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Vascular fibrosis is a major complication of hypertension and atherosclerosis, yet it is largely untreatable. Natriuretic peptides (NPs) repress fibrogenic activation of vascular smooth muscle cells (VSMCs), but the intracellular mechanism mediating this effect remains undetermined. Here we show that inhibition of RhoA through phosphorylation at Ser188, the site targeted by the NP effector cyclic GMP (cGMP)-dependent protein kinase I (cGK I), is critical to fully exert antifibrotic potential. cGK I(+/-) mouse blood vessels exhibited an attenuated P-RhoA level and concurrently increased RhoA/ROCK signaling. Importantly, cGK I insufficiency caused dynamic recruitment of ROCK into the fibrogenic programs, thereby eliciting exaggerated vascular hypertrophy and fibrosis. Transgenic expression of cGK I-unphosphorylatable RhoA(A188) in VSMCs augmented ROCK activity, vascular hypertrophy, and fibrosis more prominently than did that of wild-type RhoA, consistent with the notion that RhoA(A188) escapes the intrinsic inhibition by cGK I. Additionally, VSMCs expressing RhoA(A188) became refractory to the antifibrotic effects of NPs. Our results identify cGK I-mediated Ser188 phosphorylation of RhoA as a converging node for pro- and antifibrotic signals and may explain how diminished cGMP signaling, commonly associated with vascular malfunction, predisposes individuals to vascular fibrosis.

The mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is an immediate-early gene comprised of a dual-specificity phosphatase domain and a noncatalytic NH(2) terminus. Here, we show that the NH(2) terminus of MKP-1, containing the cdc25 homology domains A (CH2A) and B (CH2B), mediates MKP-1 nuclear targeting and modulates MAPK-mediated gene expression. An LXXLL motif which is known to mediate protein-protein interactions with nuclear-targeted hormone receptors was identified proximal to the CH2A domain of MKP-1. The NH(2) terminus alone of MKP-1 containing this LXXLL motif was sufficient to direct nuclear targeting, and mutating this motif to LXXAA resulted in the exclusion of MKP-1 from the nucleus. We found that the LXXLL motif proximal to the CH2A domain was present in other nuclear-localized MKPs but was absent in MKPs that localized to the cytoplasm. These data suggest that this LXXLL motif confers nuclear targeting properties to the MKPs. The NH(2) terminus of MKP-1 was also found to inhibit the activation of the serum response element (SRE) by preventing MAPK-mediated phosphorylation of the regulatory serine 383 residue on Elk-1. Moreover, we show that MKP-1 plays a major role in the attenuation of serum-induced SRE activity, since MKP-1 null fibroblasts exhibited enhanced SRE activity in response to serum compared with wild-type fibroblasts. The NH(2) terminus of MKP-1, when reconstituted into MKP-1 null fibroblasts to levels similar to endogenous MKP-1 following serum stimulation, reduced serum-mediated SRE activity. Collectively, these data reveal novel roles for the NH(2) terminus of MKP-1 in nuclear targeting and transcriptional regulation.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKC delta) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKC delta activity as cells age, production of the PKC delta catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKC delta in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKC delta inhibitor rottlerin and ectopic expression of a dominant negative form of PKC delta independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKC delta activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKC delta contributes to the senescent phenotype.

Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRFDelta5 to SRF. Increased levels of SRFDelta5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRFDelta5 variant relative to wild-type SRF. SRFDelta5 forms DNA binding-competent homodimers and heterodimers. SRFDelta5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal alpha-actin, cardiac alpha-actin, smooth alpha-actin, SM22alpha, and SRF promoter-luciferase reporter activities. Expression of SRFDelta5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal alpha-actin and myogenin mRNAs. SRFDelta5 repressed the serum-induced activity of the c-fos serum response element. SRFDelta5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRFDelta5, may play an important regulatory role in modulating SRF-dependent gene expression.

Serum response element binding protein (SRE BP) is a novel binding factor present in nuclear extracts of avian and NIH 3T3 fibroblasts which specifically binds to the cfos SRE within a region overlapping and immediately 3' to the CArG box. Site-directed mutagenesis combined with transfection experiments in NIH 3T3 cells showed that binding of both serum response factor (SRF) and SRE BP is necessary for maximal serum induction of the SRE. In this study, we have combined size fractionation of the SRE BP DNA binding activity with C/EBPbeta antibodies to demonstrate that homodimers and heterodimers of p35C/EBPbeta (a transactivator) and p20C/EBPbeta (a repressor) contribute to the SRE BP complex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBPbeta is dependent on SRF binding but not ternary complex factor (TCF) formation. Both p35C/EBPbeta and p20C/EBPbeta bind to SRF in vitro via a carboxy-terminal domain that probably does not include the leucine zipper. Moreover, SRE mutants which retain responsiveness to the TCF-independent signaling pathway bind SRE BP in vitro with affinities that are nearly identical to that of the wild-type SRE, whereas mutant SRE.M, which is not responsive to the TCF-independent pathway, has a nearly 10-fold lower affinity for SRE BP. We propose that C/EBPbeta may play a role in conjunction with SRF in the TCF-independent signaling pathway for SRE activation.

