Graded transcriptional response to different concentrations of a single transactivator.

Threshold mechanisms of transcriptional activation are thought to be critical for translating continuous gradients of extracellular signals into discrete all-or-none cellular responses, such as mitogenesis and differentiation. Indeed, unequivocal evidence for a graded transcriptional response in which the concentration of inducer directly correlates with the level of gene expression in individual eukaryotic cells is lacking. By using a novel binary tetracycline regulatable retroviral vector system, we observed a graded rather than a threshold mechanism of transcriptional activation in two different model systems. When polyclonal populations of cells were analyzed at the single cell level, a dose-dependent, stepwise increase in expression of the reporter gene, green fluorescent protein (GFP), was observed by fluorescence-activated cell sorting. These data provide evidence that, in addition to the generally observed all-or-none switch, the basal transcription machinery also can respond proportionally to changes in concentration of extracellular inducers and trancriptional activators.

[1]  M. Gossen,et al.  Transcriptional activation by tetracyclines in mammalian cells. , 1995, Science.

[2]  Tom Misteli,et al.  Applications of the green fluorescent protein in cell biology and biotechnology , 1997, Nature Biotechnology.

[3]  H. Blau,et al.  High-efficiency retroviral infection of primary myoblasts , 1997, Somatic cell and molecular genetics.

[4]  E. Whitelaw,et al.  A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene , 1997, Molecular and cellular biology.

[5]  H. Weintraub,et al.  Formation of stable transcription complexes as assayed by analysis of individual templates. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[6]  B. Chernov,et al.  A complex RNA sequence determines the internal initiation of encephalomyocarditis virus RNA translation. , 1991, Nucleic acids research.

[7]  P. Kantoff,et al.  Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. D. Huang,et al.  The establishment and interpretation of transcription factor gradients in the Drosophila embryo. , 1995, Biochimica et biophysica acta.

[9]  J E Ferrell,et al.  The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. , 1998, Science.

[10]  V. de Waard,et al.  Lack of gradual regulation of tetracycline‐controlled gene expression by the tetracyclin‐repressor/VP16 transactivator (tTA) in cultured cells , 1997, FEBS letters.

[11]  S. Tapscott,et al.  Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[12]  N. Shastri,et al.  Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[13]  W. Wright.  Induction of muscle genes in neural cells , 1984, The Journal of cell biology.

[14]  Michael R. Green,et al.  Binding of general transcription factor TFIIB to an acidic activating region , 1991, Nature.

[15]  M. Groudine,et al.  An upstream activator of transcription coordinately increases the level and epigenetic stability of gene expression. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[16]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[17]  M. Ko,et al.  The dose dependence of glucocorticoid‐inducible gene expression results from changes in the number of transcriptionally active templates. , 1990, The EMBO journal.

[18]  W. Wright,et al.  The suppression of myogenic functions in heterokaryons formed by fusing chick myocytes to diploid rat fibroblasts. , 1983, Cell differentiation.

[19]  H. Blau,et al.  Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy , 1994, The Journal of cell biology.

[20]  J. Ferrell.  Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs. , 1996, Trends in biochemical sciences.

[21]  H. Blau,et al.  Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  T. Graf,et al.  GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. , 1995, Genes & development.

[23]  Victor K. Lin,et al.  Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD , 1989, Cell.

[24]  D Kosman,et al.  The dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophila embryos. , 1991, Genes & development.

[25]  M. Shales,et al.  Reduced binding of TFIID to transcriptionally compromised mutants of VP16 , 1991, Nature.

[26]  M. Groudine,et al.  Enhancers increase the probability but not the level of gene expression. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[27]  H. Blau,et al.  Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. , 1991, Science.

[28]  K. Struhl,et al.  The gradient morphogen bicoid is a concentration-dependent transcriptional activator , 1989, Cell.

[29]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[30]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Helen M. Blau,et al.  Cytoplasmic activation of human nuclear genes in stable heterocaryons , 1983, Cell.

[32]  C. Nüsslein-Volhard,et al.  A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo , 1989, Cell.

[33]  Michael R. Green,et al.  Interaction between an acidic activator and transcription factor TFIIB is required for transcriptional activation , 1993, Nature.

[34]  W. Wright.  Expression of differentiated functions in heterokaryons between skeletal myocytes, adrenal cells, fibroblasts and glial cells. , 1984, Experimental cell research.

[35]  G. Nolan,et al.  Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[36]  J. T. Kadonaga,et al.  Threshold phenomena and long-distance activation of transcription by RNA polymerase II. , 1992, Science.

[37]  Michael Carey,et al.  A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives , 1990, Nature.

[38]  H. Blau,et al.  Plasticity of the differentiated state. , 1985, Science.

[39]  S. Goodbourn.  T-Cell Activation: Transcriptional regulation in activated T cells , 1994, Current Biology.

[40]  R Y Tsien,et al.  Understanding, improving and using green fluorescent proteins. , 1995, Trends in biochemical sciences.

[41]  B. Malissen,et al.  Visualizing interleukin 2 gene expression at the single cell level , 1989, European journal of immunology.

[42]  G. Nolan,et al.  Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. , 1990, Genes & development.

[43]  H. Blau,et al.  Effect of cell history on response to helix–loop–helix family of myogenic regulators , 1990, Nature.

[44]  Wanjin Hong,et al.  A role for retinoblastoma protein in potentiating transcriptional activation by the glucocorticoid receptor , 1995, Nature.

[45]  C. Ingles,et al.  Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID , 1990, Nature.

[46]  Wolfgang Driever,et al.  The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryo , 1989, Nature.

[47]  D. Scalzo,et al.  Transcriptional enhancers act in cis to suppress position-effect variegation. , 1996, Genes & development.