GAL4 Fusion Proteins Research Articles

Baculovirus-transfer vector for eukaryotic expression and immunoaffinity purification of Gal4-fusion proteins.

Baculovirus-transfer vector for eukaryotic expression and immunoaffinity purification of Gal4-fusion proteins.

CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex.

CBF1 is a member of the CSL family of DNA binding factors, which mediate either transcriptional repression or transcriptional activation. CSL proteins play a central role in Notch signaling and in Epstein-Barr virus-induced immortalization. Notch is a transmembrane protein involved in cell-fate decisions, and the cytoplasmic domain of Notch (NotchIC) targets CBF1. The Epstein-Barr virus-immortalizing protein EBNA2 activates both cellular and viral gene expression by targeting CBF1 and mimicking NotchIC. We have examined the mechanism of CBF1-mediated repression and show that CBF1 binds to a unique corepressor, CBF1 interacting corepressor (CIR). A CIR homolog is encoded by Caenorhabditis elegans, indicating that CIR is evolutionarily conserved. Two CBF1 mutants that were unable to bind CIR did not function as repressors, suggesting that targeting of CIR to CBF1 is an important component of repression. When expressed as a Gal4 fusion protein, CIR repressed reporter gene expression. CIR binds to histone deacetylase and to SAP30 and serves as a linker between CBF1 and the histone deacetylase complex.

PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins.

The PRDI-BF1/Blimp-1 protein is a transcriptional repressor required for normal B-cell differentiation, and it has been implicated in the repression of beta-interferon (IFN-beta) and c-myc gene expression. Here, we show that PRDI-BF1 represses transcription of the IFN-beta promoter and of an artificial promoter through an active repression mechanism. We also identified a minimal repression domain in PRDI-BF1 that is sufficient for transcriptional repression when tethered to DNA as a Gal4 fusion protein. Remarkably, this repression domain interacts specifically with hGrg, TLE1, and TLE2 proteins, all of which are members of the Groucho family of transcriptional corepressors. In addition, the hGrg protein itself can function as a potent repressor when tethered to DNA through the Gal4 DNA-binding domain. We also find that the amino-terminal glutamine-rich domains of hGrg and TLE1 are sufficient to mediate dimerization of the two Groucho family proteins. Proteins containing only this domain can function as a dominant-negative inhibitor of PRDI-BF1 repression, and can significantly increase the IFN-beta promoter activity after virus induction. We conclude that PRDI-BF1/Blimp-1 represses transcription by recruiting a complex of Groucho family proteins to DNA, and suggest that such corepressor complexes are required for the postinduction repression of the IFN-beta promoter.

Ser727-dependent recruitment of MCM5 by Stat1alpha in IFN-gamma-induced transcriptional activation.

Stat1alpha is a latent cytoplasmic transcription factor activated in response to interferon-gamma (IFN-gamma). The C-terminal 38 amino acids of Stat1alpha are required to trigger transcription and therefore may possibly serve as a transcription activation domain (TAD). Here we show that the C-terminus of Stat1alpha is an independent TAD which can interact with a specific group of nuclear proteins. Mutation of the Stat1 Ser727 and Leu724 decreases its transcriptional activity and affinity for the nuclear proteins. One of the interacting proteins was identified as MCM5, a member of the mini-chromosome maintenance (MCM) family involved in DNA replication. Both in vitro and in vivo interaction of Stat1alpha and MCM5 were demonstrated. Furthermore, the in vitro interaction required Ser727 and was enhanced by its phosphorylation. Transient over-expression of MCM5 enhanced transcriptional activation by Stat1alpha in a Ser727-dependent manner. Finally, changes in the level of nuclear localized MCM5 during the cell cycle correlated with the changes in transcriptional response to IFN-gamma acting through Stat1alpha. These results strongly suggest that MCM5 is recruited through interaction with Stat1alpha in a Ser727- and Leu724-dependent manner to play a role in optimal transcriptional activation.

C-Myc target gene specificity is determined by a post-DNAbinding mechanism.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

Functional analysis of ZNF85 KRAB zinc finger protein, a member of the highly homologous ZNF91 family.

