..
PRIMIG M, WINKLER H, AMMERER G: T h e DNA-binding and Oligomerisation Domain of MCMI is Sufficient for its Interaction with O t h e r Regulatory Protein. I~IBO J 1991, 10:4209-4218. Demonstration that the major functions of MCM1 require only its DNAbinding domain, and that a hybrid protein in which the MCM1 dimerization region is replaced by that of SRF will maintain cell viability but cannot interact with STE12, MAT0d and MATa2.
225
226
Gene expression and differentiation MUELLERCGF, NORDHEIMA: A Protein Domain Conserved Between Yeast MCM1 and H u m a n SRF Directs Ternary Complex Formation. EMBOJ 1991, 10:4219--4229. Use of chimeric proteins to identify residues in the SRF/MCM1 DNAbinding domain that mediate interactions with accessory proteins. 26. ..
27.
CHRIST C, TYE BK: Functional Domains of the Yeast Transcription/replication Factor MCM1. Genes Dev 1991, 5:751-763.
28.
GAUTHIER,RC, FERNANDEZ,A, L~tB, NJ: Ras-induced c-fos Expression and Proliferation in Living Rat Fibroblasts Involves C-kinase Activation and the s e r u m Response Element Pathway. E)dBO J 1990, 9:171-180.
29. •.
GAUT~IER-ROUVIEREC, BASSET M, Bt.ANCHARDJ-M, CAVADOREJC, FERNANDEZA, LAMB NJC: Casein Kinase II Induces c-fos Expression Via the Serum Response Element Pathway and p67/SRF Phophorylation in Living Fibroblasts. EMBOJ 1991, 10:2921-2930. Describes the elegant use of microinjection to study the regulation of c-fo~ Shows that immunodepletion of SRF from cell nuclei by antibody microiniection prevents SRE-dependent induction of the c-fos gene by serum or microinjected CKII. 30.
GUTMANA, W&SYLYKC, W&S~XYKB: Cell-specific Regulation of Oncogene-responsive Sequences of the c-fos Promoter. Mol Cell Biol 1991, 11:5381-5387.
31.
SARTOREI.LIV, WEBSTER KA, KEDES I- Muscle-specific Expression of the Cardiac Alpha-actin Gene Requires MyoD1, CArG-Box Binding Factor, and Spl. Genes Dev 1990, 4:1811-1822.
32. •
39.
MOHUNTJ, CHAMBERSAE, TOWERS N, TAYLORM'V: Expression of Genes Encoding the Transcription Factor SRF During Early Development of X e n o p u s Laevis: Identification of a CArG Box.binding Activity as SRF. EMBOJ 1991, 10:933-940.
40.
MANAKJR, PRYWES R: Mutation of Serum Response Factor Phosphorylation Sites and the Mechanism by w h i c h its DNA-binding Activity is Increased by Casein Kinase II. Mol Cell Biol 1991, 11:3652-3659.
41. •.
GRAHam!R, GIL~IAN M: Distinct Protein Targets for Signals Acting at the c-fos Serum Response Element. Science 1991, 251:189-192. This paper shows that in Balb/C 3T3 cells, mutations that block p62/TCF binding reduce the response of the human c-fosgene to PKCdependent signalling pathways but have no effect on PKC-independent signal transduction. 42. ,•
MA~aKRK, ROE MXV, BLACKSHEARPJ: Epidermal Growth Factor and Other Mitogens Induce Binding of a Protein C o m p l e x to the c-los s e r u m Response Element in H u m a n Astrocytoma and O t h e r Cells. J Biol Cbem 1991, 266:8576-8582. E~fidence that in several cell t3q3es binding of p62/TCF or a similar protein to the c-fos SRE is regulated by growth-factor stimulation operating by both PKC-dependent and PKC-independent pathways.
43. t.
KONIGH: Cell-type Specific Multiprotein C o m p l e x Formation Over the c-fos Serum Response Element in Viv~ Ternary C o m p l e x Formation is not Required for the Induction of c-fos. Nucleic Acids Res 1991, 19:3607-3611. A genomic footprinting analysis of the c f o s SRE is given. Shows that the p62/TCF genomic footprint is not always observed at this SRE, even in cells in which the gene is inducible by serum stimulation.
GAOTHIER-ROU',qEREC, CAVADOREJ-C, BL~d'~CHARDJ-M, L~IB NJC, FERNANDEZA: p67SRF is a Constitutive Nuclear Protein Implicated in the Modulation of Genes Required Throughout the G1 Period. Cell Regul 1991, 2:575-588. Demostrate.s the use of SRF-specific antibodies, immunofluorescence and microinjection to study the function and intracellular localization of SRF.
