1: J Biol Chem. 2005 Feb 18;280(7):6204-14. Epub 2004 Dec 2. Cell cycle-mediated regulation of smooth muscle alpha-actin gene transcription in fibroblasts and vascular smooth muscle cells involves multiple adenovirus E1A-interacting cofactors. Wang SX, Elder PK, Zheng Y, Strauch AR, Kelm RJ Jr. Department of Medicine, University of Vermont, 208 South Park Dr., Burlington, VT 05405, USA. Expression of smooth muscle alpha-actin in growth factor-induced myofibroblasts and in differentiated vascular smooth muscle cells is transcriptionally controlled by multiple positive or negative trans-acting factors interacting with distinct cis-elements in the 5'-flanking region of the gene. Because none of the transcriptional regulators reported to date is smooth muscle cell- or myofibroblast-specific per se, the dynamic interplay among many factors interacting at specific sites along the promoter appears to be a signature feature of smooth muscle alpha-actin gene regulation in these cell types. Herein, the ability of the adenovirus E1A 12 S protein to bind and functionally inactivate specific cell regulatory factors has been exploited to identify several previously unknown coactivators of the mouse smooth muscle alpha-actin promoter in rodent fibroblasts and vascular smooth muscle cells. In transient cotransfection assays, ectopic expression of wild type E1A suppressed promoter activity in a dose- and cis-element-dependent manner. In asynchronous cells, N-terminal E1A mutants defective in CREB-binding protein (CBP) and p300 binding capacity exhibited markedly reduced inhibitory activity toward a smooth muscle alpha-actin promoter driven by a composite TEF-1-, SRF-, and Sp1/3-regulated enhancer. In synchronized cells, however, a more complex mutant E1A inhibitory pattern indicated that collaboration between CBP/p300 and the retinoblastoma family of pocket proteins was required to produce a fully functional enhancer. Cotransfection experiments conducted with Rb(-/-) fibroblasts demonstrated the necessity of pRB in augmenting smooth muscle alpha-actin enhancer/promoter activity. Physical interaction studies with the use of purified wild type and mutant E1A proteins confirmed that CBP, p300, and pRB were targets of E1A binding in nuclear extracts of vascular smooth muscle cells and/or fibroblasts. Collectively, these results suggest that a repertoire of E1A-interacting proteins, namely CBP/p300 and pRB, serve to integrate the activities of multiple trans-acting factors to control smooth muscle alpha-actin gene transcription in a cell type- and cell cycle-dependent manner. PMID: 15576380 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Appl Physiol. 2004 Dec;97(6):2207-13. Epub 2004 Aug 13. Regulation of Egr-1, SRF, and Sp1 mRNA expression in contracting skeletal muscle cells. Irrcher I, Hood DA. Dept. of Kinesiology and Health Science, York University, Toronto, Ontario, Canada M3J 1P3. The early cellular signals associated with contractile activity initiate the activation and induction of transcription factors that regulate changes in skeletal muscle phenotype. The transcription factors Egr-1, Sp1, and serum response factor (SRF) are potentially important mediators of mitochondrial biogenesis based on the prevalence of binding sites for them in the promoter regions of genes encoding mitochondrial proteins, including PGC-1 alpha, the important regulator of mitochondrial biogenesis. Thus, to further define a role for transcription factors at the onset of contractile activity, we examined the time-dependent alterations in Egr-1, Sp1, and SRF mRNA and the levels in electrically stimulated mouse C(2)C(12) skeletal muscle cells. Early transient increases in Egr-1 mRNA levels within 30 min (P < 0.05) of contractile activity led to threefold increases (P < 0.05) in Egr-1 protein by 60 min. The increase in Egr-1 mRNA was not because of increased stability, as Egr-1 mRNA half-life after 30 min of stimulation showed only a 58% decline. Stimulation of muscle cells had no effect on Sp1 mRNA but led to progressive increases (P < 0.05) in SRF mRNA by 30 and 60 min. This was not matched by increases in SRF protein but occurred coincident with increases (P < 0.05) in SRF-serum response element DNA binding at 30 and 60 min as a result of SRF phosphorylation on serine-103. To assess the importance of the recovery period, 12 h of continuous contractile activity was compared with four successive 3-h bouts, with an intervening 21-h recovery period after each bout. Continuous contractile activity led to a twofold increase (P < 0.05) in Egr-1 mRNA, no change in SRF mRNA, and a 43% decrease in Sp1 mRNA expression. The recovery period prevented the decline in Sp1 mRNA, produced a decrease in Egr-1 mRNA, and had no effect on SRF mRNA. Thus continuous and intermittent contractile activity evoked different specific transcription factor expression patterns, which may ultimately contribute to divergent qualitative, or temporal patterns of, phenotypic adaptation in muscle. PMID: 15310743 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Genome Res. 2004 Mar;14(3):451-8. Eukaryotic regulatory element conservation analysis and identification using comparative genomics. Liu Y, Liu XS, Wei L, Altman RB, Batzoglou S. Stanford Medical Informatics, Stanford University, Stanford, California 94305, USA. Comparative genomics is a promising approach to the challenging problem of eukaryotic regulatory element identification, because functional noncoding sequences may be conserved across species from evolutionary constraints. We systematically analyzed known human and Saccharomyces cerevisiae regulatory elements and discovered that human regulatory elements are more conserved between human and mouse than are background sequences. Although S. cerevisiae regulatory elements do not appear to be more conserved by comparison of S. cerevisiae to Schizosaccharomyces pombe, they are more conserved when compared with multiple other yeast genomes (Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces bayanus). Based on these analyses, we developed a sequence-motif-finding algorithm called CompareProspector, which extends Gibbs sampling by biasing the search in regions conserved across species. Using human-mouse comparison, CompareProspector identified known motifs for transcription factors Mef2, Myf, Srf, and Sp1 from a set of human-muscle-specific genes. It also discovered the NFAT motif from genes up-regulated by CD28 stimulation in T-cells, which implies the direct involvement of NFAT in mediating the CD28 stimulatory signal. Using Caenorhabditis elegans-Caenorhabditis briggsae comparison, CompareProspector found the PHA-4 motif and the UNC-86 motif. CompareProspector outperformed many other computational motif-finding programs, demonstrating the power of comparative genomics-based biased sampling in eukaryotic regulatory element identification. PMID: 14993210 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Med Virol. 