We previously identified the ZNF85 (HPF4) KRAB zinc finger gene, a member of the human ZNF91 family. Here, we show that the ZNF85 gene is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line. Immunocytochemical localization of a panel of beta-Gal/ZNF85 fusion proteins revealed that ZNF85 contains at least one nuclear localization signal located in the spacer region connecting the KRAB domain with the zinc finger repeats. Bacterially expressed ZNF85 zinc finger domain bound strongly and exclusively to DNA in vitro in a zinc-dependent manner. The KRAB(A) domain of the ZNF85 protein and of several other members of the ZNF91 family exhibited repressing activity when tested in Gal4 fusion protein assays. The repression was significantly enhanced by the addition of the KRAB (B) domain, whereas further addition of other conserved regions had no effect. The ZNF85 KRAB(A) and (B) domains in vitro bound several nuclear proteins that might constitute critical cofactors for repression.

Phosphorylation and spatiotemporal distribution of KW8 (NDRF/NeuroD2), a NeuroD family basic helix–loop–helix protein

KW8, a NeuroD family basic helix–loop–helix protein, was initially cloned during the course of screening for the genes related to long term potentiation in rat hippocampal slice. Its homologue NDRF/NeuroD was also reported. In this report its phosphorylation and spatiotemporal distribution was studied. KW8 was expressed not only during embryonic and neonatal periods but also in adults. In adult, KW8 was expressed only in brain tissues, such as the cerebral cortex, hippocampus and cerebellum. Immunohistological studies revealed that KW8 was localized in the nuclei of neurons. On immunoblotting of rat brain tissue, COS-1 cells and Neuro2A cells overexpressing KW8, this protein was detected as several diffuse bands. Alkaline phosphatase treatment reduced the molecular weights of these bands. Metabolic labeling with 32 Pi in COS-1 cells confirmed that the KW8 protein was phosphorylated in vivo. Some of the physiological functions of KW8 might be regulated by this phosphorylation. In yeast, the GAL4 fusion protein containing the C-terminal region of KW8 activated transcription of the reporter gene, suggesting that KW8 had transcriptional activity.

TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4.

TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.

Regulation of theDrosophilabHLH-PAS Protein Sima by Hypoxia: Functional Evidence for Homology with Mammalian HIF-1α

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric complex of two basic-helix–loop-helix proteins of the PAS family which is critical for oxygen-dependent expression of many mammalian genes. Regulation is mediated by the alpha subunit (HIF-1α) and sequences from HIF-1α can confer hypoxia-inducible activity on a Gal4 fusion protein. To analyse conservation of this system of gene regulation betweenDrosophilaand mammalian cells we constructed Gal4 fusions with a series ofDrosophilabasic-helix–loop-helix PAS (bHLH-PAS) proteins and tested for hypoxia inducibility in transfected Hep3B cells. We found that Gal4 fusions with Similar (Sima) but not otherDrosophilabHLH-PAS proteins showed inducible activity following exposure to stimuli which classically activate mammalian HIF-1: hypoxia, cobaltous ions, and desferrioxamine. We also found that Sima protein accumulated inDrosophilaSL2 cells following hypoxia. Together these findings indicate the existence of functional homologies between Sima and HIF-1α, and that conservation is such as to enable Sima to interact with the hypoxia signal transduction system in mammalian cells.

Transcriptional activation domains stimulate initiation and elongation at different times and via different residues.

Transcriptional activators can stimulate multiple steps in the transcription process. We have used GAL4 fusion proteins to characterize the ability of different transcriptional activation domains to stimulate transcriptional elongation on the hsp70 gene in vitro. Stimulation of elongation apparently occurs via a mechanistic pathway different from that of stimulation of initiation: the herpes simplex virus VP16, heat shock factor 1 (HSF1) and amphipathic helix (AH) activation domains all stimulate initiation, but only VP16 and HSF1 stimulate elongation; and mutations in hydrophobic residues of the HSF1 activation domains impair stimulation of elongation but not of initiation, while mutations in adjacent acidic residues impair stimulation of initiation more than of elongation. Experiments in which activators were exchanged between initiation and elongation demonstrate that the elongation function of HSF1 will stimulate RNA polymerase that has initiated and is transcriptionally engaged. Transcriptional activators thus appear to have at least two distinct functions that reside in the same domain, and that act at different times to stimulate initiation and elongation.

