1: Oncogene. 2005 Jun 16;24(26):4193-205. Mitogen regulated induction of FRA-1 proto-oncogene is controlled by the transcription factors binding to both serum and TPA response elements. Adiseshaiah P, Peddakama S, Zhang Q, Kalvakolanu DV, Reddy SP. Department of Environmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA. FRA-1, a member of the FOS family of transcription factors, is overexpressed in a variety of human tumors, and contributes to tumor progression. In addition to mitogens, various toxicants and carcinogens persistently induce FRA-1 expression in vitro and in vivo. Although the mitogen induced expression of c-FOS is relatively well understood, it is poorly defined in the case of FRA-1. Our recent analysis of the FRA-1 promoter has shown a critical role for a TRE located at -318 in mediating the TPA-induced expression. The -379 to -283 bp promoter segment containing a critical TRE (-318), however, is insufficient for the induction of FRA-1 promoter. Here, we show that a 40-bp (-276/-237) segment, comprising a TCF binding site and the CArG box (collectively known as serum response element, SRE), and an ATF site, is also necessary for the FRA-1 induction by TPA and EGF. Interestingly, the -283 to +32 bp FRA-1 promoter fragment containing an SRE and an ATF site alone was also insufficient to confer TPA sensitivity to a reporter gene. However, in association with the -318 TRE, the SRE and ATF sites imparted a strong TPA-inducibility to the reporter. Similarly, EGF also required these motifs for the full induction of this gene. Using ChIP assays we show that, in contrast to c-Jun, SRF, Elk1, ATF1 and CREB proteins bind to SRE and ATF sites of the FRA-1 promoter, constitutively. RNAi-mediated knockdown of endogenous SRF, ELK1 and c-JUN protein expression significantly reduced TPA-stimulated FRA-1 promoter activity. Thus, a bipartite enhancer formed by an upstream TRE and the downstream SRE and ATF sites and the cognate factors is necessary and sufficient for the regulation of FRA-1 in response to mitogens. PMID: 15806162 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: BMC Mol Biol. 2004 Aug 25;5:13. Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent. Selvaraj A, Prywes R. Department of Biological Sciences, Columbia University, New York, New York, USA. as814@columbia.edu BACKGROUND: Serum Response Factor (SRF) is a transcription factor that is required for the expression of many genes including immediate early genes, cytoskeletal genes, and muscle-specific genes. SRF is activated in response to extra-cellular signals by its association with a diverse set of co-activators in different cell types. In the case of the ubiquitously expressed immediate early genes, the two sets of SRF binding proteins that regulate its activity are the TCF family of proteins that include Elk1, SAP1 and SAP2 and the myocardin-related MKL family of proteins that include MKL1 and MKL2 (also known as MAL, MRTF-A and -B and BSAC). In response to serum or growth factors these two classes of co-activators are activated by different upstream signal transduction pathways. However, it is not clear how they differentially activate SRF target genes. RESULTS: In order to identify the serum-inducible SRF target genes that are specifically dependent on the MKL pathway, we have performed microarray experiments using a cell line that expresses dominant negative MKL1. This approach was used to identify SRF target genes whose activation is MKL-dependent. Twenty-eight of 150 serum-inducible genes were found to be MKL-dependent. The promoters of the serum-inducible genes were analyzed for SRF binding sites and other common regulatory elements. Putative SRF binding sites were found at a higher rate than in a mouse promoter database but were only identified in 12% of the serum-inducible promoters analyzed. Additional partial matches to the consensus SRF binding site were found at a higher than expected rate in the MKL-dependent gene promoters. The analysis for other common regulatory elements is discussed. CONCLUSIONS: These results suggest that a subset of immediate early and SRF target genes are activated by the Rho-MKL pathway. MKL may also contribute to the induction of other SRF target genes however its role is not essential, possibly due to other activation mechanisms such as MAPK phosphorylation of TCFs. PMID: 15329155 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Genesis. 2004 Feb;38(2):87-92. Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange. Cesari F, Rennekampff V, Vintersten K, Vuong LG, Seibler J, Bode J, Wiebel FF, Nordheim A. Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome. Copyright 2004 Wiley-Liss, Inc. Publication Types: Letter PMID: 14994271 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Neurobiol. 2003 Nov;57(2):204-20. Erratum in: J Neurobiol. 