Physical interaction between the MADS box of serum response factor and the TEA/ATTS DNA-binding domain of transcription enhancer factor-1.

Serum response factor is a MADS box transcription factor that binds to consensus sequences CC(A/T)(6)GG found in the promoter region of several serum-inducible and muscle-specific genes. In skeletal myocytes serum response factor (SRF) has been shown to heterodimerize with the myogenic basic helix-loop-helix family of factors, related to MyoD, for control of muscle gene regulation. Here we report that SRF binds to another myogenic factor, TEF-1, that has been implicated in the regulation of a variety of cardiac muscle genes. By using different biochemical assays such as affinity precipitation of protein, GST-pulldown assay, and coimmunoprecipitation of proteins, we show that SRF binds to TEF-1 both in in vitro and in vivo assay conditions. A strong interaction of SRF with TEF-1 was seen even when one protein was denatured and immobilized on nitrocellulose membrane, indicating a direct and stable interaction between SRF and TEF-1, which occurs without a cofactor. This interaction is mediated through the C-terminal subdomain of MADS box of SRF encompassing amino acids 204-244 and the putative 2nd and 3rd alpha-helix/beta-sheet configuration of the TEA/ATTS DNA-binding domain of TEF-1. In the transient transfection assay, a positive cooperative effect of SRF and TEF-1 was observed when DNA-binding sites for both factors, serum response element and M-CAT respectively, were intact; mutation of either site abolished their synergistic effect. Similarly, an SRF mutant, SRFpm-1, defective in DNA binding failed to collaborate with TEF-1 for gene regulation, indicating that the synergistic trans-activation function of SRF and TEF-1 occurs via their binding to cognate DNA-binding sites. Our results demonstrate a novel association between SRF and TEF-1 for cardiac muscle gene regulation and disclose a general mechanism by which these two super families of factors are likely to control diversified biological functions.

GATA-4 and serum response factor regulate transcription of the muscle-specific carnitine palmitoyltransferase I beta in rat heart.

Transcriptional regulation of nuclear encoded mitochondrial proteins is dependent on nuclear transcription factors that act on genes encoding key components of mitochondrial transcription, replication, and heme biosynthetic machinery. Cellular factors that target expression of proteins to the heart have been well characterized with respect to excitation-contraction coupling. No information currently exists that examines whether parallel transcriptional mechanisms regulate nuclear encoded expression of heart-specific mitochondrial isoforms. The muscle CPT-Ibeta isoform in heart is a TATA-less gene that uses Sp-1 proteins to support basal expression. The rat cardiac fatty acid response element (-301/-289), previously characterized in the human gene, is responsive to oleic acid following serum deprivation. Deletion and mutational analysis of the 5'-flanking sequence of the carnitine palmitoyltransferase Ibeta (CPT-Ibeta) gene defines regulatory regions in the -391/+80 promoter luciferase construct. When deleted or mutated constructs were individually transfected into cardiac myocytes, CPT-I/luciferase reporter gene expression was significantly depressed at sites involving a putative MEF2 sequence downstream from the fatty acid response element and a cluster of heart-specific regulatory regions flanked by two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and competition specificity (or supershifts) with oligonucleotides and antibodies. Individual expression vectors for Nkx2.5, serum response factor (SRF), and GATA4 enhanced CPT-I reporter gene expression 4-36-fold in CV-1 cells. Although cotransfection of Nkx and SRF produced additive luciferase expression, the combination of SRF and GATA-4 cotransfection resulted in synergistic activation of CPT-Ibeta. The results demonstrate that SRF and the tissue-restricted isoform, GATA-4, drive robust gene transcription of a mitochondrial protein highly expressed in heart.

Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators.

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.

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice.

The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Dominant negative murine serum response factor: alternative splicing within the activation domain inhibits transactivation of serum response factor binding targets.

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.

A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells.

Coronary artery smooth muscle (SM) cells originate from proepicardial cells that migrate over the surface of the heart, undergo epithelial to mesenchymal transformation and invade the subepicardial and cardiac matrix. Prior to contact with the heart, proepicardial cells exhibit no expression of smooth muscle markers including SMalphaactin, SM22alpha, calponin, SMgammaactin or SM-myosin heavy chain detectable by RT-PCR or by immunostaining. To identify factors required for coronary smooth muscle differentiation, we excised proepicardial cells from Hamburger-Hamilton stage-17 quail embryos and examined them ex vivo. Proepicardial cells initially formed an epithelial colony that was uniformly positive for cytokeratin, an epicardial marker. Transcripts for flk-1, Nkx 2.5, GATA4 or smooth muscle markers were undetectable, indicating an absence of endothelial, myocardial or preformed smooth muscle cells. By 24 hours, cytokeratin-positive cells became SMalphaactin-positive. Moreover, serum response factor, undetectable in freshly isolated proepicardial cells, became strongly expressed in virtually all epicardial cells. By 72 hours, a subset of epicardial cells exhibited a rearrangement of cytoskeletal actin, focal adhesion formation and acquisition of a motile phenotype. Coordinately with mesenchymal transformation, calponin, SM22alpha and SMgammaactin became expressed. By 5-10 days, SM-myosin heavy chain mRNA was found, by which time nearly all cells had become mesenchymal. RT-PCR showed that large increases in serum response factor expression coincide with smooth muscle differentiation in vitro. Two different dominant-negative serum response factor constructs prevented the appearance of calponin-, SM22alpha- and SMgammaactin-positive cells. By contrast, dominant-negative serum response factor did not block mesenchymal transformation nor significantly reduce the number of cytokeratin-positive cells. These results indicate that the stepwise differentiation of coronary smooth muscle cells from proepicardial cells requires transcriptionally active serum response factor.

