Abstract
BackgroundIn 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.ResultsUsing 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.ConclusionsThus, 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.
Highlights
IntroductionIn 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)
In interferon-γ-stimulated cells, the dimeric transcription factor signal transducer and activator of transcription 1 (STAT1) recognizes semi-palindromic motifs in the promoter regions of cytokine-driven target genes termed gamma-activated sites (GAS)
Structural data of the DNA-bound STAT1 dimer revealed that the carboxyl group of E411 has a distance of 5.7 Å from the phosphodiester backbone of the cocrystallized DNA double helix and that there is no other residue in the STAT1 molecule to prevent its free access to DNA (Figure 1A)
Summary
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). A variety of different transcription factors are involved in the execution of genetic programs, and sequencespecific DNA binding is a hallmark of transcriptional regulation. Central to their role as gene-specific transcription factors is their ability to recognize distinct elements in the promoter regions of responsive genes. There is often some flexibility in the nucleotide sequence that is recognized, certain key bases are crucial for interactions with the DNA-binding fold and is required for the binding of tyrosinephosphorylated STAT dimers to semi-palindromic DNA sequences termed gamma-activated sites (GAS) [12]. The carboxy terminus containing the transactivation domain is most variable in size and sequence among the different STAT family members and is frequently subjected to alternative splicing [16]
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