YY1 is a multifunctional transcription factor that acts as an activator or repressor in different contexts. YY1 binds to multiple sites in the mouse c-fos promoter, inducing at each site a sharp DNA bend. Binding of YY1 to a site situated between the cyclic AMP response element (CRE) and the TATA box bends the DNA in a way that interferes with the interaction of proteins bound at the CRE and TATA elements, resulting in repression of transcription. Here, we show that binding of YY1 to a different site in the c-fos promoter has a different result. Binding of YY1 to the c-fos serum response element (SRE) enhances the binding of serum response factor (SRF). This enhancement requires the binding of YY1 to SRE DNA. YY1 and SRF can cooccupy the SRE at least transiently. In the region of overlapping contact, YY1 contacts DNA in the major groove, while SRF contacts DNA in the minor groove. YY1 also enhances the association of SRF with the SRE in transfected insect cells. Thus, although YY1 induces similar structural changes in DNA at different binding sites, it can have distinct local effects on protein-DNA and protein-protein interactions. These data support a general role for YY1 in the building of highly organized promoter complexes.

Induction of the primary response gene egr-1 occurs rapidly following antigen receptor cross-linking in B lymphocytes. Antisense studies have demonstrated that this induction is necessary for their subsequent activation to this signal. The present study examines the molecular mechanism whereby the receptor-generated signals interact with the egr-1 promoter to elicit transcription. Deletion mapping and point mutations have indicated that two of the five serum response elements (SREs) in the egr-1 promoter can mediate induction. Of the two critical SREs, both are capable of mediating maximal induction even in the absence of the other SRE. Our results also indicate that adjacent Ets motifs are necessary for induction. Like the c-fos SRE, the egr-1 SRE/Ets sites are occupied by a multiprotein (ternary) complex containing a homodimer of serum response factor and an unidentified member of the Ets family of transcription factors. The identification of a ternary complex-dependent mechanism of egr-1 induction, along with selective utilization of SREs in B lymphocytes, suggests that a complicated array of signaling cascades interacts with unique combinations of regulatory elements in the egr-1 promoter in different cell types.

The c-fos serum response element (SRE) is necessary and sufficient for induction of the c-fos gene in response to serum and growth factors. This activation is dependent upon serum response factor (SRF), a transcriptional activator which binds the SRE. A factor, p62TCF, which binds in conjunction with SRF to the SRE and which is activated by mitogen-activated protein kinase, has also been implicated in c-fos regulation. By using a reporter gene system with weak SRE mutations that is dependent upon overexpression of SRF for serum induction, we have found that there are at least two pathways for serum induction that converge on the SRE. Loss of TCF binding by mutations in SRF and the SRE did not reduce serum induction of the reporter genes. We have found a pathway for serum induction that is sensitive to mutations in the A/T-containing central sequence of the SRE and which is independent of TCF. When this pathway was mutated, activation was dependent upon TCF binding, demonstrating that TCF can also function in serum induction. Both of the signalling pathways required a minimal domain of SRF. This domain, spanning SRF's DNA binding domain, was sufficient for serum induction when fused to a heterologous transcriptional activation domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

Cardiac myocytes undergo a major genetic switch within the first week of postnatal development, when cell division ceases terminally and many cardiac genes are either activated or silenced. We have developed stage-specific cardiocyte cultures to analyze transcriptional control of the rat atrial natriuretic factor (ANF) gene to identify the mechanisms underlying tissue-specific and developmental regulation of this gene in the heart. The first 700 bp of ANF flanking sequences was sufficient for cardiac muscle- and stage-specific expression in both atrial and ventricular myocytes, and a cardiac muscle-specific enhancer was localized between -136 and -700 bp. Deletion of this enhancer markedly reduced promoter activity in cardiac myocytes and derepressed ANF promoter activity in nonexpressing cells. Two distinct domains of the enhancer appeared to contribute differentially to cardiac specificity depending on the differentiation stage of the myocytes. DNase I footprinting of the enhancer domain active in differentiated cells revealed four putative regulatory elements including an A+T-rich region and a CArG element. Deletion mutagenesis and promoter reconstitution assays revealed an important role for the CArG-containing element exclusively in cardiac cells, where its activity was switched on in differentiated myocytes. Transcriptional activity of the ANF-CArG box correlated with the presence of a cardiac- and stage-specific DNA-binding complex which was not recognized by the c-fos serum response element. Thus, the use of this in vitro model system representing stage-specific cardiac development unraveled the presence of different regulatory mechanisms for transcription of the ANF gene during cardiac differentiation and may be useful for studying the regulatory pathways of other genes that undergo switching during cardiac myogenesis.