CDNA cloning and characterization of mouse DTEF-1 and ETF, members of the TEA/ATTS family of transcription factors.

Members of the TEA/ATTS family of transcription factors have been found in most representative eukaryotic organisms. In vertebrates, the TEA family contains at least four members, which share overlapping DNA-binding specificity and have similar transcriptional activation properties. In this article, we describe the cDNA cloning and characterization of the murine TEA proteins DTEF-1 (mDTEF-1) and ETF. Using in situ hybridization analysis of mouse embryos, we found that mDTEF-1 and ETF transcript distributions substantially overlap. ETF is expressed throughout the embryo except in the myocardium early in development, whereas late in development, it is enriched in lung and neuroectoderm. Mouse DTEF-1 is expressed at a much lower level throughout development and is substantially enriched in ectoderm and skin, as well as in the developing pituitary at midgestation. Northern blot analysis of adult mouse tissue total RNA showed that both ETF and mDTEF-1 are abundant in uterus and lung relative to other tissues. Using gel mobility shift assays and GAL4-fusion protein analysis, we demonstrated that the full coding sequences of ETF and mDTEF-1 encode M-CAT/GT-IIC-binding proteins containing activation domains.

Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation.

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-kappaB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IkappaB molecule, which in turn would bind and remove activated, DNA-bound NF-kappaB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-kappaB-dependent transcription, without changing the expression level of IkappaB. Neither the mRNA of IkappaB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-kappaB p65 subunit.

Interaction of the Human Protein Kinase PKR with the Mouse PKR Homolog Occurs via the N-Terminal Region of PKR and Does Not Inactivate Autophosphorylation Activity of Mouse PKR

The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon. Mutant forms of human PKR display a transdominant behavior when expressed in transfected cells. The potential for the human PKR protein to physically interact with the mouse PKR homolog has therefore been examined. The yeast two-hybrid system was used to probe the association between mouse and human PKR proteins as measured by activation of two Gal4-responsive reporter genes,HIS3 andlacZ. Expression of full-length wild-type mouse PKR(1-515)WT as a Gal4 fusion protein did not exhibit the growth suppression phenotype in yeast characteristic of wild-type human PKR(1-551)WT. Coexpression of mouse PKR(1-515)WT as a Gal4 DNA-binding domain fusion with either the catalytic-deficient human PKR(1-551) K296R mutant, the RNA-binding-deficient human PKR(1-551)K64E/K296R double mutant, or wild-type mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusions resulted in activation of theHIS3 andlacZ reporters. The N-terminal RNA-binding region of human PKR, both WT and the K64E RNA-binding-deficient mutant, also interacted with mouse PKR(1-515)WT sufficiently to activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathioneS-transferase (GST) fusions inEscherichia coliwere purified on Sepharose beads. Using GST-PKR fusion chromatography, direct physical interaction between the mouse and human PKR homologs was established. Intraspecies PKR interactions were more efficient than interspecies PKR interactions, and interactions between RNA-binding-sufficient PKR proteins were more efficient than those involving an RNA-binding mutant as measured by binding to GST-PKR protein Sepharose beads. The N-terminal region of human PKR within amino acids 1–184 was sufficient for binding mouse PKR. Purified mouse full-length PKR(1-515)WT GST fusion protein retained kinase activity on Sepharose beads, but the activity was not impaired by association with either the full-length or the N-terminal region of human PKR.

Interaction between the Amino- and Carboxyl-terminal Regions of the Rat Androgen Receptor Modulates Transcriptional Activity and Is Influenced by Nuclear Receptor Coactivators