2003 Sep;71(1):94-104. Differential patterns of human cytomegalovirus gene expression in various T-cell lines carrying human T-cell leukemia-lymphoma virus type I: role of Tax-activated cellular transcription factors. Beck Z, Bacsi A, Liu X, Ebbesen P, Andirko I, Csoma E, Konya J, Nagy E, Toth FD. Institute of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary. Replication of human cytomegalovirus (HCMV) was investigated in various T-cell lines expressing the tax gene product of human T-cell leukemia-lymphoma virus type I (HTLV-I). Differential patterns of HCMV replication were found in HTLV-I-carrying cell lines. HCMV gene expression was restricted to the immediate-early genes in MT-2 and MT-4 cells, whereas full replication cycle of the virus was observed in C8166-45 cells. Productive HCMV infection induced a cytopathic effect resulting in the lysis of infected cells. The results of electrophoretic mobility shift assay (EMSA) showed high levels of NF-kappaB-, CREB/ATF-1-, and SRF-specific DNA binding activity in all Tax-positive cell lines. In contrast, SP1 activity could be detected only in C8166-45 cells. Using an inducible system (Jurkat cell line JPX-9), a dramatic increase in NF-kappaB, CREB/ATF-1, SRF, and SP1 binding activity, as well as productive HCMV infection, were observed upon Tax expression. Overexpression of SP1 in MT-2 and MT-4 cells converted HCMV infection from an abortive to a productive one. These data suggest that the stimulatory effect of Tax protein on HCMV in T cells is accomplished through at least five host-related transcription factor pathways. The results of this study provide possible mechanisms whereby HCMV infections might imply suppression of adult T-cell leukemia. Copyright 2003 Wiley-Liss, Inc. PMID: 12858414 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: ScientificWorldJournal. 2002 May 29;2:1469-83. Sarco(endo)plasmic reticulum Ca2+-ATPase-2 gene: structure and transcriptional regulation of the human gene. Zarain-Herzberg A, Alvarez-Fernandez G. Laboratorio de Biologia Molecular, Departamento de Bioquimica, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Mexico DF. angelz@bq.unam.mx The sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) belong to a family of active calcium transport enzymes encoded by the SERCA1, 2, and 3 genes. In this study, we describe the complete structure of the human SERCA2 gene and its 5 -regulatory region. The hSERCA2 gene is located in chromosome 12 position q24.1 in Contig NT_009770.8, spans 70 kb, and is organized in 21 exons intervened by 20 introns. The last two exons of the pre-mRNA produce by alternatively splicing the cardiac/slow-twitch muscle-specific SERCA2a isoform and the ubiquitous SERCA2b isoform. The sequence of the proximal 225-bp regulatory region of the SERCA2 genes is 80% G+C-rich and is conserved among human, rabbit, rat, and mouse species. It contains a TATA-like-box, an E-box/USF sequence, a CAAT-box, four Sp1 binding sites, and a thyroid hormone responsive element (TRE). There are two other conserved regulatory regions located between positions -410 to -661 bp and from -919 to -1410 bp. Among the DNA cis-elements present in these two regulatory regions there are potential binding sites for: GATA-4, -5, -6, Nkx-2.5/Csx, OTF-1, USF, MEF-2, SRF, PPAR/RXR, AP-2, and TREs. Upstream from position -1.5 kb, there is no significant homology among the SERCA2 genes cloned. In addition, the human gene has several repeated sequences mainly of the Alu and L2 type located upstream from position -1.7 kb, spanning in a continuous fashion for more than 40 kb. In this study, we report the cloning of 2.4 kb of 5-regulatory region and demonstrate that the proximal promoter region is sufficient for expression in cardiac myocytes, and the region from -225 to -1232 bp contains regulatory DNA elements which down-regulate the expression of the SERCA2 gene in neonatal cardiomyocytes. PMID: 12805933 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Physiol Pharmacol. 2002 Jun;53(2):147-57. Serum response factor: discovery, biochemistry, biological roles and implications for tissue injury healing. Chai J, Tarnawski AS. Department of Medicine/Gastroenterology, VA Long Beach Healthcare System, CA, USA. Serum response factor (SRF) is a transcription factor, which binds to a serum response element (SRE) associated with a variety of genes including immediate early genes such as c-fos, fosB, junB, egr-1 and -2, neuronal genes such as nurr1 and nur77 and muscle genes such as actins and myosins. By regulating expression of these genes, SRF controls cell growth and differentiation, neuronal transmission as well as muscle development and function. SRF can be activated by a variety of agents, including serum, lysophosphatidic acid (LPA), lipopolysaccharide (LPS), 12-O-tetradecanoylphorbol-13-acetate (TPA), cytokines, tumor necrosis factor-alpha (TNFalpha), agents that increase intracellular Ca2+, T-cell virus1 activator protein, hepatitis B virus activator proteins pX, activated oncogenes and protooncogenes as well as extracellular stimuli such as antioxidant and UV light. SRF itself is regulated by both cellular signal transduction pathways and interaction with other transcription factors e.g. Sp1, ATF6 and myogenic regulatory factors. Its biological function is best elucidated for myocardium. Specific cardiac SRF transgenesis demonstrated that overexpression