ATF5 has been shown to be a critical regulator of cell proliferation and survival; however, the underlying mechanism remains largely unknown. We demonstrate here that ATF5 interacts with the transcriptional coactivator p300, which acetylates ATF5 at lysine-29 (K29), which in turn enhances the interaction between ATF5 and p300 and binding of the ATF5/p300 complex to the ATF5 response element (ARE) region of the Egr-1 promoter. ARE-bound ATF5/p300 acetylates lysine-14 (K14) of nucleosomal histone H3 at both the ARE and serum response element (SRE) of the Egr-1 promoter, which facilitates binding of extracellular signal-regulated kinase (ERK)-phosphorylated Elk-1 to the SRE, activating the Egr-1 promoter. Interference of p300-dependent acetylation of ATF5 or nucleosomal histone H3 or blockade of ERK-dependent Elk-1 phosphorylation abrogates ATF5-dependent Egr-1 activation and cell proliferation and survival. These findings assign a central role for the ATF5/p300 complex in ATF5 function and suggest that coordinated actions by ATF5, p300, Elk-1, and ERK/mitogen-activated protein kinase (MAPK) are essential for ATF5-dependent Egr-1 activation and cell proliferation and survival.

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

Unlike that of skeletal muscle cells in which growth and differentiation appear mutually exclusive, growth stimulation of cardiac cells is characterized by transient expression of early response nuclear proto-oncogenes as well as induction of several cardiac-specific markers. This observation led to the speculation that these proto-oncogenes, particularly c-fos and c-jun, might act as positive regulators of cardiac transcription. We have examined the role of c-jun and c-fos in basal and growth-stimulated cardiac transcription, using the cardiac-specific atrial natriuretic factor (ANF) gene as a marker. The results indicate that c-jun and c-fos are negative regulators of ANF transcription. Inducers of jun and fos activity, such as mitogens and growth factors, inhibited endogenous ANF transcripts. In transient cotransfection assays, jun and fos were able to trans-repress the ANF promoter in both quiescent and alpha 1-adrenergic stimulated myocytes. This repression was specific to myocyte cultures and was not observed in nonmuscle cells. Deletion analysis indicated that repression does not require typical AP-1-binding sites (tetradecanoyl phorbol acetate response elements) or serum response elements but is targeted at a cardiac-specific element within the ANF promoter. Various Fos-related proteins, including Fra-1, Fos B, and v-Fos, were able to trans-repress ANF transcription. In addition, C-terminal c-fos mutants which no longer repress transcription of such early growth response genes as c-fos and EGR-1 retained the ability to repress ANF transcription. Repression by c-jun occurs via the N-terminal activation domain and does not require the DNA-binding domain, suggesting that proto-oncogene repression involves interaction with one or more limiting cardiac-specific coactivators.

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

Induction of c-fos transcription by serum growth factors requires the serum response element (SRE). The SRE is a multifunctional element which responds to several positively and negatively acting signals. To identify cellular proteins that might mediate functions of the SRE, we screened a human cDNA expression library with an SRE probe. We report the isolation and characterization of SRE-ZBP, a previously unidentified SRE-binding protein. SRE-ZBP is a member of the C2H2 zinc finger family of proteins exemplified by TFIIIA and the Drosophila Krüppel protein. The seven tandemly repeated zinc finger motifs in SRE-ZBP are sufficient for high-affinity binding to the SRE. We show that SRE-ZBP is a nuclear protein and identify a candidate cellular protein encoded by the SRE-ZBP gene. Because we cannot detect any DNA-binding activity attributable to the endogenous protein, we propose that SRE-ZBP activity may be subject to posttranslational regulation. Like c-fos mRNA, SRE-ZBP mRNA is serum inducible in HeLa cells, but with slower kinetics. The role of SRE-ZBP in the regulation of c-fos transcription remains unestablished, but this protein binds to a region of the SRE where mutations lead to derepression.