Identical N-terminal deletions in the wild-type rat androgen receptor (rAR) and a constitutively active rAR (ARDelta641-902) devoid of the ligand-binding domain (LBD) resulted in dissimilar consequences in transcriptional activation: deletion of residues 149-295 abolished wild-type AR activity, but did not influence that of ARDelta641-902. The activity of the N-terminal transactivation domain is thus controlled by the hormone-occupied LBD, suggesting that the N- and C-terminal regions of rAR communicate. Consistent with this idea, a strong androgen-dependent interaction between the N-terminal region and LBD was demonstrated in a mammalian two-hybrid system using GAL4 and VP16 fusion proteins. This interaction can be direct or indirect. Several nuclear receptor coactivators (CBP, F-SRC-1, SRC-1, and RIP140) that interact with other steroid receptors were tested as potential mediators of the N- and C-terminal interaction of rAR using the mammalian two-hybrid system. CBP or F-SRC-1 not only enhanced AR-mediated transactivation, but also facilitated the androgen-dependent interaction between the N- and C-terminal domains, implying that part of the coactivator-dependent transcriptional activation occurs via this mechanism. In contrast, SRC-1, a coactivator for the progesterone receptor, inhibited both AR-mediated transactivation and interaction between the N and C termini. Recruitment of coregulators may involve AR domains other than the LBD, as F-SRC-1 and CBP enhanced, but SRC-1 repressed, the transcriptional activity of ARDelta641-902. Collectively, interplay between the N-terminal region and LBD of rAR results in the formation of a transactivation complex that includes coregulators and that is mandatory for optimal activation of androgen-induced promoters.

P32/TAP, a Cellular Protein That Interacts with EBNA-1 of Epstein–Barr Virus

The Epstein–Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors. EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters. The mechanisms by which EBNA-1 performs these functions are not known. Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1. We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa. It has been previously reported that P32/TAP is expressed on the cell surface. Our transient expression assays detected full-length P32/TAP (1–282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus. Three lines of evidence support P32/TAP interaction with EBNA-1. First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40–60 and aa 325–376, each of which contains arginine–glycine repeats. These regions interact with the C-terminal half of P32/TAP. Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm. Third, P32/TAP co-immunoprecipitated with EBNA-1. We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244–282) transactivates expression from a reporter containing upstream Gal4-binding sites. Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays. Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation.

The SYT protein involved in the t(X;18) synovial sarcoma translocation is a transcriptional activator localised in nuclear bodies.

The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to either of two closely related genes SSX1 and SSX2 on chromosome X. The resulting chimaeric genes express SYT-SSX1 or SYT-SSX2 fusion proteins in which the C-terminal amino acids of SYT are replaced by amino acids from the C-terminus of the SSX proteins. Using green fluorescent protein fusions we demonstrate that the SYT, SSX and the SYT-SSX proteins are nuclear proteins. We demonstrate that whilst the SSX1 protein has a uniform nuclear distribution the SYT protein has a speckled distribution in the cell nucleus, and this distribution is retained with the SYT-SSX2 fusion protein. Since the SYT speckles do not co-localise with PML-containing bodies (PODs) or spliceosomes it is possible that they represent a novel nuclear structure. Transfection of constructs expressing GAL4 fusion proteins demonstrate that the SYT domains present in the SYT-SSX fusion proteins can activate transcription of a luciferase reporter. It is proposed that the t(X;18) translocation results in the generation of an SYT-SSX transcriptional co-activator in which the addition of the C-terminal SSX domain to SYT provides a new interacting domain that redirects the SYT activation domain to different target promoters.

Transactivation and Inhibitory Domains of Hypoxia-inducible Factor 1α: MODULATION OF TRANSCRIPTIONAL ACTIVITY BY OXYGEN TENSION

Hypoxia-inducible factor 1 (HIF-1) binds to cis-acting hypoxia-response elements within the erythropoietin, vascular endothelial growth factor, and other genes to activate transcription in hypoxic cells. HIF-1 is a basic helix-loop-helix transcription factor composed of HIF-1alpha and HIF-1beta subunits. Here, we demonstrate that HIF-1alpha contains two transactivation domains located between amino acids 531 and 826. When expressed as GAL4 fusion proteins, the transcriptional activity of these domains increased in response to hypoxia. Fusion protein levels were unaffected by changes in cellular O2 tension. Two minimal transactivation domains were localized to amino acid residues 531-575 and 786-826. The transcriptional activation domains were separated by amino acid sequences that inhibited transactivation. Deletion analysis demonstrated that the gradual removal of inhibitory domain sequences (amino acids 576-785) was associated with progressively increased transcriptional activity of the fusion proteins, especially in cells cultured at 20% O2. Transcriptional activity of GAL4/HIF-1alpha fusion proteins was increased in cells exposed to 1% O2, cobalt chloride, or desferrioxamine, each of which also increased levels of endogenous HIF-1alpha protein but did not affect fusion protein levels. These results indicate that increased transcriptional activity mediated by HIF-1 in hypoxic cells results from both increased HIF-1alpha protein levels and increased activity of HIF-1alpha transactivation domains.