of SRF caused hypertrophic cardiomyopathy in mouse and the mouse died of heart failure within 6 months after birth. Other transgenic data suggested that sufficient SRF was needed for embryogenesis and early development. Since SRF is important regulator of numerous genes involved in cell growth and differentiation, including muscle and neural components, SRF may also play a crucial role in tissue injury and ulcer healing, e.g. healing of gastrointestinal ulcers. Publication Types: Review Review, Tutorial PMID: 12120892 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Biol Chem. 2001 Jan 12;276(2):1026-33. GATA-4 and serum response factor regulate transcription of the muscle-specific carnitine palmitoyltransferase I beta in rat heart. Moore ML, Wang GL, Belaguli NS, Schwartz RJ, McMillin JB. Department of Pathology and Laboratory Medicine, Medical School, University of Texas-Houston Health Science Center, Baylor College of Medicine, Houston, Texas 77030, USA. 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. PMID: 11038368 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Oncogene. 1999 Dec 2;18(51):7319-27. Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in response to serum growth signals. Spencer JA, Misra RP. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, WI 53226, USA. Serum Response Factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth, differentiation, and development. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied, however, less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including an ETS domain binding site, an overlapping Sp1/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces the SRF gene by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation by cells by lysophosphatidic acid (LPA) or whole serum, the SRF promoter is upregulated by a bipartite pathway that requires both an Sp1 factor binding site and the CArG motifs for maximal stimulation. The CArG box-dependent component of this pathway is targeted by Rho mediated signals, and the Sp1 binding site dependent component is targeted by Ras mediated signals. PMID: 10602487 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Anticancer Drug Des. 1999 Jun;14(3):179-86. Effect of ecteinascidin-743 on the interaction between DNA binding proteins and DNA. Bonfanti M, La Valle E, Fernandez Sousa Faro JM, Faircloth G, Caretti G, Mantovani R, D'Incalci M. Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy. Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, a tunicate growing in mangrove roots in Caribbean. It has been shown to bind in the minor groove of DNA forming covalent adducts by reaction of the N2 of guanine with the carbinolamine moiety. We investigated ET-743 ability to inhibit the binding of different transcription factors to their consensus sequences by using gel shift assays. We have selected three types of factors: (i) oncogene products such as MYC, c-MYB and Maf; (ii) transcriptional activators regulated during the cell cycle as E2F and SRF; and (iii) general transcription factors such as TATA binding protein (TBP), Sp1 and NF-Y. We observed no inhibition of the binding of Sp1, Maf, MYB and MYC. Inhibition of DNA binding was observed for TBP, E2F, SRF at ET-743 concentrations ranging from 50 to 300 microM. The inhibition of binding of NF-Y occurs at even lower concentrations (i.e. 10-30 microM) when the recombinant subunits of NF-Y are preincubated with the drug, indicating that the inhibition of NF-Y binding does not require previous ET-743 DNA binding. Since NF-Y is a trimer containing two subunits with high resemblance to histones H2B and H2A, we have investigated the effect of ET-743 on nucleosome reconstitution. ET-743 caused a decrease of the nucleosomal band at 100 nM, with the complete disappearance of the band at 3-10 microM. These data suggest that the mode of action of this novel anticancer drug is related to its ability to modify the interaction between some DNA binding proteins and DNA. PMID: 10500494 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Cancer Res. 1999 Aug 1;59(15):3795-802. Transformation blocks differentiation-induced inhibition of serum response factor interactions with serum response elements. Ding W, Witte MM, Scott RE. Department of Pathology, University of Tennessee Medical Center, Memphis 38163, USA. The differentiation of nontransformed 3T3T mesenchymal stem cells is a multistep process that is associated with the progressive repression of mitogenic responsiveness to serum growth factors that ultimately results in expression of the terminally differentiated adipocyte phenotype. The repression of serum-induced mitogenesis by differentiation correlates with repression of the serum-inducible transcription of junB and c-fos. In contrast, the differentiation of neoplastically transformed cells does not repress mitogenic responsiveness or junB or c-fos inducibility. Because the junB and c-fos promoters both contain serum response elements (SREs), the current studies tested the possibility that differentiation might repress the ability of serum response factor (SRF) to bind to the SRE in normal cells but not in transformed cells. We now report that differentiation represses SRE serum inducibility using nontransformed cells transiently transfected with pjunB SRE thymidine kinase/chloroamphenicol acetyltransferase (SREtk/CAT) or pc-fos SREtk/CAT containing an intact SRF-binding domain. Adipocyte differentiation of nontransformed cells also markedly represses the ability of SRF to bind to the junB SRE, the c-fos SRE, and other SREs, as determined by mobility shift and gel supershift assays, without affecting the DNA binding characteristics of the nuclear protein SP-1. By comparison, the ability of SRF to bind SRE is not repressed by the differentiation of SV40 large T antigen-transformed 3T3T cells. The results further establish that adipocyte differentiation blocks the nuclear localization of SRF, thus preventing its interaction with SREs in nontransformed cells but not in transformed cells. PMID: 10446998 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Mol Cell Biol. 1999 Apr;19(4):2577-84. Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac alpha-actin promoter. Biesiada E, Hamamori Y, Kedes L, Sartorelli V. Institute for Genetic Medicine and Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles, California, USA. Activation of the human cardiac alpha-actin (HCA) promoter in skeletal muscle cells requires the integrity of DNA binding sites for the serum response factor (SRF), Sp1, and the myogenic basic helix-loop-helix (bHLH) family. In this study we report that activation of the HCA correlates with formation of a muscle-specific multiprotein complex on the promoter. We provide evidence that proteins eluted from the multiprotein complex specifically react with antibodies directed against myogenin, Sp1, and SRF and that the complex can be assembled in vitro by using the HCA promoter and purified MyoD, E12, SRF, and Sp1. In vitro and in vivo assays revealed a direct association of Sp1 and myogenin-MyoD mediated by the DNA-binding domain of Sp1 and the HLH motif of myogenin. The results obtained in this study indicate that protein-protein interactions and the cooperative DNA binding of transcriptional activators are critical steps in the formation of a transcriptionally productive multiprotein complex on the HCA promoter and suggest that the same mechanisms might be utilized to regulate the transcription of muscle-specific and other genes. PMID: 10082523 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Biol Chem. 1999 Jan 15;274(3):1801-13. Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation. Townsend KJ, Zhou P, Qian L, Bieszczad CK, Lowrey CH, Yen A, Craig RW. Departments of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835, USA. Proliferation, differentiation, and apoptosis are tightly regulated during hematopoiesis, allowing amplification along specific lineages while preventing excessive proliferation of immature cells. The MCL1 member of the BCL2 family is up-regulated during the induction of monocytic differentiation (approximately 10-fold with 12-O-tetradecanoylphorbol 13-acetate (TPA)). MCL1 has effects similar to those of BCL2, up-regulation promoting viability, but differs from BCL2 in its rapid inducibility and its pattern of expression. Nuclear factors that regulate MCL1 transcription have now been identified, extending the previous demonstration of signal transduction through mitogen-activated protein kinase. A 162-base pair segment of the human MCL1 5'-flank was found to direct luciferase reporter activity, allowing approximately 10-fold induction with TPA that was suppressible upon inhibition of the extracellular signal-regulated kinase (ERK) pathway. Serum response factor (SRF), Elk-1, and Sp1 bound to cognate sites within this segment, SRF and Elk-1 acting coordinately to affect both basal activity and TPA inducibility, whereas Sp1 affected basal activity only. Thus, the mechanism of the TPA-induced increase in MCL1 expression seen in myelomonocytic cells at early stages of differentiation involves signal transduction through ERKs and transcriptional activation through SRF/Elk-1. This finding provides a parallel to early response genes (e.g. c-FOS and EGR1) that affect maturation commitment in these cells and therefore suggests a means through which enhancement of cell viability may be linked to the induction of differentiation. PMID: 9880563 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Cancer Lett. 1997 Nov 11;119(2):137-41. Activation of serum response factor in the liver of Long-Evans Cinnamon (LEC) rat. Maeda Y, Taira T, Haraguchi K, Hirose K, Kazusaka A, Fujita S. Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. We have studied the DNA binding activities of transcription factors in the liver of Long-Evans Cinnamon (LEC) rats, an animal model of Wilson's disease. Owing to a genetic defect, this strain of rats accumulates excessive copper in the liver and develops severe hepatitis and hepatocellular carcinoma. We found that the DNA binding activity of the serum response factor (SRF) was higher in the liver of LEC rats (approximately 2-fold) than in that of Wistar rats. There was a close correlation between the intensity of the activity and the concentrations of copper in the nuclear protein. The DNA binding activity of Sp1, on the other hand, showed similar levels in both LEC and Wistar rats. SRF may play an important role in the development of hepatocellular carcinoma in LEC rats by mediating the proto-oncogene c-fos induction. We suggest that the copper in nuclear protein may be involved in the activation of SRF. PMID: 9570363 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: J Biol Chem. 1997 Nov 21;272(47):29842-51. Interaction of CArG elements and a GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells. Madsen CS, Regan CP, Owens GK. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA. We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions. PMID: 9368057 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: J Biol Chem. 1997 Jul 18;272(29):18222-31. Organization and myogenic restricted expression of the murine serum response factor gene. A role for autoregulation. Belaguli NS, Schildmeyer LA, Schwartz RJ. Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA. 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. PMID: 9218459 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: J Biochem (Tokyo). 