Organization and myogenic restricted expression of the murine serum response factor gene. A role for autoregulation.

Serum response factor (SRF), a member of an ancient family of DNA-binding proteins, is generally assumed to be a ubiquitous transcription factor involved in regulating growth factor-responsive genes. However, avian SRF was recently shown (Croissant, J. D., Kim, J.-H., Eichele, G., Goering, L., Lough, J., Prywes, R., and Schwartz, R. J. (1996) Dev. Biol. 177, 250-264) to be preferentially expressed in myogenic lineages and is required for regulating post-replicative muscle gene expression. Given the central importance of SRF for the muscle tissue-restricted expression of the striated alpha-actin gene family, we wanted to determine how SRF might contribute to this muscle-restricted expression. Here we have characterized the murine SRF genomic locus, which has seven exons interrupted by six introns, with the entire locus spanning 11 kilobases. Murine SRF transcripts were processed to two 3'-untranslated region polyadenylation signals, yielding 4.5- and 2.5-kilobase mRNA species. Murine SRF mRNA levels were the highest in adult skeletal and cardiac muscle, but barely detected in liver, lung, and spleen tissues. During early mouse development, in situ hybridization analysis revealed enrichment of SRF transcripts in the myotomal portion of somites, the myocardium of the heart, and the smooth muscle media of vessels of mouse embryos. Likewise, murine SRF promoter activity was tissue-restricted, being 80-fold greater in primary skeletal myoblasts than in liver-derived HepG2 cells. In addition, SRF promoter activity increased 6-fold when myoblasts withdrew from the cell cycle and fused into differentiated myotubes. A 310-base pair promoter fragment depended upon multiple intact serum response elements in combination with Sp1 sites for maximal myogenic restricted activity. Furthermore, cotransfected SRF expression vector stimulated SRF promoter transcription, whereas dominant-negative SRF mutants blocked SRF promoter activity, demonstrating a positive role for an SRF-dependent autoregulatory loop.

Identification and location of human papillomavirus type 16 antisense early promoter and characterisation of antisense RNA.

Antisense RNA sequences of various regions of human papillomavirus type 16 (HPV 16) were previously found in a number of cervical lesions, but the viral or cellular promoter has not been identified. HPV 16 E7 oncogene antisense transcripts expressed from an antisense promoter in viral DNA were found in the present study by RNase protection assays for total and cytoplasmic RNA. The antisense promoter for these transcripts was located within HPV 16 nt 4030-4230 by deletion analyses. The results also suggested that most of the antisense RNA was relatively short. The antisense promoter of HPV 16 was functional for expression of antisense RNA of a heterologous gene. Antisense-sense double-stranded E7 RNA was detected, and the sense RNA of this duplex was apparently inefficient for splicing or cleavage/poly(A) addition. These results show that HPV 16 can produce early region antisense RNA, which is from a promoter within a defined region of the viral genome. The possible importance of these transcripts for the regulation of episomal HPV 16 gene expression in infected and premalignant lesions and the possible importance of their deregulation for expression in malignant lesions are discussed.

Serum response factor mediates AP-1-dependent induction of the skeletal alpha-actin promoter in ventricular myocytes.

"Fetal" gene transcription, including activation of the skeletal alpha-actin (SkA) promoter, is provoked in cardiac myocytes by mechanical stress and trophic ligands. Induction of the promoter by transforming growth factor beta or norepinephrine requires serum response factor (SRF) and TEF-1; expression is inhibited by YY1. We and others postulated that immediate-early transcription factors might couple trophic signals to this fetal program. However, multiple Fos/Jun proteins exist, and the exact relationship between control by Fos/Jun versus SRF, TEF-1, and YY1 is unexplained. We therefore cotransfected ventricular myocytes with Fos, Jun, or JunB, and SkA reporter genes. SkA transcription was augmented by Jun, Fos/Jun, Fos/JunB, and Jun/JunB; Fos and JunB alone were neutral or inhibitory. Mutation of the SRF site, SRE1, impaired activation by Jun; YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred Jun activation to a heterologous promoter, as did the c-fos SRE. Deletions of DNA binding, dimerization, or trans-activation domains of Jun and SRF abolished activation by Jun and synergy with SRF. Neither direct binding of Fos/Jun to SREs, nor physical interaction between Fos/Jun and SRF, was detected in mobility-shift assays. Thus, AP-1 factors activate a hypertrophy-associated gene via SRF, without detectable binding to the promoter or to SRF.

