Abstract
Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.
Highlights
Signal-dependent transcription factors that bind to DNA sequences distant from the transcriptional initiation site are required for inducible gene expression and act in concert with components of the general transcriptional machinery to recruit the DNA-dependent RNA polymerase II to transcription start sites
STAT1 dimers are released as import cargo and bind to palindromic gamma-activated site (GAS) elements with the consensus sequence 59-TTC(N)3-4GAA-93 in the promoter regions of IFNc-responsive genes, where they function as cytokine-regulated transcription factors [14,15,16]
We show that, in combination with a key lysine residue, two negatively charged amino acid residues located in the STAT1 linker domain allow for the spatial orientation of the DNA double helix in relation to transcriptional active STAT1 dimers which are required for GAS recognition and gene expression
Summary
Signal-dependent transcription factors that bind to DNA sequences distant from the transcriptional initiation site are required for inducible gene expression and act in concert with components of the general transcriptional machinery to recruit the DNA-dependent RNA polymerase II to transcription start sites. Activated JAK1 and JAK2 phosphorylate the intracellular domain of IFNGR1, thereby creating phospho-tyrosine docking sites that recruit STAT1 proteins via their Src-homology-2-(SH2) domain to the carboxy-terminal receptor tail [4]. STAT1 dimers are released as import cargo and bind to palindromic gamma-activated site (GAS) elements with the consensus sequence 59-TTC(N)3-4GAA-93 in the promoter regions of IFNc-responsive genes, where they function as cytokine-regulated transcription factors [14,15,16]. Independent of cytokine stimulation, unphosphorylated STAT molecules translocate constitutively between the cytoplasm and the nucleus in both directions [21] This transport pathway occurs at high exchange rates and is mediated through direct contacts of residues in the STAT1 linker domain with nucleoporins located in the nuclear pore complex [22]
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