1997 Jul;122(1):157-67. Structure and expression of the human SM22alpha gene, assignment of the gene to chromosome 11, and repression of the promoter activity by cytosine DNA methylation. Yamamura H, Masuda H, Ikeda W, Tokuyama T, Takagi M, Shibata N, Tatsuta M, Takahashi K. Department of Medicine, Osaka Medical Center for Cancer and Cardiovascular Diseases. To investigate the molecular mechanisms that control expression of smooth muscle cell (SMC) differentiation genes, we have isolated the human SM22a gene, which is composed of five exons and four introns, spanning an approximately 6-kilobase (kb) genomic DNA at chromosome region 11q23.2. Expression of the SM22a messenger RNA was detected in serum-stimulated cell cultures including SMC, undifferentiated skeletal muscle-lineage cells, and fibroblasts, and it was down-regulated in SMC of balloon-injured atheromatous human vessels. A major transcription start site of the SM22alpha gene is located at 75 base-pairs (bp) upstream of the ATG start codon. Analysis of the 2.6 kb 5'-upstream sequence demonstrated that two CArG/SRF-boxes and two GC-box/Sp1-binding sites were present at bp -147 and -274, and at bp -233 and -1635, respectively. The nucleotide sequences of the two CArG/SRF-boxes and the proximal GC-box/Sp1 binding site are 100% conserved with those of the murine SM22alpha genes [Solway, J., Seltzer, J., Samaha, F.F., Kim, S., Alger, L.E., Niu, Q., Morriesey, E.E., Ip, H.S., and Parmacek, M.S. (1995) J. Biol. Chem. 270, 13460-13469; Kemp, P.R., Osbourn, J.K., Grainger, D.J., and Metcalf, C. (1995) Biochem. J. 310, 1037-1043]. Cell transfection assays using a luciferase reporter gene construct containing the 455-bp 5'-flanking region (positions -26 to -480) showed that methylation of the CpG dinucleotides within this segment reduces its transcriptional activity. The results imply a novel mechanism for transcriptional control of the SMC differentiation-specific gene promoter. PMID: 9276683 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Mol Cell Biochem. 1997 Apr;169(1-2):61-72. The anti-cancer agent distamycin A displaces essential transcription factors and selectively inhibits myogenic differentiation. Taylor A, Webster KA, Gustafson TA, Kedes L. Department of Biological Sciences, Wichita State University, KS 67208, USA. The anticancer drug, distamycin A, alters DNA conformation by binding to A/T-rich domains. We propose that binding of the drug to DNA alters transcription factor interactions and that this may alter genetic regulation. We have analyzed the effects of distamycin A upon expression of the muscle-specific cardiac and skeletal alpha-actin genes which have A/T-rich regulatory elements in their promoters. Distamycin A specifically inhibited endogenous muscle genes in the myogenic C2 cell line and effectively eliminated the myogenic program. Conversely, when 10T1/2C18 derived pleuripotential TA1 cells were induced to differentiate in the presence of distamycin A, adipocyte differentiation was enhanced whereas the numbers of cells committing to the myogenic program decreased dramatically. Using the mobility shift assay distamycin A selectively inhibited binding of two important transcription factors, SRF and MEF2, to their respective A/T-rich elements. The binding of factors Sp1 and MyoD were not affected. The inhibition of factor binding correlated with a repression of muscle-specific promoter activity as assayed by transient transfection assays. Co-expression of the myoD gene, driven by a distamycin A-insensitive promoter, failed to relieve the inhibition of these muscle-specific promoters by distamycin A. Additionally, SRF and MEF2 dependent promoters were selectively down regulated by distamycin A. These results suggest that distamycin A may inhibit muscle-specific gene expression by selectively interfering with transcription factor interactions and demonstrate the importance of these A/T-rich elements in regulating differentiation of this specific cell type. PMID: 9089632 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Exp Cell Res. 1996 Dec 15;229(2):432-7. Age-associated changes in basal c-fos transcription factor binding activity in rat hearts. Tsou H, Azhar G, Lu XG, Kovacs S, Peacocke M, Wei JY. Gerontology Division, Beth Israel Hospital, Boston, Massachusetts, 02215, USA. The early response proto-oncogene c-fos is expressed at very low levels in the mammalian heart at baseline. To further investigate the mechanism of altered c-fos expression with age, we studied in the basal state the binding of five transcription proteins to their cognate sites in the c-fos promoter/enhancer region, in adult and old F344 rats. Our results show a reduced binding of E2F and AP1 proteins to the c-fos promoter in aging hearts. The major calcium/cyclic AMP response element (CRE) and SP1 binding was unchanged. The only increase seen with age was in the serum response element (SRE) binding proteins. SRE is the point of convergence of different signal transduction pathways (via MAP kinases and the Rho family of GTPases) at the c-fos promoter. Increased SRE binding may reflect a compensation for a decreased binding of other transcription proteins to the c-fos promoter, alteration in the phosphorylation status of SRF, or a change in the ternary complex factors Elk 1 or SAP 1. Other possibilities include defects in the signal transduction pathways with aging, which combine to produce an overall negative balance in the function of the c-fos promoter despite the increased SRE binding activity. Both in vitro and in vivo experiments have shown decreased c-fos expression with age. This may be due partly to alterations in the basal levels of transcription factor binding. PMID: 8986626 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: J Biol Chem. 1996 Jul 12;271(28):16535-43. Expression of the serum response factor gene is regulated by serum response factor binding sites. Spencer JA, Misra RP. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. The serum response factor (SRF) is a ubiquitous transcription factor that plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. Notably, SRF has been found to be a key regulator of members of a class of cellular response genes termed immediate-early genes (IEGs), many of which are believed to be involved in regulating cell growth and differentiation. The mechanism by which SRF activates transcription of IEGs in response to mitogenic agents has been extensively studied. Significantly less is known about how expression of the SRF gene itself is mediated. We and others have previously shown that the SRF gene is itself transiently induced by a variety of mitogenic agents and belongs to a class of "delayed" early response genes. We have cloned the SRF promoter and in the present study have analyzed the upstream regulatory sequences involved in mediating serum responsiveness of the SRF gene. Our analysis indicates that inducible SRF expression requires both SRF binding sites located within the first 63 nucleotides upstream from the start site of transcriptional initiation and an Sp1 site located 83 nucleotides upstream from the start site. Maximal transcriptional activity of the promoter also requires two CCAATT box sites located 90 and 123 nucleotides upstream of the start site. PMID: 8663310 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: J Biol Chem. 1996 May 3;271(18):10827-33. Serum response factor mediates AP-1-dependent induction of the skeletal alpha-actin promoter in ventricular myocytes. Paradis P, MacLellan WR, Belaguli NS, Schwartz RJ, Schneider MD. Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA. "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. PMID: 8631897 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Biol Signals. 1996 May-Jun;5(3):170-9. Role of transcription factors in the age-dependent regulation of the androgen receptor gene in rat liver. Supakar PC, Roy AK. Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio 78284-7762, USA. Androgen receptor (AR) is a ligand-activated transcription factor involved in mediating male reproductive functions. The high expression of the AR gene in target tissues of young-adult animals is generally followed by an age-dependent decline during the postreproductive life. The liver of male rats shows about a 50- to 100-fold decline in androgen sensitivity during old age due to a concomitant decline of the AR gene expression. This decline corresponds to changes in the nuclear level of several transcription factors that bind to the AR gene promoter. The positively acting factors that control the AR gene and undergo an age-dependent decline include the age-dependent transcription factor (ADF), Sp1 and the serum response factor (SRF). Nuclear factor kappa B, which functions as a negative regulator of the AR promoter, undergoes about a 10-fold increase during the age-dependent loss of the hepatic androgen sensitivity. Additionally, AP3, which can potentially function as a regulator of the AR gene, shows a marked increase during old age. Thus, a coordinated interaction among a number of positive and negative regulators appears to guide the downregulation of the AR gene during aging. Publication Types: Review Review, Tutorial PMID: 8864062 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: DNA Cell Biol. 1995 Jul;14(7):609-18. The nucleotide sequence, structure, and preliminary studies on the transcriptional regulation of the bovine alpha skeletal actin gene. Davey HW, Kelly JK, Wildeman AG. Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada. The promoters of mammalian striated muscle actin gene contain binding sites for a number of transcription factors. Examples are the CArG boxes, which bind a protein identical to or related to serum response factor (SRF), E boxes, which bind myogenic determination factors such as MyoD and myogenin, and -CCGCCC- motifs, which bind the transcription factor Sp1. To date, the only mammalian sequences isolated and analyzed are from rodent and human. We have now isolated and sequenced the bovine gene encoding alpha skeletal actin, including almost 3 kb of 5'-flanking region. When compared to the human and rodent genes (the only ones previously cloned and for which 5'-flanking sequences to only approximately -750 are known), there was the expected conservation in the coding region. A comparison of the promoter regions indicated that the bovine gene has three CArG boxes in the 5'-flanking region in positions identical to those in other species. The bovine proximal promoter is unique from those of human and rodent in that it has only one E box in the vicinity of the TATA box, near -350, whereas the other mammals have three. Far upstream sequences reveal clusters of E boxes near -2,500 and -1,500. A minimal promoter element, to -297, which has no E boxes, is sufficient to activate transcription in myotubes derived from rat L6 and mouse C2C12 myoblasts. PMID: 7626220 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Nature. 1995 Feb 16;373(6515):632-5. Interaction with RAP74 subunit of TFIIF is required for transcriptional activation by serum response factor. Joliot V, Demma M, Prywes R. Department of Biological Sciences, Columbia University, New York, New York 10027. A few general transcription factors, in particular TFIID and TFIIB, have been found to bind transcriptional activators. Here we show that the general transcription factor TFIIF is also a target for a transcriptional activator, namely serum response factor (SRF), which binds to the c-fos promoter. Using a yeast interaction assay, we find that SRF binds the RAP74 subunit of TFIIF and that SRF's transcriptional activation domain is the region involved in this binding. Further, RAP74's central charged cluster domain is required for binding to SRF's activation domain. Deletion of this domain impairs RAP74's ability to support SRF-activated transcription in vitro but has little effect on the protein's basal transcription activity or its ability to support SP1-activated transcription. The correlation of SRF-RAP74 binding with transcriptional activation suggests that RAP74 is a critical target for SRF-activated transcription. PMID: 7854423 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Mol Reprod Dev. 1994 Sep;39(1):112-7. Control of cardiac gene transcription by fibroblast growth factors. Schneider MD, Kirshenbaum LA, Brand T, MacLellan WR. Molecular Cardiology Unit, Baylor College of Medicine, Houston, Texas 77030. Skeletal alpha-actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, downregulated after birth, and reactivated by tropic signals including basic fibroblast growth factor (FGF-2) and type beta transforming growth factors (TGF beta). To investigate the molecular basis for cardiac-restricted and growth factor-induced SkA transcription, we have undertaken a mutational analysis of the SkA promoter in neonatal ventricular myocytes, with emphasis on the role of three nominal serum response elements. Serum response factor (SRF) and the bifunctional factor YY1 are the predominant cardiac proteins contacting the proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1. or SRF alone, virtually abolish SkA transcription; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases expression, substantiating the predicted role of YY1 as an inhibitor of SRF effects. SkA transcription requires combinational action of SRE1 with consensus sites for Sp1 and the SV40 enhancer binding protein, TEF-1. As an isolated motif, SRE1 can confer responsiveness to both FGF-2 and TGF beta to a heterologous promoter. Whether TEF-1 binding sites likewise can function as FGF response elements is unknown. Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has largely been undertaken to date in neonatal ventricular myocytes, as the adult ventricular myocyte has been refractory to conventional procedures for gene transfer. To circumvent expected limitations of other methods, we have used replication-deficient adenovirus to achieve efficient gene transfer to adult cardiac cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS) Publication Types: Review Review, Tutorial PMID: 7528025 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: J Biol Chem. 1994 Jun 17;269(24):16754-60. Transforming growth factor-beta response elements of the skeletal alpha-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1. MacLellan WR, Lee TC, Schwartz RJ, Schneider MD. Department of Medicine, Baylor College of Medicine, Houston, Texas 77030. Skeletal alpha-actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, down-regulated after birth, and reactivated by trophic signals including type beta-transforming growth factors (TGF beta). To investigate the molecular basis for cardiac-restricted and TGF beta-induced SkA transcription, we have undertaken a mutational analysis of the SkA promoter in ventricular myocytes, with emphasis on the role of three nominal serum response elements. Serum response factor (SRF) and the bifunctional factor YY1 are the predominant cardiac proteins contacting the proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1, or SRF alone, virtually abolish SkA transcription in both TGF beta- and vehicle-treated cells; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases both basal and TGF beta-dependent expression, substantiating the predicted role of YY1 as an inhibitor of SRF effects. However, efficient SkA transcription requires combinatorial action of SRE1 with consensus sites for Sp1 and the SV40 enhancer-binding protein, TEF-1. As isolated motifs, either SRE1- or TEF-1-binding sites function as TGF beta response elements. Induction of the SkA promoter by TGF beta required SRF and TEF-1 in concert, unlike other pathways for TGF beta-dependent gene expression. PMID: 8206998 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Biol Chem. 1994 Feb 4;269(5):3489-97. Role of transcription factor TFIIF in serum response factor-activated transcription. Zhu H, Joliot V, Prywes R. Department of Biological Sciences, Columbia University, New York, New York 10027. We have found that the general transcription factor TFIIF has an important role in serum response factor (SRF)-activated transcription in vitro. A low amount of TFIIF was sufficient for basal transcription, whereas higher amounts were required for SRF, but not Sp1, activation. High TFIIF levels also increased activation by GAL4-VP16, whereas none of the other general transcription factors had these properties. TFIIF could also relieve squelching by SRF in vitro, suggesting that SRF may directly bind TFIIF. We found more direct evidence for SRF-TFIIF interaction by DNA binding assays where the RAP74 subunit of TFIIF bound DNA in conjunction with SRF, but not alone. RAP74 also bound DNA with GAL4-VP16, but not with Sp1 or the DNA binding domain of GAL4. These results suggest that the mechanism of transcriptional activation by SRF, and perhaps some other activators, involves their interaction with TFIIF. PMID: 8106390 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Proc Natl Acad Sci U S A. 1992 May 1;89(9):4047-51. Myocardial activation of the human cardiac alpha-actin promoter by helix-loop-helix proteins. Sartorelli V, Hong NA, Bishopric NH, Kedes L. Institute for Genetic Medicine, University of Southern California, School of Medicine, Los Angeles 90033. The cardiac alpha-actin gene is expressed in both heart and skeletal muscle. In skeletal myogenic cells, the 177-base-pair promoter of the human cardiac alpha-actin (HCA) gene requires three transcription factors for activation: Sp1, serum response factor (SRF), and MyoD. However, MyoD is undetectable in heart. To search for a functional equivalent of MyoD, we analyzed the transcriptional regulation of the HCA promoter in primary cultures of rat cardiac myocytes. The same DNA sequence elements recognized by SRF, Sp1, and MyoD and required for HCA transcription in skeletal muscle cells were also found to be necessary for expression in cardiomyocytes. Overexpression of Id, a negative regulator of basic helix-loop-helix proteins, selectively attenuated expression of the HCA promoter. Cardiomyocyte nuclei contain a protein complex that specifically interacts with the same required sequence (E box) in the HCA promoter that is bound by MyoD in skeletal myogenic cells. Furthermore, these complexes contain a peptide that is a member of the E2A family of basic helix-loop-helix proteins. Cardiomyocyte nuclei appear to be enriched for a protein that can bind to the E-box site as dimers with the E12 protein. These results suggest that a member of the basic helix-loop-helix family, together with SRF and Sp1, activates the HCA promoter in heart. Alternative strategies for myocardial transcription of HCA are discussed. PMID: 1570331 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Nucleic Acids Res. 1991 Oct 25;19(20):5739-42. O6-methylguanine inhibits the binding of transcription factors to DNA. Bonfanti M, Broggini M, Prontera C, D'Incalci M. Laboratory of Cancer Chemotherapy, Istituto di Richerche Farmacologiche Mario Negri, Milan, Italy. To study the effect of methylation of O6-guanine on the binding of cellular factors to different DNA sequences, modified oligonucleotides were constructed, in which O6-Methylguanine (O6-MeG) replaced some guanines. The DNA sequences utilized were: the region of the c-fos promoter containing the binding site for serum response factor (SRF); the region of the HIV LTR containing two binding sites for the transcription factor NF kappa B; the region of the HIV LTR containing three binding sites for the cellular factor sp1. After incubation of labeled oligonucleotides, either unmodified or containing O6-MeG, with nuclear extracts obtained from different cell lines, gel retardation assays indicated that the presence of O6-MeG resulted in inhibition of binding of cellular factors to DNA sequences located in the promoter regions of genes. This inhibition was not the same for all modified oligonucleotides but dependent on the position in which O6-MeG was located. The results obtained indicate that alkylation of O6-guanine affects the binding of transcription factors and thereby possibly the regulation of genes expression. PMID: 1945851 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Genes Dev. 1990 Oct;4(10):1811-22. Muscle-specific expression of the cardiac alpha-actin gene requires MyoD1, CArG-box binding factor, and Sp1. Sartorelli V, Webster KA, Kedes L. Program in Molecular Biology and Genetics, University of Southern California School of Medicine, Los Angeles 90033. Expression of the human cardiac alpha-actin gene (HCA) depends on the interactions of multiple transcriptional regulators with promoter elements. We report here that the tissue-specific expression of this promoter is determined by the simultaneous interaction of at least three specific protein-DNA complexes. The myogenic determinant gene MyoD1 activated the transcription of transfected HCA-CAT promoter constructs in nonmuscle cells, including CV-1 and HeLa cells. Gel mobility-shift and footprinting assays revealed that MyoD1 specifically interacted with a single consensus core sequence, CANNTG, at -50. Previously characterized sites interact with a protein identical with or related to the serum response factor (SRF) at -100 and Sp1 at -70. All three elements must be intact to support transcription in muscle cells: site-specific mutation within any one of these three elements eliminated transcriptional expression by the promoter. Furthermore, expression of the promoter in embryonic Drosophila melanogaster cells that lack MyoD1 and Sp1 is strictly dependent on all three sites remaining intact and on the presence of exogenously supplied Sp1 and MyoD1. These experiments suggest that the presence of three sequence-specific binding proteins, including MyoD1, and their intact target DNA sequences are minimal requirements for muscle-specific expression of the HCA gene. PMID: 2123467 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: New Biol. 1990 Jul;2(7):642-7. Sp1 represses IL-2 receptor alpha chain gene expression. Roman DG, Toledano MB, Leonard WJ. Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892. Sp1 is a DNA-binding protein that acts as a positive regulator of eukaryotic gene expression. The interleukin-2 receptor alpha chain (IL2R alpha) gene 5' regulatory region contains a single Sp1 consensus motif that overlaps a CArG box capable of binding serum response factor (SRF). The CArG box has previously been shown to be important for IL2R alpha gene expression. In this study, the results of competition experiments suggest that Sp1 and SRF compete for binding to the CArG region. Site-directed mutagenesis and transient transfection assays indicate that the IL2R alpha gene Sp1 serves the unusual role of repressing gene expression, most likely by competing for binding of nuclear factor(s) to the CArG box. PMID: 2083254 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Mol Cell Biol. 1989 Aug;9(8):3269-83. Identification of multiple proteins that interact with functional regions of the human cardiac alpha-actin promoter. Gustafson TA, Kedes L. Department of Medicine, Stanford University School of Medicine, Palo Alto, California. 5' Sequences of the human cardiac alpha-actin gene are involved in the tissue-specific and developmental regulation of the gene. Deletion analyses combined with transient expression experiments in muscle cells have demonstrated three primary regions of functional importance (A. Minty and L. Kedes, Mol. Cell. Biol. 6:2125-2136, 1986; T. Miwa and L. Kedes, Mol. Cell. Biol. 7:2803-2813, 1987), and we have previously demonstrated binding of a protein indistinguishable from serum response factor (SRF) to the most proximal region (T.A. Gustafson, T. Miwa, L.M. Boxer, and L. Kedes, Mol. Cell. Biol. 8:4110-4119, 1988). In this report, we examine protein interaction with the remainder of the promoter. Gel shift and footprinting assays revealed that at least seven distinct nuclear proteins interacted with known and putative regulatory regions of the promoter. The transcription factor Sp1 bound to eight sites, as demonstrated by footprinting assays and gel shift analysis with purified Sp1. Purified CCAAT box-binding transcription factor CTF/NF-I and Sp1 were shown to interact with the far-upstream regulatory element at -410, and footprint analysis showed extensive overlap of these two sites. Two unidentified proteins with similar but distinct footprints interacted with the second region of functional importance at -140, which contains the second CArG motif [CC(A + T rich)6GG], and these proteins were shown to be distinct from SRF. SRF was found to bind to the remaining three CArG boxes, two of which were closely interdigitated with Sp1 sites. In addition, CArG box 4 was found to interact with SRF and another distinct protein whose footprint was contained within the SRF-binding site. Sequences surrounding the TATA box were also shown to bind proteins. Sp1 was shown to bind to a site immediately downstream from the TATA box and to a site within the first exon. Thus, each of the three functional upstream regions, as defined by transfection assays, was shown to interact with five factors: Sp1 and CTF/NF-I at the upstream site, two unidentified proteins at the central site, and SRF at the most proximal site. These results suggest that expression of the cardiac actin gene in muscle cells is controlled by complex interactions among multiple upstream and intragenic elements. PMID: 2796988 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------