2004 May;59(2):259. Ying, Ju-Sung [corrected to Sung, Ying-Ju]. Rapid electrical and delayed molecular signals regulate the serum response element after nerve injury: convergence of injury and learning signals. Lin H, Bao J, Sung YJ, Walters ET, Ambron RT. Department of Anatomy and Cell Biology, 1201 Black Building, Columbia University, West 168th Street, New York, New York 10032, USA. Axotomy elicits changes in gene expression, but little is known about how information from the site of injury is communicated to the cell nucleus. We crushed nerves in Aplysia californica and the sciatic nerve in the mouse and found short- and long-term activation of an Elk1-SRF transcription complex that binds to the serum response element (SRE). The enhanced short-term binding appeared rapidly and was attributed to the injury-induced activation of an Elk1 kinase that phosphorylates Elk1 at ser383. This kinase is the previously described Aplysia (ap) ERK2 homologue, apMAPK. Nerve crush evoked action potentials that propagated along the axon to the cell soma. Exposing axons to medium containing high K(+), which evoked a similar burst of spikes, or bathing the ganglia in 20 microM serotonin (5HT) for 20 min, activated the apMAPK and enhanced SRE binding. Since 5HT is released in response to electrical activity, our data indicate that the short-term process is initiated by an injury-induced electrical discharge that causes the release of 5HT which activates apMAPK. 5HT is also released in response to noxious stimuli for aversive learning. Hence, apMAPK is a point of convergence for injury signals and learning signals. The delay before the onset of the long-term SRE binding was reduced when the crush was closer to the ganglion and was attributed to an Elk1 kinase that is activated by injury in the axon and retrogradely transported to the cell body. Although this Elk1 kinase phosphorylates mammalian rElk1 at ser383, it is distinct from apMAPK. Copyright 2003 Wiley Periodicals, Inc. PMID: 14556286 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Neuroimmunol. 2003 Sep;142(1-2):58-66. Sublytic terminal complement attack induces c-fos transcriptional activation in myotubes. Badea TD, Park JH, Soane L, Niculescu T, Niculescu F, Rus H, Shin ML. Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Sublytic C5b-9 alters the molecular phenotype of myotubes by inhibiting muscle-specific gene expression. Here, we showed that C5b-9 induced c-fos mRNA and transcription. Using c-fos promoter-CAT constructs and electrophoretic mobility shift assay (EMSA), the minimal c-fos promoter activity was shown to increase within 30-min exposure to serum C5b-9, which also induced the binding of serum response factor (SRF), along with ternary complex factor (TCF) Elk1 and Sap1a to the serum response element. C5b-9 activated ERK1, which in turn activated Elk1 in myotubes. We propose that c-fos gene transcription associated with myotube dedifferentiation is induced by C5b-9 through ERK1-mediated assembly of serum response factor-ternary complex. PMID: 14512164 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Infect Dis. 2003 Mar 1;187(5):820-8. Epub 2003 Feb 24. Synergistic activation of the serum response element-dependent pathway by hepatitis B virus x protein and large-isoform hepatitis delta antigen. Goto T, Kato N, Yoshida H, Otsuka M, Moriyama M, Shiratori Y, Koike K, Matsumura M, Omata M. Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Hepatitis delta virus (HDV) is a naturally occurring satellite of hepatitis B virus (HBV). There are few studies of the effects of the combination of HBV and HDV proteins (HDV antigens [HDAgs]) on intracellular signaling pathways. To understand the influence of HBV and HDV coinfection on hepatocytes, we investigated the effect of HBV proteins and HDAgs on the serum response element (SRE)-dependent pathway. Reporter assays revealed that only HBV X protein (HBx), alone or with the large isoform of HDAg (LHDAg), synergistically activated the SRE-dependent pathway. The effect of HBx and LHDAg on Elk1 or serum response factor (SRF) was examined, because both proteins bind to the SRE. HBx activated the transcriptional ability of Elk1, whereas LHDAg activated the transcriptional ability of SRF. Thus, HBx and LHDAg synergistically activated the SRE-dependent pathway. These results may help us understand clinical phenomena in patients coinfected with HBV and HDV. PMID: 12599056 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Gastroenterology. 2002 Dec;123(6):1962-71. Helicobacter pylori CagA protein activates serum response element-driven transcription independently of tyrosine phosphorylation. Hirata Y, Maeda S, Mitsuno Y, Tateishi K, Yanai A, Akanuma M, Yoshida H, Kawabe T, Shiratori Y, Omata M. Department of Gastroenterology, University of Tokyo, Japan. HIRATAY-INT@h.u-tokyo.ac.jp BACKGROUND & AIMS: Infection with Helicobacter pylori possessing the cag pathogenicity island (PAI) is associated with severe gastritis and gastric cancer. CagA protein, one of the products of cag PAI, is translocated into epithelial cells, where cytoskeletal rearrangements occur as a result of CagA tyrosine (Tyr) phosphorylation. Here we identify a new role for CagA protein as an activator of host cell signaling. METHODS: We transfected CagA into epithelial cells and analyzed its effect on transcription by reporter assays. The mechanism of reporter activation was assessed by electrophoretic mobility shift assays (EMSA) and immunoblots. Responsible regions of CagA for