DNA elements with the CC(A/T)6GG, or CArG, motif occur in promoters that are under different regulatory controls. CArG elements from the skeletal actin, c-fos, and myogenin genes were tested for their abilities to confer tissue-specific expression on reporter genes when the individual elements were situated immediately upstream from a TATA element. The c-fos CArG element, also referred to as the serum response element (SRE), conferred basal, constitutive expression on the test promoter. The CArG motif from the myogenin gene was inactive. The skeletal actin CArG motif functioned as a muscle regulatory element (MRE) in that basal expression was detected only in muscle cultures. Muscle-specific expression from the 28-bp MRE and the 2.3-kb skeletal actin promoter was trans repressed by the Fos and Jun proteins. The expression and factor-binding properties of a series of synthetic CArG elements were analyzed. Muscle-specific expression was conferred by perfect 28-bp palindromes on the left and right halves of the skeletal actin MRE. Chimeric elements of the skeletal actin MRE and the c-fos SRE differed in their expression properties. Muscle-specific expression was observed when the left half of the MRE was fused to the right half of the SRE. Constitutive expression was conferred by a chimera with the right half of the MRE fused to the left half of the SRE and by chimeras which exchanged the central CC(A/T)6GG sequences. At least three distinct proteins specifically bound to these CArG elements. The natural and synthetic CArG elements differed in their affinities for these proteins; however, muscle-specific expression could not be attributed to differences in the binding of a single protein. Furthermore, the MRE did not bind MyoD or the myogenin-E12 heterodimer, indicating that muscle-specific expression from this element does not involve a direct interaction with these helix-loop-helix proteins. These data demonstrate that the conserved CArG motifs form the core of a family of functionally different DNA regulatory elements that may contribute to the tissue-specific expression properties of their cognate promoters.

Three upstream CBAR cis-acting promoter elements, containing the inner core CC(A/T)6GG of the serum response element (SRE), are required for myogenic cell type-restricted expression of the avian skeletal alpha-actin gene (K.L. Chow and R.J. Schwartz, Mol. Cell. Biol. 10:528-538, 1990). These actin SRE elements display differential binding properties with two distinct nuclear proteins, serum response factor (SRF) and another factor described here as F-ACT1. SRF is able to bind to all actin SREs with various affinities. This multisite interaction is marked by cooperative binding events in that the two high-affinity proximal and distal SREs facilitate the weak central-site interaction with SRF, leading to the formation of a higher-order SRF-promoter complex. Functional analyses reveal that undisrupted multiple SRF-DNA interactions are absolutely essential for promoter activity in myogenic cells. F-ACT1, present at higher levels in nonmyogenic cells and replicating myoblasts than in myotubes, binds solely to the proximal SRE, and its binding is mutually exclusive with that of SRF owing to their overlapping base contacts. The cooperative promoter binding by SRF, however, can effectively displace prebound F-ACT1. In addition, an intact F-ACT1 binding site acts as a negative promoter element by restricting developmentally timed expression in myoblasts. F-ACT1 may therefore act as a repressor of skeletal alpha-actin gene transcription. This interplay between F-ACT1 and SRF may constitute a developmental as well as a physiologically regulated mechanism which modulates sarcomeric actin gene expression.

We have identified oncogene-responsive sequences in the human c-fos promoter that mediate induction of transcription by several nonnuclear oncoproteins and the tumor promoter TPA. These sequences are regulated in a cell-specific manner. (i) In NIH 3T3 cells, the CArG box of the c-fos promoter is sufficient to mediate activation by oncogenes. (ii) In contrast, in HeLa cells, additional flanking sequences are also required, including the outer arm of the serum response element and the FAP site. We also show that the serum response factor, which binds to the CArG box, activates transcription in vivo in NIH 3T3 cells but not in HeLa cells. Finally, we present evidence that the intracellular level of the c-Fos protein could be a major determinant of cell-specific regulation of these oncogene-responsive elements of the c-fos promoter.

Growth factor regulation of c-fos proto-oncogene transcription is mediated by a 20-bp region of dyad symmetry, termed the serum response element. The inner core of this element binds a 67-kDa phosphoprotein, the serum response factor (SRF), that is thought to play a pivotal role in the c-fos transcriptional response. To investigate the mechanism by which SRF regulates c-fos expression, we generated polyclonal anti-SRF antibodies and used these antibodies to analyze the biochemical properties of SRF. These studies indicate that the synthesis of SRF is transient, occurring within 30 min to 4 h after serum stimulation of quiescent fibroblasts. Newly synthesized SRF is transported to the nucleus, where it is increasingly modified by phosphorylation during progression through the cell cycle. Within 2 h of serum stimulation, differentially modified forms of SRF can be distinguished on the basis of the ability to bind a synthetic serum response element. SRF protein exhibits a half-life of greater than 12 h and is predominantly nuclear, with no change occurring in its localization upon serum stimulation. We find that the induction of SRF synthesis is regulated at the transcriptional level and that cytoplasmic SRF mRNA is transiently expressed with somewhat delayed kinetics compared with c-fos mRNA expression. These features of SRF expression suggest a model whereby newly synthesized SRF functions in the shutoff of c-fos transcription.

