Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors

Using in vivo dimethylsulfate footprinting, we have analyzed protein-DNA interactions within two regions upstream of the tyrosine aminotransferase (TAT) gene that are characterized by an altered chromatin structure in TAT-expressing as compared to nonexpressing cells. All the identified protein contacts to DNA are found exclusively in the TAT-expressing hepatoma cells. In vitro analyses of specific DNA-binding factors in crude nuclear extracts yield DNAase I footprints that correlate well with the binding sites in vivo. Surprisingly, all DNA-binding activities are present in nuclei of TAT-expressing and nonexpressing cells, indicating that the mere presence of factors is not sufficient for their interaction with a binding site in vivo. Genomic sequencing reveals methylation of CpG dinucleotides in the regions analyzed in nonexpressing cells, whereas no methylation is found in TAT-expressing cells. In vitro methylation at a cytosine residue within a footprint region prevents the interaction of a factor with its binding site.

[1]  G. Church,et al.  Cell-type-specific contacts to immunoglobulin enhancers in nuclei , 1985, Nature.

[2]  A. Bird.  CpG-rich islands and the function of DNA methylation , 1986, Nature.

[3]  T. Maniatis,et al.  Regulation of inducible and tissue-specific gene expression. , 1987, Science.

[4]  W. Gilbert,et al.  An amino-terminal fragment of lac repressor binds specifically to lac operator. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[5]  W. Schaffner,et al.  Cell type‐specificity elements of the immunoglobulin heavy chain gene enhancer. , 1987, The EMBO journal.

[6]  P. Sharp,et al.  A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes , 1986, Nature.

[7]  O. Greengard.  CHAPTER 2 – The Developmental Formation of Enzymes in Rat Liver , 1970 .

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

[9]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Vieira,et al.  A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. , 1982, Gene.

[11]  U. Pettersson,et al.  Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis). , 1984, Biochimica et biophysica acta.

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

[13]  G. Rubin,et al.  The effect of chromosomal position on the expression of the drosophila xanthine dehydrogenase gene , 1983, Cell.

[14]  U. Schibler,et al.  Tissue-specific in vitro transcription from the mouse albumin promoter , 1986, Cell.

[15]  S. Gluecksohn-waelsch.  Regulatory genes in development , 1987 .

[16]  D. Crothers,et al.  Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. , 1981, Nucleic acids research.

[17]  B. Gloss,et al.  In vivo protein–DNA interactions in a glucocorticoid response element require the presence of the hormone , 1986, Nature.

[18]  R. Kornberg,et al.  Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones , 1987, Cell.

[19]  David Baltimore,et al.  Multiple nuclear factors interact with the immunoglobulin enhancer sequences , 1986, Cell.

[20]  H. Richard-Foy,et al.  Sequence‐specific positioning of nucleosomes over the steroid‐inducible MMTV promoter. , 1987, The EMBO journal.

[21]  J. Jiricny,et al.  Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Ptashne,et al.  Specific DNA binding of GAL4, a positive regulatory protein of yeast , 1985, Cell.

[23]  G. Church,et al.  Genomic sequencing. , 1993, Methods in molecular biology.

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

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  R. Tjian,et al.  Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements , 1987, Cell.

[27]  H. Zachau,et al.  Sequences closely related to an immunoglobulin gene promoter/enhancer element occur also upstream of other eukaryotic and of prokaryotic genes. , 1986, Nucleic acids research.

[28]  H. Cedar,et al.  Muscle-specific activation of a methylated chimeric actin gene , 1986, Cell.

[29]  W. Doerfler,et al.  DNA methylation and gene activity. , 1983, Annual review of biochemistry.

[30]  G. Hager,et al.  Steroid-dependent interaction of transcription factors with the inducible promoter of mouse mammary tumor virus in vivo , 1987, Cell.

[31]  G. H. Thomas,et al.  Selected topics in chromatin structure. , 1985, Annual review of genetics.

[32]  D. Kioussis,et al.  β-Globin gene inactivation by DNA translocation in γβ-thalassaemi , 1983, Nature.

[33]  P. Sharp,et al.  Distinct factors bind to apparently homolgous sequences in the immunoglobulin heavy-chain enhancer , 1986, Nature.

[34]  M. Karin,et al.  A pituitary-specific trans-acting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells , 1987, Cell.

[35]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[36]  W. Gilbert,et al.  Detection of cytosine methylation in the maize alcohol dehydrogenase gene by genomic sequencing , 1986, Nature.

[37]  M. M. Garner,et al.  A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system , 1981, Nucleic Acids Res..

[38]  G. Church,et al.  B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo. , 1985, Science.

[39]  Carl Wu,et al.  Induction of sequence-specific binding of Drosophila heat shock activator protein without protein synthesis , 1987, Nature.

[40]  K. Martin,et al.  The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[41]  D. Jump,et al.  Transcriptional activation of the rat liver S14 gene during postnatal development. , 1988, The Journal of biological chemistry.

[42]  T. Maniatis,et al.  Detection of factors that interact with the human β-interferon regulatory region in vivo by DNAase I footprinting , 1986, Cell.

[43]  K. Yamamoto,et al.  Steroid receptor regulated transcription of specific genes and gene networks. , 1985, Annual review of genetics.

[44]  P. Tucker,et al.  Interaction of cell-type-specific nuclear proteins with immunoglobulin VH promoter region sequences , 1986, Nature.

[45]  J. Svoboda.  Presence of Chicken Tumour Virus in the Sarcoma of the Adult Rat inoculated after Birth with Rous Sarcoma Tissue , 1960, Nature.

[46]  Robert Tjian,et al.  Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins , 1985, Nature.

[47]  G. Stein,et al.  Protein-DNA interactions in vivo upstream of a cell cycle-regulated human H4 histone gene. , 1987, Science.

[48]  G. Schütz,et al.  Tissue‐specific DNaseI hypersensitive sites in the 5′‐flanking sequences of the tryptophan oxygenase and the tyrosine aminotransferase genes. , 1984, The EMBO journal.

[49]  G. Schütz,et al.  Transcription activation of the tyrosine aminotransferase gene by glucocorticoids and cAMP in primary hepatocytes. , 1987, European journal of biochemistry.

[50]  P. Chambon,et al.  Stimulation of in vitro transcription from the SV40 early promoter by the enhancer involves a specific trans‐acting factor. , 1984, The EMBO journal.

[51]  M. Karin,et al.  Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor , 1987, Cell.

[52]  H. Zentgraf,et al.  Isolation and characterization of the rat tyrosine aminotransferase gene. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[53]  H. Schöler,et al.  Nuclear factors binding specific sequences within the immunoglobulin enhancer interact differentially with other enhancer elements. , 1986, The EMBO journal.

[54]  G. Felsenfeld,et al.  A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[55]  P. Chambon,et al.  The mouse immunoglobulin heavy‐chain enhancer: effect on transcription in vitro and binding of proteins present in HeLa and lymphoid B cell extracts. , 1986, The EMBO journal.

[56]  W. Gilbert,et al.  Detection in vivo of protein-DNA interactions within the lac operon of Escherichia coli , 1985, Nature.

[57]  B. Gloss,et al.  Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene , 1987, Cell.