reporter activation were determined by truncation and mutagenesis of cagA gene. RESULTS: In HeLa cells, expression of CagA increased serum response element (SRE)-driven and serum response factor (SRF)-driven transcription by 40-fold and 3.3-fold, respectively, but did not affect nuclear factor kappaB- or AP-1-driven transcription. CagA-mediated SRE activation was also observed in gastric cell lines. Immunoblotting and EMSA revealed that transfection of CagA enhanced phosphorylation of and DNA binding by Elk1. Furthermore, involvement of Ras and MEK in CagA-mediated Elk1 phosphorylation was observed. SRE activation was dependent on several regions within the C-terminal portion of CagA (CagA(873-1002)), and independent of Tyr phosphorylation. CONCLUSIONS: The C-terminal portion of CagA enhances SRE-driven transcription by activating an upstream signaling cascade without requiring CagA Tyr phosphorylation. This result suggests that translocated CagA regulates 2 distinct cellular responses: phosphorylation-dependent cytoskeletal rearrangement and phosphorylation-independent transcriptional activation. PMID: 12454853 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Gerontol A Biol Sci Med Sci. 1998 Jan;53(1):B3-10. SRF binding to SRE in the rat heart: influence of age. Lu XG, Azhar G, Liu L, Tsou H, Wei JY. Gerontology Division, Beth Israel Deaconess Medical Center, New York, New York, USA. One important promoter element at the 5' end of the c-fos gene is the serum response element (SRE). SRE is the site of attachment of the 67-kDa protein serum response factor (SRF) and several accessory proteins (Elk1, SAP1, SAP2/NET), termed the ternary complex factors. The binding of SRF to SRE plays an integral role in c-fos transcription and may occur independently of the association of the ternary complex factors. In the current study, we found that SRF protein expression was increased in the hearts of the old vs young adult rats in the basal condition. The hearts of old rats may have posttranslationally modified SRF proteins that are different compared to that of the young adults. The SRF increase was present both in the cytoplasm as well as in the nucleus in the old hearts. To test whether SRF protein levels in response to acute stress might be altered with age, we studied hearts of young adult and old rats during myocardial infarction. The young adult rat hearts responded to acute ischemic stress with an increase in both p62 and p67 SRF. The hearts of the old rats, however, did not exhibit a significant change in SRF protein expression. These findings demonstrate qualitative as well as quantitative age differences in SRF protein levels, both at baseline and following stimulation. The reduced SRF expression in response to acute cardiac ischemic stress in the old rats might contribute to the observed age-related decrease in the induction of immediate early genes such as c-fos in the heart. PMID: 9467416 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Oncogene. 1997 Jan 16;14(2):213-21. FLI1 and EWS-FLI1 function as ternary complex factors and ELK1 and SAP1a function as ternary and quaternary complex factors on the Egr1 promoter serum response elements. Watson DK, Robinson L, Hodge DR, Kola I, Papas TS, Seth A. Center for Molecular and Structural Biology, Medical University of South Carolina, Charleston 29425, USA. The ETS gene products are a family of transcriptional regulatory proteins that contain a highly conserved and structurally unique DNA binding domain, termed the ETS domain. Several ETS proteins bind to DNA as monomers, however it has been shown that the DNA binding activity is enhanced or modulated in the presence of other factors. By differential display and whole genome PCR techniques, we have recently shown that the Erg1 gene is a target for ETS proteins. The Egr1 promoter contains multiple ETS binding sites, three of which exist as parts of two serum response elements (SREI and SREII). The SRE is a cis-element that regulates the expression of many growth factor responsive genes. ELK1 and SAP1a have been shown to form ternary complexes with SRF on the SRE located in the c-fos promoter. Similarly, we examined whether the ELK1, SAP1a, FLI1, EWS-FLI1, ETS1, ETS2, PEA3 and PU.1 proteins can form ternary complexes with SRF on the Egr1 SREI and II. Our results demonstrate that indeed ELK1, SAPla, FLI1 and EWS-FLI1 are able to form ternary complexes with SRF on Egr1 SREs. In addition, ELK1 and SAP1a can also form quarternary complexes on the Egr1 SREI. However, the proteins ETS1, ETS2, PEA3 and PU.1 were unable to form ternary complexes with SRF on either the Egr1 or c-fos SREs. Our data demonstrate that FLI1 and EWS-FLI1 constitute new members of a subgroup of ETS proteins that can function as ternary complex factors and further implicate a novel function for these ETS transcription factors in the regulation of the Egr1 gene. By amino acid sequence comparison we found that, in fact, 50% of the amino acids present in the B-box of SAP1a and ELK1, which are required for interaction with SRF, are identical to those present in both FLI1 (amino acids 231- 248) and EWS-FLI1 proteins. This B-box is not present in