The serum response element (SRE) is essential for serum and growth factor stimulation of the c-fos gene. We have examined the nuclear proteins, obtained from tissues with elevated expression of the c-fos gene (proliferating rat liver and hepatocarcinoma), that bind to the SRE sequence. A synthetic oligonucleotide containing the SRE sequence from the mouse c-fos gene promoter (-299 to -322) was radioactively labeled, used as a probe for the mobility shift assay and Southwestern (DNA-protein) blotting, and also used for sequence-specific affinity chromatography. We have identified a group of nuclear proteins of molecular sizes 36, 45, 62, 67, 72, and 112 kDa capable of interacting with the SRE sequence. The 36-, 67-, and 112-kDa proteins have DNA-binding properties, but the presence of the others in the SRE-protein complex could be the result of protein-protein interaction. All of these protein factors were present in nuclei obtained from intact and proliferating rat liver as well as from 5123tc Morris hepatoma. The DNA-binding activity (on Southwestern blots) of the 67- and 112-kDa proteins was not affected by alkaline phosphatase treatment, but the ability of the dephosphorylated nuclear proteins to form the complex with the SRE sequence under gel shift assay conditions was severely impaired. The same alkaline phosphatase treatment completely abolished the DNA-binding properties of the c-fos cyclic AMP-responsive element-specific proteins. Therefore, transcriptional activation of the c-fos gene at the SRE must require the presence of a multiprotein complex the formation of which is governed by phosphorylation. The binding of the 67- and 62-kDa proteins to the c-fos SRE has been previously reported; however, the 36-. 45-, 72-, and 112-kDa proteins are novel factors involved in the multifaceted regulation of c-fos gene expression in vivo.

Three cis-acting domains that contribute to the basal promoter activity of the human c-fos gene were identified. One encompasses the serum response element and has been previously described. Another spans an NF1-like site situated at -170. Mutations and in vitro protein binding assays pinpoint this site as the sole basal element of the medial domain. The third, or promoter-proximal, domain can be divided into several distinct sites, one containing a directly repeated GC-rich element and the other consisting of partially overlapping recognition sites for transcription factors ATF/CREB and MLTF/USF. Each of these sites contributes to basal activity as assayed by transient transfections and by in vitro transcription. Consistent with this, several complexes could be visualized between this region and nuclear proteins in vitro and genomic footprinting demonstrated that both elements are constitutively bound in vivo. On the basis of these results, we conclude that all three domains are necessary for full c-fos promoter function.

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.

The transcription factor Elk-1 belongs to the ternary complex factor (TCF) subfamily of Ets proteins. TCFs interact with serum response factor to bind jointly to serum response elements in the promoters of immediate-early genes (IEGs). TCFs mediate the rapid transcriptional response of IEGs to various extracellular stimuli which activate mitogen-activated protein kinase signaling. To investigate physiological functions of Elk-1 in vivo, we generated Elk-1-deficient mice by homologous recombination in embryonic stem cells. These animals were found to be phenotypically indistinguishable from their wild-type littermates. Histological analysis of various tissues failed to reveal any differences between Elk-1 mutant and wild-type mice. Elk-1 deficiency caused no changes in the proteomic displays of brain or spleen extracts. Also, no immunological defects could be detected in mice lacking Elk-1, even upon infection with coxsackievirus B3. In mouse embryonic fibroblasts, Elk-1 was dispensable for c-fos and Egr-1 transcriptional activation upon stimulation with serum, lysophosphatidic acid, or tetradecanoyl phorbol acetate. However, in brains of Elk-1-deficient mice, cortical and hippocampal CA1 expression of c-fos, but not Egr-1 or c-Jun, was markedly reduced 4 h following kainate-induced seizures. This was not accompanied by altered patterns of neuronal apoptosis. Collectively, our data indicate that Elk-1 is essential neither for mouse development nor for adult life, suggesting compensatory activities by other TCFs.

