Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro

We have characterized an in vitro transcription system derived from uninfected HeLa cells that accurately initiates RNA synthesis at the herpes virus thymidine kinase (TK) promoter. Analysis of linker-scanning, single-site, and promoter-inversion mutants reveals that the TK upstream elements previously mapped in vivo are accurately recognized in vitro. A protein fraction required for TK transcription in reconstitution experiments was found to contain multiple protein species that bind specifically to the TK promoter. DNAase I footprint experiments with wild-type and mutant promoters reveal that the TK upstream elements contain three distinctive protein binding sites, two of which appear to be recognized by the Sp1 transcription factor and one which interacts with a cellular protein that binds to "CCAAT" sequences. Optimal expression of the thymidine kinase gene appears to require the coordinate interaction of these two types of transcription factors with the three upstream elements of the promoter.

[1]  J. Reiser,et al.  Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit β-globin gene in mouse 3T6 cells , 1983, Cell.

[2]  R. Tjian,et al.  Sp1 binds to promoter sequences and activates herpes simplex virus ‘immediate-early’ gene transcription in vitro , 1985, Nature.

[3]  S. McKnight.  Functional relationships between transcriptional control signals of the thymidine kinase gene of herpes simplex virus , 1982, Cell.

[4]  S. Silverstein,et al.  "Transactivation" control signals in the promoter of the herpesvirus thymidine kinase gene. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. Sharp,et al.  DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. Grosschedl,et al.  Identification of regulatory sequences in the prelude sequences of an H2A histone gene by the study of specific deletion mutants in vivo. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S. McKnight,et al.  Transcriptional control signals of a eukaryotic protein-coding gene. , 1982, Science.

[8]  P. Chambon,et al.  The repeated GC-rich motifs upstream from the TATA box are important elements of the SV40 early promoter. , 1983, Nucleic acids research.

[9]  K. Yamamoto,et al.  DNA sequences bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo , 1983, Cell.

[10]  G. Hayward,et al.  Evidence for a direct role for both the 175,000- and 110,000-molecular-weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters , 1985, Journal of virology.

[11]  Tom Maniatis,et al.  The structure and evolution of the human β-globin gene family , 1980, Cell.

[12]  P. Gruss,et al.  Simian virus 40 tandem repeated sequences as an element of the early promoter. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[13]  P. Lebowitz,et al.  Initiation and regulation of simian virus 40 early transcription in vitro , 1982, Journal of virology.

[14]  B. Roizman,et al.  Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional viral polypeptides. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Maniatis,et al.  Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells , 1985, Molecular and cellular biology.

[16]  M. Fromm,et al.  Transcription in vivo from SV40 early promoter deletion mutants without repression by large T antigen. , 1983, Journal of molecular and applied genetics.

[17]  Pierre Chambon,et al.  In vivo sequence requirements of the SV40 early promoter region , 1981, Nature.

[18]  G. Hayward,et al.  Regulation of herpesvirus macromolecular synthesis: evidence for multilevel regulation of herpes simplex 1 RNA and protein synthesis. , 1975, Cold Spring Harbor symposia on quantitative biology.

[19]  R. Dixon,et al.  Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type 1 immediate early protein VP175 , 1980, Journal of virology.

[20]  R. Axel,et al.  Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: Identification of an upstream control region , 1981, Cell.

[21]  B. Roizman,et al.  Regulation of Herpesvirus Macromolecular Synthesis I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins , 1974, Journal of virology.

[22]  B. Mcauslan,et al.  Regulation of herpes simplex virus-induced thymidine kinase. , 1974, Biochemical and biophysical research communications.

[23]  R. Tjian,et al.  Multiple specific contacts between a mammalian transcription factor and its cognate promoters , 1984, Nature.

[24]  S. McKnight,et al.  The distal transcription signals of the herpesvirus tk gene share a common hexanucleotide control sequence , 1984, Cell.

[25]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[26]  H. Pelham,et al.  A synthetic heat‐shock promoter element confers heat‐inducibility on the herpes simplex virus thymidine kinase gene. , 1982, The EMBO journal.

[27]  S. McKnight,et al.  Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes , 1985, Molecular and cellular biology.

[28]  C Benoist,et al.  The ovalbumin gene-sequence of putative control regions , 1980, Nucleic Acids Res..

[29]  P. Chambon,et al.  The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. , 1981, Nucleic acids research.

[30]  J. Banerji,et al.  Expression of a β-globin gene is enhanced by remote SV40 DNA sequences , 1981, Cell.

[31]  T. Maniatis,et al.  Human β-interferon gene expression is regulated by an inducible enhancer element , 1985, Cell.

[32]  R. Palmiter,et al.  A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[33]  R. Everett.  Trans activation of transcription by herpes virus products: requirement for two HSV‐1 immediate‐early polypeptides for maximum activity. , 1984, The EMBO journal.

[34]  R. Tjian,et al.  Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II , 1983, Cell.

[35]  R. Myers,et al.  SV40 gene expression is modulated by the cooperative binding of T antigen to DNA , 1981, Cell.

[36]  D. Galas,et al.  DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. , 1978, Nucleic acids research.

[37]  R. Tjian,et al.  The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter , 1983, Cell.

[38]  R. Flavell,et al.  Sequence requirements for the transcription of the rabbit β-globin gene in vivo: -80 region , 1982 .

[39]  R. Watson,et al.  Temporal regulation of herpes simplex virus type 1 transcription: location of transcripts on the viral genome , 1977, Cell.

[40]  R. Tjian,et al.  A novel strategy for constructing clustered point mutations. , 1985, Nucleic acids research.

[41]  D. Spandidos,et al.  Transcriptional regulation of a herpes simplex virus immediate early gene is mediated through an enhancer‐type sequence. , 1984, The EMBO journal.