ETS1, ETS2, PEA3 or PU.1 and these proteins were unable to form ternary complexes with SRF and Egrl-SREs or c-fos SRE. Furthermore, deletion of 194 amino terminal amino acids of FLI1 did not interfere with its ability to interact with SRF, in fact, this truncation increased the stability of the ternary complex. The FLI1 protein has a unique R-domain located next to the DNA binding region. This R-domain may modulate the interaction with SRF, providing a mechanism that would be unique to FLI1 and EWS-FLI1, thus implicating a novel function for these ETS transcription factors in the regulation of the Egr1 gene. PMID: 9010223 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Mol Cell Biol. 1995 Jun;15(6):3318-26. Sequence-specific targeting of nuclear signal transduction pathways by homeodomain proteins. Grueneberg DA, Simon KJ, Brennan K, Gilman M. Cold Spring Harbor Laboratory, New York 11724, USA. Cells translate extracellular signals into specific programs of gene expression that reflect their developmental history or identity. We present evidence that one way this interpretation may be performed is by cooperative interactions between serum response factor (SRF) and certain homeodomain proteins. We show that human and Drosophila homeodomain proteins of the paired class have the ability to recruit SRF to DNA sequences not efficiently recognized by SRF on its own, thereby imparting to a linked reporter gene the potential to respond to polypeptide growth factors. This activity requires both the DNA-binding activity of the homeodomain and putative protein-protein contact residues on the exposed surfaces of homeodomain helices 1 and 2. The ability of the homeodomain to impart signal responsiveness is DNA sequence specific, and this specificity differs from the simple DNA-binding specificity of the homeodomain in vitro. The homeodomain imparts response to a spectrum of signals characteristic of the natural SRF-binding site in the c-fos gene. Response to some of these signals is dependent on the secondary recruitment of SRF-dependent ternary complex factors, and we show directly that a homeodomain can promote the recruitment of one such factor, Elk1. We infer that SRF and homeodomains interact cooperatively on DNA and that formation of SRF-homeodomain complexes permits the recruitment of signal-responsive SRF accessory proteins. The ability to route extracellular signals to specific target genes is a novel activity of the homeodomain, which may contribute to the identity function displayed by many homeodomain genes. PMID: 7760827 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Genomics. 1994 Oct;23(3):710-1. Mapping of the human SAP1 (SRF accessory protein 1) gene and SAP2, a gene encoding a related protein, to chromosomal bands 1q32 and 12q23, respectively. Shipley J, Sheer D, Dalton S, Treisman R, Patel K. Human Cytogenetics Laboratory, Imperial Cancer Research Fund, London, United Kingdom. SAP1, SAP2, and ELK1 form a related subgroup of ETS-domain proteins that can form ternary complexes with the transcription factor SRF at the c-fos serum response element (SRE). SAP1 was identified by a genetic screen for proteins interacting with SRF expressed in yeast, and SAP2 by its homology with SAP1; ELK1 was previously identified by its homology to the ETS domain. cDNA probes were used to isolate cosmid and phage clones harboring genes encoding SAP1 and SAP2. These clones were subsequently used to map the genes to 1q32 and 12q23, respectively, by fluorescence in situ hybridization. PMID: 7851904 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Genes Dev. 1994 Jul 1;8(13):1502-13. Net, a new ets transcription factor that is activated by Ras. Giovane A, Pintzas A, Maira SM, Sobieszczuk P, Wasylyk B. Centre National de la Recherche Scientifique-Laboratoire de Genetique Moleculaire des Eucaryotes (CNRS-LGME), Institut National de la Sante et de la Recherche Medicale (INSERM)-U. 184, Faculte de Medecine, Strasbourg, France. Ras signaling appears to be mediated in part by transcription factors that belong to the ets gene family. To identify downstream targets for the Ras signal transduction pathway, we have used Ras-transformed mouse fibroblasts to isolate a new member of the ets gene family, net. Net has sequence similarity in three regions with the ets factors Elk1 and SAP1, which have been implicated in the serum response of the fos promoter. Net shares various properties with these proteins, including the ability to bind to ets DNA motifs through the Ets domain of the protein and form ternary complexes with the serum response factor SRF on the fos serum response element, SRE. However, Net differs from Elk1 and SAP1 in a number of ways. The pattern of net RNA expression in adult mouse tissues is different. Net has negative effects on transcription in a number of assays, unlike Elk1. Strikingly, Ras, Src, and Mos expression switch Net activity to positive. The study of Net should help in understanding the interplay between Net and other members of the Elk subfamily and their contribution to signal transduction through Ras to the nucleus. PMID: 7958835 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------