Receptor-mediated tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) is required for the propagation of many of insulin's biological effects. The amino-terminal pleckstrin homology (PH) domain of IRS-1 plays a pivotal role in promoting insulin receptor (IR)-IRS-1 protein interactions. We have recently reported the isolation of a PH domain-interacting protein, PHIP, which selectively binds to the IRS-1 PH domain and is stably associated with IRS-1 in mammalian cells. Here we demonstrate that overexpression of PHIP in fibroblasts enhances insulin-induced transcriptional responses in a mitogen-activated protein kinase-dependent manner. In contrast, a dominant-negative mutant of PHIP (DN-PHIP) was shown to specifically block transcriptional and mitogenic signals elicited by insulin and not serum. In order to examine whether PHIP/IRS-1 complexes participate in the signal transduction pathway linking the IR to GLUT4 traffic in muscle cells, L6 myoblasts stably expressing a myc-tagged GLUT4 construct (L6GLUT4myc) were transfected with either wild-type or dominant-interfering forms of PHIP. Whereas insulin-dependent GLUT4myc membrane translocation was not affected by overexpression of PHIP, DN-PHIP caused a nearly complete inhibition of GLUT4 translocation, in a manner identical to that observed with a dominant-negative mutant of the p85 subunit of phosphatidylinositol 3-kinase (Deltap85). Furthermore, DN-PHIP markedly inhibited insulin-stimulated actin cytoskeletal reorganization, a process required for the productive incorporation of GLUT4 vesicles at the cell surface in L6 cells. Our results are consistent with the hypothesis that PHIP represents a physiological protein ligand of the IRS-1 PH domain, which plays an important role in insulin receptor-mediated mitogenic and metabolic signal transduction.

Hypertrophic growth of cardiac muscle cells is induced by a variety of physiological and pathological stimuli and is associated with a number of changes, including activation of genes such as atrial natriuretic factor. We found that two serum response element (SRE)-like DNA elements, one of which does not meet the consensus sequence and binds serum response factor (SRF) with low affinity, regulate the activity of this promoter. Surprisingly, the ability to induce the promoter by two different physiologic stimuli, as well as various activated transcription factors, including SRF-VP16, was primarily dependent upon the nonconsensus rather than the consensus SRE. This SRE controls the induction of gene expression via an unusual mechanism in that it is required to allow some, but not all, active transcription factors at unrelated sites on the promoter to stimulate gene expression. Thus, in addition to regulation of SRF activity by growth stimuli, regulation of a low-affinity SRE element controls inducible gene expression by modulating the ability of other transcription factors to stimulate the transcription machinery.

The human homeodomain protein Phox1 can impart serum-responsive transcriptional activity to the c-fos serum response element (SRE) by interacting with serum response factor (SRF). This activity is shared with other Paired class homeodomains but not with more distantly related homeodomains. To understand the mechanism of action of Phox1 at the SRE and the basis for the selective activity of Paired class homeodomains in this context, we performed a detailed mutagenesis of the Phox1 homeodomain. We found that amino acid residues that contact the major groove of the DNA are required for SRE activation in vivo, suggesting an in vivo requirement for major-groove DNA contact by the homeodomain. In contrast, substitution of a lysine residue in the N-terminal arm of the Phox1 homeodomain appeared to abolish DNA binding without affecting activity in vivo. Certain substitutions on the exposed surfaces of helices 1 and 2, not required for DNA binding, abolished activity in vivo, suggesting that these surfaces contact an accessory protein(s) required for this activity. We also found that transfer of a single amino acid residue from the surface of Phox1 helix 1 to the corresponding position in the distantly related Deformed (Dfd) homeodomain imparts to Dfd the ability to activate the SRE in vivo. We propose that Phox1 interacts with one or more factors at the SRE, in addition to SRF, and that the specificity of this interaction is determined by residues on the surfaces of helices 1 and 2.

Endothelin-1 (ET-1) triggers poorly understood nuclear signaling cascades that control gene expression, cell growth, and differentiation. To better understand how ET-1 regulates gene expression, we asked whether voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases (CaMKs) propagate signals from ET-1 receptors to the c-fos promoter in mesangial cells. Ca2+ influx through voltage-insensitive Ca2+ channels, one of the earliest postreceptor events in ET-1 signaling, mediated induction of c-fos mRNA and activation of the c-fos promoter by ET-1. A CaMK inhibitor (KN-93) blocked activation of the c-fos promoter by ET-1. Ectopic expression of CaMKII potentiated stimulation by ET-1, providing further evidence that CaMKs contribute to c-fos promoter activation by ET-1. The c-fos serum response element was necessary but not sufficient for CaMKII to activate the c-fos promoter. Activation of the c-fos promoter by ET-1 and CaMKII also required the FAP cis element, an AP-1-like sequence adjacent to the serum response element. Thus, voltage-insensitive Ca2+ channels and CaMKs apparently propagate ET-1 signals to the c-fos promoter that require multiple, interdependent cis elements. Moreover, these experiments suggest an important role for voltage-insensitive Ca2+ channels in nuclear signal transduction in nonexcitable cells.