Splice sites of human papillomavirus type 16 E6 gene or heterologous gene required for transformation by E7 and accumulation of E7 RNA.

Transformation of primary baby rat kidney cells by the human papillomavirus type 16 (HPV 16) E7 gene and efficient accumulation of E7 RNA have been shown by this laboratory to depend on the integrity of the nucleotide position (nt) 880 splice donor site. Here, the splice sites within the HPV 16 E6 open reading frame (ORF) and the sites of the SV40 splicing unit were examined for an ability to provide this requirement. Constructs containing the HPV 16 E6 sites and the SV40 splice site sequences were used for transformation and RNase protection assays. E6 splice sites supported a low level of transformation, in assays for complete HPV 16 early region constructs containing loss-of-function mutations of the nt 880 site. Using constructs with wild-type E6 or SV40 splice sites showed that both splice sites could substitute similarly for the requirement in cis of the nt 880 site for transformation. HPV 16 E6 mutated splice site and SV40 splice site in reverse, nonfunctional orientation relative to the promoter, were not transformation competent. The HPV 16 E7 RNA levels for the E6 splice site constructs correlated closely with the transformation frequency. The SV40 splice sites were required for E7 transcript accumulation. The results showed E6 splice site function and evidence for enhanced exon skipping from E6 splice donor site to acceptor sites 3' of the E7 ORF. This was shown with constructs containing loss-of-function mutations of the nt 880 site. These results confirmed the function of the splice sites by the transformation competent constructs and suggested lower transformation frequency than for wild type was due to skipping of the E7 exon. These patterns of transcripts may have a role in the regulation of gene expression during progression to malignancy. The combined results revealed that the general presence of a functional splice donor site was absolutely required for transformation by HPV 16 E7 and accumulation of E7 RNA.

Glucocorticoid requirement for growth of human papillomavirus 16-transformed primary rat kidney epithelial cells: correlation of development of hormone resistance with viral RNA expression and processing.

The establishment of transformation of primary baby rat kidney epithelial cells by human papillomavirus type 16 DNA requires glucocorticoid hormones (Pater et al., Nature (Lond.), 335: 832-835, 1988). In this report we provide evidence that growth of transformed baby rat kidney cells in culture also requires glucocorticoids. However, transformed cells for which growth does not require hormone readily arise after a brief period of crisis, if cultured without added hormone. No reduction of glucocorticoid receptor was evident in non-hormone-requiring cells. The expression of human papillomavirus 16 RNA in these cells was analyzed by Northern blot, primer extension, and RNase protection analysis. Cells that do not require hormone had greatly reduced levels of transcripts initiated from the viral P97 promoter. However, there is evidence for compensating alterations to allow more efficient expression of E7 mRNA, since the growth of these cells is correlated with altered patterns of viral RNA expression and processing.

Glucocorticoid-dependent transformation by human papillomavirus type 16 E7 coding and 3' noncoding sequences.

The establishment of transformation of primary rodent cells by human papillomavirus (HPV) type 16 DNA requires glucocorticoid hormones (Pater et al., Nature 335, 832-835, 1988). Here we provide evidence by mutational analysis that, in the context of the hormone-regulated HPV 16 promoter/enhancer, the only protein coding sequences of HPV 16 required are those of the E7 gene. Moreover, additional sequences adjacent to the 3' end of E7 coding sequences are also essential for the establishment of the transformed phenotype. Splice donor sites, especially an E7 ORF 3' proximal one, are implicated for this cis-acting function, since specific deletion mutations of these splice sites greatly or completely reduced the frequency of transformation and the level of E7 RNA.

Nucleotide 880 splice donor site required for efficient transformation and RNA accumulation by human papillomavirus type 16 E7 gene.

Mutations within coding sequences of the various human papillomavirus type 16 (HPV-16) genes have been used to demonstrate that the HPV-16 E7 gene is necessary and sufficient for transformation of rodent cells. We now provide evidence that, in addition to E7 coding sequences, a small cis-acting region immediately flanking the 3' end of E7 coding sequences is also required for transformation. This was shown by translation termination linker insertion, progressive deletion analysis, and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within the 3'-flanking region by deletion of as few as 4 nt or substitution of 3 nt totally abolished transformation. Regeneration of the wild-type sequence in a previously transformation-incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in a stronger transformation phenotype, while mutating the +2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the amount of E7 mRNA in transformation-deficient splice site mutants was much lower. Nuclear runoff experiments revealed that there was no change in the rate of synthesis of E7 message in the nt 880 splice site mutant. Furthermore, mutations of HPV-16 sequences indicated that the two other early region splice donor sites have no more than minor roles in transformation and efficient RNA accumulation. These results indicate that the specific integrity of the nt 880 splice donor site is essential for both accumulation of E7 RNA and efficient E7-mediated transformation.