Differential in vivo activation of the class II and class III snRNA genes by the POU-specific domain of Oct-1.

Many snRNA genes contain binding sites for the ubiquitous transcription factor Oct-1. In vitro studies have shown that this factor potentiates binding of an essential transcription factor (PTF) to the proximal sequence element (PSE) of snRNA genes, and activates transcription. Using Gal4 fusion proteins, I show here that the POU-specific region of the DNA-binding domain of Oct-1 is sufficient both to potentiate PTF binding in vitro and to transactivate pol II- and pol III-dependent snRNA genes in vivo . A single amino acid change in this domain abrogates both activation and interaction with PTF. The N- and C-terminal regions of Oct-1 also activate transcription of both classes of snRNA genes. Wild-type levels of Pol II-dependent U2 expression require all activation domains, whereas efficient activation of the pol III-dependent 7SK and U6 genes is effected by the POU-specific domain alone. These results indicate that contacts between PTF and amino acids in the POU-specific domain of Oct-1 are critical for efficient transactivation of snRNA genes in vivo. The POU-specific domain of Oct-2A also activates these genes, but the N- and C-terminal domains are relatively inactive.

Subunit Interactions in the Na,K-ATPase Explored with the Yeast Two-hybrid System

Subunit interactions of the alpha1- and beta1-subunits of the chicken Na,K-ATPase were explored with the yeast two-hybrid system. Gal4-fusion proteins containing domains of the alpha1- and beta1-subunits were designed for examining both intersubunit and intrasubunit protein-protein interactions. Regions of the alpha- and beta-subunits known to be involved in alpha-beta-subunit assembly were positive in two-hybrid assay, supporting the validity of the assays. A library of beta-subunit ectodomains with C-terminal truncations was screened to find the maximal truncation retaining an interaction with the alpha-subunit extracellular H7H8 loop (where H7 refers to the seventh membrane span, and so on). The maximal truncation removed all the cysteines involved in disulfide bridges, leaving only 63 amino acids of the beta-subunit ectodomain. Scanning alanine mutagenesis led to identification of an evolutionarily conserved sequence of four amino acids (SYGQ) in the extracellular H7H8 loop of the alpha-subunit that is crucial to alpha-beta-intersubunit interactions. Oligomerization studies with single domains failed to detect self-association of either of the two large cytosolic loops (H2H3 and H4H5) within the alpha-subunit. However, evidence was found for an interaction between these two cytoplasmic loops.

The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein kinase pathway.

Mutation of Saccharomyces cerevisiae RLM1, which encodes a MADS-box transcription factor, confers resistance to the toxic effects of constitutive activity of the Mpk1 mitogen-activated kinase (MAPK) pathway. The Rlm1 DNA-binding domain, which is similar to that of the metazoan MEF2 transcription factors, is also closely related to that of a second S. cerevisiae protein, Smp1 (second MEF2-like protein), encoded by the YBR182C open reading frame (N. Demolis et al., Yeast 10:1511-1525, 1994; H. Feldmann et al., EMBO J. 13:5795-5809, 1994). We show that Rlm1 and Smp1 have MEF2-related DNA-binding specificities: Rlm1 binds with the same specificity as MEF2, CTA(T/A)4TAG, while SMP1 binds a more extended consensus sequence, ACTACTA(T/A)4TAG. The two DNA-binding domains can heterodimerize with each other and with MEF2A. Deletion of RLM1 enhances resistance to cell wall disruptants, increases saturation density, reduces flocculation, and inactivates reporter genes controlled by the Rlm1 consensus binding site. Deletion of SMP1 neither causes these phenotypes nor enhances the Rlm1 deletion phenotype. However, overexpression of the DNA-binding domain of either protein causes an osmoremedial phenotype. Synthetic and naturally occurring MEF2 consensus sequences exhibit strong RLM1- and MPK1-dependent upstream activation sequence activity. Transcriptional activation by Rlm1 requires its C-terminal sequences, and Gal4 fusion proteins containing Rlm1 C-terminal sequences also act as MPK1-dependent transcriptional activators. These results establish the Rlm1 C-terminal sequences as a target for the Mpk1 MAPK pathway.