Transcriptional induction of the c-fos gene in response to epidermal growth factor stimulation is mediated in part by a ternary nucleoprotein complex within the promoter consisting of serum response factor (SRF), p62TCF/Elk-1 and the serum response element (SRE). Both SRF and p62TCF/Elk-1 contact the DNA and bind in a cooperative manner to the SRE. In this study, we demonstrate that SRF and Elk-1 interact directly in the absence of the SRE. A 30-amino-acid peptide from Elk-1 (B-box) is both necessary and sufficient to mediate protein-protein contacts with SRF. Moreover, the Elk-1 B-box is necessary to enable SRF-dependent binding of an alternative ETS domain (from the transcription factor PU.1) to the c-fos SRE. Mutations in either the Elk-1 B-box or the C-terminal half of the SRF DNA-binding domain (coreSRF) which show reduced ability to form ternary complexes also show greatly reduced protein-protein interactions in the absence of the SRE. Our results clearly demonstrate that direct protein-protein interactions between the transcription factors Elk-1 and SRF, in addition to DNA contacts, contribute to the formation of a ternary complex on the c-fos SRE. We discuss the wider applicability of our results in describing specific protein-protein interactions between short well-defined transcription factor domains.

Transcriptional control of the cardiac/slow skeletal alkali myosin light-chain (MLC1c/1s) gene is mediated, in part, by two highly conserved AT-rich cis-acting elements present in the immediate 5' flanking region. These elements cooperate to form an enhancer that can impart tissue specificity to heterologous promoters that are themselves not tissue specific in their pattern of expression. In the chicken, one of these elements matches the binding site for myocyte-specific enhancer-binding factor 2, while the other is a cis-acting element present in the transcriptional control regions of all striated alkali MLC genes (except MLC3f) and is referred to as the MLC box. The central decanucleotide core region of the MLC box closely resembles the CArG (CC[A/T]6GG) box of the serum response element, and the binding of muscle nuclear protein complexes to this element can be competed for with a synthetic serum response element. On the basis of their competition profiles and requirements for nonspecific competitor, two nuclear protein complexes, which compete for binding to the CArG-like region of the MLC box, have been identified. One of the complexes binds to a mutation of the CArG-like region that inactivates transcription of a linked reporter gene, while binding of the other complex is inhibited by this mutation. This latter complex reacts with an antibody to serum response factor (SRF) and exhibits the same binding characteristics as purified SRF. These results demonstrate that transcriptional control of the chicken MLC1c/1s gene resides in an upstream enhancer that is composed of two separate AT-rich elements, both of which are required to drive expression of a linked reporter gene. The binding of a nuclear protein complex containing SRF to one of these elements, the MLC box, is required for gene activation and apparently inhibited by other nuclear factors whose binding overlaps that of the SRF complex.

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription.

The zif268 gene, which encodes a protein with three typical zinc finger sequences, is induced in mouse 3T3 cells by serum, phorbol 12-myristate 13-acetate platelet-derived growth factor, and fibroblast growth factor. The induction is coordinate with that of c-fos. The 5'-flanking region of zif268 contains sequences that resemble known regulatory elements, including four CC(A or T)6GG sequences similar to the core serum response elements (SREs) found upstream of c-fos and actin genes. To determine whether the zif268 SRE-like elements mediate induction, CAT (chloramphenicol acetyltransferase) plasmids with different lengths of zif268 upstream sequences were tested for inducibility in 3T3 cells by serum, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. In addition, double-stranded oligonucleotides corresponding to each of the four zif268 putative SREs were tested individually for responsiveness when placed upstream of a thymidine kinase gene promoter. Each of the four SREs conferred inducibility by the agents tested, and multiple SREs resulted in greater inducibility than did a single element. Each of the zif268 SREs also competed with the c-fos SRE for binding by serum response factor present in HeLa cell nuclear extract. We conclude that the zif268 SRE-like sequences are functional and probably account for the coordinate induction of zif268 and c-fos.

In vitro mutagenesis of a 61-base-pair DNA sequence element that is necessary for induction of the c-fos proto-oncogene by growth factors revealed that a small region of dyad symmetry within the sequence element is critical for c-fos transcriptional activation. The same c-fos dyad symmetry element was found to bind a nuclear protein in vitro, causing a specific mobility shift of this c-fos regulatory sequence. An analysis of insertion and deletion mutants established a strict correlation between the ability of the dyad symmetry element to promote serum activation of c-fos transcription and in vitro nuclear protein binding. These experiments suggest that the DNA mobility shift assay detects a nuclear protein that mediates growth factor stimulation of c-fos expression. In vitro competition experiments indicate that the c-fos regulatory factor also binds to sequences within another growth factor-inducible gene, the beta-actin gene.

We have demonstrated that two sequence elements in the c-fos promoter can mediate the response of the gene to epidermal growth factor and the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). The first is the previously described serum response element. The second is a sequence element highly homologous to the consensus binding site for the HeLa cell transcription factor AP1. Although recent reports have shown that fos protein binds to AP1-binding sites through an interaction with AP1 protein and have raised the speculation that fos protein may negatively regulate expression of the c-fos gene via this interaction, we found no role for the AP1 consensus homology in the downregulation of c-fos expression following induction by epidermal growth factor and TPA.

Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.

We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.

Transcription of the c-fos proto-oncogene is rapidly induced in the rat pheochromocytoma PC12 cell line by a wide variety of stimuli, including polypeptide growth factors, phorbol esters, and calcium ion fluxes. We have mapped the upstream sequence requirements for this activation in PC12 cells by analysis of promoter deletion mutants in a transient expression assay. Two distinct pathways of c-fos induction are defined that differ in their requirement for cis-acting DNA sequences. Calcium activation of c-fos transcription is dependent on a DNA element located approximately 60 base pairs upstream of the transcription start site. This region is highly conserved between human, mouse, and chicken c-fos genes and contains a sequence that resembles the consensus for a cyclic AMP response element. The dyad symmetry element at position -300, which is necessary for serum responsiveness of c-fos, appears to be unimportant for calcium activation of the gene. The dyad symmetry element is, however, an essential cis-acting sequence for c-fos inducibility by nerve growth factor, epidermal growth factor, fibroblast growth factor, and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate. Studies in vivo and in vitro with various mutants of the dyad symmetry element indicate that c-fos activation by polypeptide growth factors and 12-O-tetradecanoyl activation by polypeptide growth factors and 12-O-tetradecanoyl phorbol-13-acetate is mediated by a common transcription factor, and that this factor is identical to the previously described serum response factor. In vitro DNA-binding assays suggest that the quantity of serum response factor-binding activity remains unchanged during c-fos transcriptional activation.

The c-fos serum response element (SRE) and a sarcomeric actin promoter element (CArG box) are similar in sequence and are recognized, respectively, by the serum response factor (SRF) and the CArG-binding factor (CBF). Although the transcriptional controls for the c-fos and sarcomeric actin genes are rather different, SRF and CBF have been found to be indistinguishable by all criteria tested. They exhibited similar chromatographic properties, sedimentation rates, and temperature stabilities. In mobility shift assays, the SRE competed more strongly than the actin CArG box for formation of either the SRF-SRE or the CBF-CArG complex. The symmetric inverted repeat of the left side of the Xenopus cytoskeletal actin SRE also competed, even more strongly, for each complex. The site-specific binding of each protein was inhibited both by orthophenanthroline, whose effects were reversed by zinc addition, and by treatment with potato acid phosphatase. Furthermore, immune serum raised against the c-fos SRF also recognized the actin CBF. We discuss how transcriptional control of these diverse genes might be obtained with a single similar factor.

A conserved 28-base-pair element in the skeletal actin promoter was sufficient to activate muscle-specific expression when placed upstream of a TATA element. This muscle regulatory element (MRE) is similar in structure to the serum response element (SRE), which is present in the promoters of the c-fos proto-oncogene and the nonmuscle actin genes. The SRE can function as a constitutive promoter element. Though the MRE and SRE differed in their tissue-specific expression properties, both elements bound to the same protein factors in vitro. These proteins are the serum response factor (SRF) and the muscle actin promoter factors 1 and 2 (MAPF1 and MAPF2). The SRF and MAPF proteins were resolved by chromatographic procedures, and they differed in their relative affinities for each element. The factors were further distinguished by their distinct, but overlapping, methylation interference footprint patterns on each element. These data indicate that the differences in tissue-specific expression may be due to a complex interaction of protein factors with these sequences.

We have investigated the sequence requirements for induction of the human c-fos gene by epidermal growth factor (EGF), 12-O-tetradecanoyl-13-acetate (TPA), and the calcium ionophore A23187 by transfecting c-fos promoter mutants into HeLa and A431 cells. Induction by both EGF and TPA in HeLa cells required the presence of the c-fos enhancer located at -317 to -298 relative to the mRNA cap site. A23187, however, did not induce expression of the transfected gene, even though it strongly induced expression of the endogenous gene, suggesting that it has different requirements for induction than do EGF and TPA. We have also investigated the role of promoter sequences downstream of the enhancer in general expression and induction of c-fos. A sequence between -97 and -76, which includes an 8-base-pair perfect direct repeat, was needed for efficient general expression but not for induction of the gene. A factor in nuclear extracts that bound specifically to this sequence was detected by a gel mobility shift assay. A 7-base-pair sequence, located between -63 and -57 relative to the mRNA cap site and previously shown to be important for general expression of mouse c-fos, was also important for general expression of the human gene. In addition, this element was important for inducibility by EGF and TPA, since induction was significantly reduced when internal deletion mutants that retained the enhancer but lacked the -63 to -57 sequence element were analyzed in transfecting assays.