Two glutamic acid residues in the DNA-binding domain are engaged in the release of STAT1 dimers from DNA

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.

A Conserved Motif in the Linker Domain of STAT1 Transcription Factor Is Required for Both Recognition and Release from High-Affinity DNA-Binding Sites

“…The glutamic acid residues in the linker domain described here have similar functions to two other key residues (E411 and E421), which we have recently identified in the DNA-binding domain to be engaged in the release of dimeric STAT1 from DNA [43]. While E559 and E563 are positioned at the back in the carboxy-terminal core of the STAT1 dimer, the residues E411 and E421 are located anteriorly in relation to the DNA double helix.…”

Toxoplasma gondii Inhibits Gamma Interferon (IFN-γ)- and IFN-β-Induced Host Cell STAT1 Transcriptional Activity by Increasing the Association of STAT1 with DNA

“…If IFN-␥ is still present and the receptors and JAKs are still active when STAT1 is exported back into the cytoplasm, STAT1 will be reactivated and cycle back to the nucleus to induce further transcription (19,20). However, when IFN-␥ is removed its signaling pathway is downregulated through dephosphorylation of the JAKs and internalization of the IFN-␥ receptor (43,46).…”

“…These steps must occur in this order since DNA-bound STAT1 is protected from tyrosine phosphatases, and only unphosphorylated STAT1 can be exported back into the cytoplasm (19). This cycling is required for full STAT1 transcriptional activity; STAT1 mutants that have a decreased dissociation rate from DNA (20) or that are defective in nuclear export (21) have decreased transcriptional output. Two other STAT family members, STAT6 (22) and STAT3 (23), also need to undergo this cycling on and off DNA to produce their full transcriptional output.…”

“…During IFN-␥ stimulation, STAT1 undergoes nuclear-cytoplasmic cycling in order to constantly monitor the activity of the IFN-␥ receptor and the JAKs (19,20). After DNA binding and transcriptional activation, STAT1 dissociates from the DNA, is dephosphorylated at tyrosine 701, and is exported back into the cytoplasm.…”

“…In the nucleus, STAT1 binds to gamma-activated sequence (GAS) sites in the DNA, leading to its serine phosphorylation at residue 727 (17). This additional serine phosphorylation is required for maximal STAT1 activity (18).During IFN-␥ stimulation, STAT1 undergoes nuclear-cytoplasmic cycling in order to constantly monitor the activity of the IFN-␥ receptor and the JAKs (19,20). After DNA binding and transcriptional activation, STAT1 dissociates from the DNA, is dephosphorylated at tyrosine 701, and is exported back into the cytoplasm.…”