title: Two glutamic acid residues in the DNA-binding domain are engaged in the release of STAT1 dimers from DNA.
abstract: BACKGROUND
  In interferon-γ-stimulated cells, the dimeric transcription factor STAT1 (signal transducer and activator of transcription 1) recognizes semi-palindromic motifs in the promoter regions of cytokine-driven target genes termed GAS (gamma-activated sites). However, the molecular steps that facilitate GAS binding and the subsequent liberation of STAT1 homodimers from these promoter elements are not well understood.
  
  
  RESULTS
  Using a mutational approach, we identified two critical glutamyl residues within the DNA-binding domain adjacent to the phosphodiester backbone of DNA which efficiently release phospho-STAT1 from DNA. The release of STAT1 dimers from DNA enhances transcriptional activity on both interferon-driven reporter and endogenous target genes. A substitution of either of the two glutamic acid residues broadens the repertoire of putative binding sites on DNA and enhances binding affinity to GAS sites. However, despite elevated levels of tyrosine phosphorylation and a prolonged nuclear accumulation period, the STAT1 DNA-binding mutants show a significantly reduced transcriptional activity upon stimulation of cells with interferon-γ. This reduced transcriptional response may be explained by the deposition of oligomerized STAT1 molecules outside GAS sites.
  
  
  CONCLUSIONS
  Thus, two negatively charged amino acid residues in the DNA-binding domain are engaged in the liberation of STAT1 from DNA, resulting in a high dissociation rate from non-GAS sites as a key feature of STAT1 signal transduction, which positively regulates cytokine-dependent gene expression probably by preventing retention at transcriptionally inert sites.
authors.affiliation: Klinik für Kardiologie, Philipps-Universität Marburg, Marburg, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Koch
authors.forename: Julia
authors.initials: J
authors.lastname: Staab
authors.forename: Volker
authors.initials: V
authors.lastname: Ruppert
authors.forename: Thomas
authors.initials: T
authors.lastname: Meyer
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D018698:Glutamic Acid / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050794:STAT1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D018698:Glutamic Acid; D007371:Interferon-gamma; D004247:DNA