Interferon γ (IFN-γ) induces rapid tyrosine phosphorylation of the latent cytoplasmic transcription factor, Stat1, which then forms homodimers, translocates to the nucleus and participates in IFN-γ-induced transcription. However, little is known of the interactions between Stat1 and the general transcription machinery during transcriptional activation. We show here that Stat1 can directly interact with the CREB-binding protein (CBP)/p300 family of transcriptional coactivators. Specifically, two interaction regions were identified: the amino-terminal region of Stat1 interacts with the CREB-binding domain of CBP/p300 and the carboxyl-terminal region of Stat1 interacts with the domain of CBP/p300 that binds adenovirus E1A protein. Transfection experiments suggest a role for these interactions in IFN-γ-induced transcription. Because CBP/p300-binding is required for the adenovirus E1A protein to regulate transcription of many genes during viral replication and cellular transformation, it is possible that the anti-viral effect of IFN-γ is based at least in part on direct competition by nuclear Stat1 with E1A for CBP/p300 binding.

STATs (signal transducers and activators of transcription) are a family of transcription factors that are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. The crystal structure of an NH ₂ -terminal conserved domain (N-domain) comprising the first 123 residues of STAT-4 was determined at 1.45 angstroms. The domain consists of eight helices that are assembled into a hook-like structure. The N-domain has been implicated in several protein-protein interactions affecting transcription, and it enables dimerized STAT molecules to polymerize and to bind DNA cooperatively. The structure shows that N-domains can interact through an extensive interface formed by polar interactions across one face of the hook. Mutagenesis of an invariant tryptophan residue at the heart of this interface abolished cooperative DNA binding by the full-length protein in vitro and reduced the transcriptional response after cytokine stimulation in vivo.

STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine‐phosphorylated transcription factor. Here, we identify and characterize a non‐transferable nuclear targeting sequence in the STAT1 DNA‐binding domain. This conserved signal is critical for the interferon‐γ (IFN‐γ)‐induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN‐γ stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive towards wheat germ agglutinin and to occur independently of the import receptor p97. Loss‐of‐function mutations of the dimer‐specific import signal block nuclear entry of tyrosine‐phosphorylated STAT1, which in turn also prevents induction of cytokine‐inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1‐dependent constitutive expression of caspases and the tumor necrosis factor‐α‐mediated induction of apoptosis proceed unaltered. Thus, tyrosine‐phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes.

Stat5a and Stat5b are rapidly activated by a wide range of cytokines and growth factors, including interleukin-2 (IL-2). We have previously shown that these signal transducers and activators of transcription (STAT proteins) are key regulatory proteins that bind to two tandem gamma interferon-activated site (GAS) motifs within an IL-2 response element (positive regulatory region III [PRRIII]) in the human IL-2Rα promoter. In this study, we demonstrate cooperative binding of Stat5 to PRRIII and explore the molecular basis underlying this cooperativity. We demonstrate that formation of a tetrameric Stat5 complex is essential for the IL-2-inducible activation of PRRIII. Stable tetramer formation of Stat5 is mediated through protein-protein interactions involving a tryptophan residue conserved in all STATs and a lysine residue in the Stat5 N-terminal domain (N domain). The functional importance of tetramer formation is shown by the decreased levels of transcriptional activation associated with mutations in these residues. Moreover, the requirement for STAT protein-protein interactions for gene activation from a promoter with tandemly linked GAS motifs can be relieved by strengthening the avidity of protein-DNA interactions for the individual binding sites. Taken together, these studies demonstrate that a dimeric but tetramerization-deficient Stat5 protein can activate only a subset of target sites. For functional activity on a wider range of potential recognition sites, N-domain-mediated oligomerization is essential.

Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNγ-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNγ. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.