Abstract
Defective cooperative DNA binding of STAT1 (signal transducer and activator of transcription 1) transcription factor has impact on interferon-γ(IFNγ)-regulated transcriptional responses. In this study, we generated N-terminal gain-of-function mutants of this protein which exhibited hyperactive cooperativity and assessed their functional consequences on gene expression. Our data show that four negatively charged, surface-exposed amino acid residues in the N-terminal domain dimer are engaged in the disassembly of tyrosine-phosphorylated tetrameric complexes on DNA and prevent the occurrence of higher-order STAT1 oligomers on low-affinity DNA binding sites. Owing to their improved tetramer stability, the N-terminal mutants showed relaxed sequence requirements for the binding to DNA as compared to the wild-type protein. Similarly to a STAT1 mutant with impaired tetramerization, the N-terminal gain-of-function mutants showed elevated tyrosine-phosphorylation levels and prolonged nuclear accumulation upon stimulation of cells with IFNγ. However, in contrast to the global impairment of IFNγ signalling in tetramerization-deficient mutants, the transcriptional consequences of the N-terminal gain-of-function mutants are rather distinct and affect gene expression locally in a promoter-specific manner. Thus, we conclude that the STAT1 N-domain acts as a double-edged sword: while one interface is crucial for the formation of tetrameric complexes on IFNγ-regulated promoters, the opposite interface harbours an inhibitory mechanism that limits the accumulation of higher-order oligomers simply by disrupting cooperative DNA binding.