44.
GUT~N A, W&SYL','K B: The Collagenase Gene Promoter Contains a TPA and Oncogene-responsive Unit Encompassing the PEA3 and AP-1 Binding Sites. F_A,IBOJ 1990, 9:2241-2246.
45.
Eu3LE R, TYE B-K: Both Activation and Repression of aMating-type-specific Genes in Yeast Require Transcription Factor M c m l . Proc Nail Acad Sci USA 1991, 88:10966-10970.
33. •
46.
TAN S, RICHMOND TJ: DNA-binding-induced Conformational Change of the Yeast Transcriptional Activator PRTF. Cell 1990, 62:367-377.
47.
JAR\qSEE, PL.~.GEN DC, SPRAGUE GJ: Identification of a DNA s e g m e n t that is Necessary and Sufficient for Alpha-specific Gene Control in Saccharomyces Cerevisiae: Implications for Regulation of Alpha.specific and a-specific Genes. Mol Cell Biol 1988, 8:309-320.
48.
ERREDEB, AMMERER G: STE12, a Protein Involved in Celltype-specific Transcription and Signal Transduction in Yeast, is Part of Protein-DNA Complexes. Genes Dev 1989, 3:1349-1361.
MISRARP, RWERAV/vl, \Vth'qGJM, FAN PD, GREENBERG ME: The Serum Response Factor is Extensively Modified by Phosphorylation Following its Synthesis in Serum-stimulated Fibroblasts. Mol Cell Biol 1991, 11:4545-4554. This work identifies the gene encoding SRF as an immediate early gene, and demonstrates that the protein encoded is extensively modified following sTnthesis. 34.
35.
36.
BOXER LM, MIWA T, GUSTAFSON TA, KEDES lz Identification and Characterization of a Factor that Binds to Two Human Sarcomeric Actin Promoters. J Biol Cbem 1989, 264:1284-1292. PRYWESR, DLrYI'AA, CROMHSHJA, ROEDER RG: Phosphorylation of Serum Response Factor, a Factor that Binds to the Serum Response Element of the c-FOS Enhancer. Proc Nail Acad Sci USA 1988, 85:7206-7210. SCHAL&~AG, DOPPLER C: Inhibition of c-fos Transcription and Phosphorylation of the Serum Response Factor by an Inhibitor of Phospholipase C-type Reactions. Mol Cell Biol 1990, 10:5558-5561.
37. •
MANAKJR, DE BN, KRIS RM, PRYWES R: Casein Kinase It Enhances the DNA-binding Activity of Serum Response Factor. Genes Det, 1990, 4:955-967. Demonstration that CKII phosphorylation results in a conformational change in the SRF DNA-binding domain. MARAISRM, HSUAN JJ, McGuIGAN CM, WYNNE J, TREISMAN R: Casein Kinase II Phosphorylation Increases the Rate of Serum Response Factor-binding Site Exchange. EMBO J 1992, 11:97-105. The use of recombinant SRF produced in insect cells to show that the effect of CKII phosphorylation is to accelerate the rate rather than the extent of SRF DNA binding.
49. SENGUPTAP, COCHRAN BH: MATed can Mediate Gene Acti•, vation by a Mating Factor. Genes Dev 1991, 5:1924-1934. Use of Lex.A gene fusions to demonstrate that MAT~xl but not MCM1 can mediate a response to mating pheromone, and that MATcxl contains a powerful actix~tion domain. 50.
YOAN YO, FIELDS S: Properties of the DNA.binding Domain of t h e S. cerevisiae STE12 Protein. Mol Cell Biol 1991, 11:5910-5918.
51.
HAGEN DC, MCCAFFREY G, SPRAGUE GJ: P h e r o m o n e Response Elements are Necessary and Sufficient for Basal and P h e r u m o n e - i n d u c e d Transcription of the FUS1 Gene of Saccharomyces cerevisiae. Mol Cell Biol 1991, 11:2952-2961.
38. •
R Treisman, Imperial Cancer Research Fund, PO Box 123, Lincolns Inn Fields, London WC2A 3PX, UK. G Ammerer, Forschunginstitut fur Molekular Pathologie (IMP), A-1090 Vienna, and lnstitut fUr Algemeine Biochemie and Ludwig Boltzmann Forschungstelle, University of Vienna, A-1090 Vienna, Austria.
