STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling

STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon–mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon–dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin–proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs

Nucleocytoplasmic transport and gene induction mediated by the transcription factor STAT1

1\. Titelseite, Inhalt, Summary, Danksagung, Abkürzungen 2\. Einleitung 1 3\. Material und Methoden 20 4\. Ergebnisse 53 5\. Diskussion 100 6\. Literaturverzeichnis 115Der Transkriptionsfaktor STAT1 befindet sich vor der Stimulation mit IFNγ preferentiell im Cytosol der Zellen. Nach Stimulation wird STAT1 durch Jak- Kinasen phosphoryliert und sehr schnell in den Zellkern transloziert. In dieser Arbeit wurden der IFNγ-abhängige nukleäre Import und Export von STAT1 untersucht. Es wurde beobachtet, daß die Behandlung von 293T-Zellen mit dem Exportinhibitor LMB keinen Einfluß auf die nukleäre Verteilung von STAT1 in unstimulierten Zellen hatte, aber die Phase der nukleären Akkumulation von STAT1 nach einer IFNγ-Stimulation verlängerte. Im weiteren Verlauf dieser Arbeit wurde ein konserviertes leuzinreiches Segment im 4-Helix-Bündel von STAT1 identifiziert, welches für den effektiven nukleären Export von STAT1 notwendig ist. Durch Mutation von zwei Leuzinresten wird der Rücktransport von STAT1 ins Cytosol vermindert. In gleicher Weise wird die Akkumulationsdauer von STAT1αWT durch LMB-Behandlung verlängert. Diese Resultate zeigen deutlich, daß STAT1 über einen CRM1-abhängigen Weg in das Cytosol exportiert wird. Die Identifizierung eines konservierten NES in dem 4-Helix-Bündel von STAT1 offenbart erstmalig die Existenz eines aktiven nukleären Exports in der Familie der STAT-Proteine. Eine reduzierte Rate des Rücktransports von STAT1 in das Cytosol der Zellen ist mit einer geringeren transkriptionellen Antwort verbunden. Diese Ergebnisse legen nahe, daß STAT-Proteine über verschiedene unabhängige Exportwege aus dem Zellkern exportiert werden und daß diese Exportwege mit der transkriptionellen Aktivität von STAT-Proteinen in einer engen Verbindung stehen. Es wurde in dieser Arbeit auch eine leuzinreiche Zielsequenz in der DNA-Bindedomäne näher charakterisiert. Dieses konservierte Signal ist von zentraler Bedeutung für den IFNγ-induzierten nukleären Import von phosphorylierten STAT1-Dimeren. Eine Mutation von zwei Leuzinresten in der NLS-Sequenz blokkiert den nukleären Import von tyrosinphosphorylierten STAT1-Proteinen vollständig. Der inhibierte nukleäre Import von phosphoryliertem STAT1 führt zu einer dramatischen Abnahme der Aktivierung IFNγ-sensitver Zielgene durch STAT1. Der Kernimport von unphosphoryliertem STAT1 wird durch Mutationen in der NLS-Sequenz nicht beeinflußt. Es wurde ebenfalls gezeigt, daß eine C-terminale Kopplung von STAT1 mit der Farnesylierungssequenz CAAX zu einer Verankerung von STAT1 mit der Plasmamembran führt. Die kovalente Bindung von STAT1 an die Plasmamembran ist mit einer vollständigen Inhibierung der IFNγ\- abhängigen Zielgenaktivierung verbunden. Die Induzierung der TNFα-vermittelten Apoptose wird durch die Verankerung von STAT1 an der Plasmamembran nicht wesentlich beeinflußt. Interessanterweise wird die STAT1-abhängige Expression der Caspasen ICE und ICH-1 und die TNFα-vermittelte Apoptose nicht durch die Mutation des NLS beeinträchtigt. Diese Ergebnisse legen nahe, daß STAT1-Dimere in den Zellkern importiert werden, damit es zu einer IFNγ\- abhängigen Zielgenaktivierung durch STAT1 kommen kann. Die nukleäre Akkumulation von STAT1 ist aber nicht für die STAT1-abhängige Aktivierung der Caspasegene und für die TNFα\- vermittelte Induktion der Apoptose notwendig. Die tyrosinphosphorylierten und unphosphorylierten STAT1-Proteine werden auf unabhängigen Wegen zwischen Cytosol und Zellkern rasch hin und her bewegt und verursachen in beiden Kompartimenten unterschiedliche Funktionen.Before stimulation signal transducers and activators of transcription (STATs) reside in the cytoplasm. STATs are phosphorylated by Janus kinases in response to cytokine stimulation and there upon rapidly translocate into the nucleus. In this thesis the IFNγ-dependent nuclear import and export of STAT1 was examined. It was found that treatment of 293T cells with the export inhibitor leptomycin B does not induce nuclear build-up of STAT1 in resting cells, but prolongs the nuclear accumulation phase in IFNγ-stimulated cells. In the course of this work a conserved leucine-rich helical segment in the coiled- coil domain of STAT1 was identified, which is responsible for the efficient nuclear export of this protein. Mutation of two leucines within this segment greatly attenuates the back transport of STAT1 into the cytoplasm. When fused to a carrier protein, the STAT1 export sequence can mediate nuclear export after intranuclear microinjection. This result indicates that STAT1 returns to the cytoplasm via a CRM1-dependent pathway. The identification of a conserved NES motif in the coiled-coil domain of STAT1 reveals for the first time the existence of an active nuclear export for a member of the STAT family of transcription factors. Notably, a reduced rate of back transport of STAT1 into the cytoplasm results in a aborted transcriptional response to stimulation with IFNγ. These data suggest that STAT proteins are actively exported from the nucleus via several separate pathways and link their activity to transcriptional activation. In addition, a nuclear targeting sequence in the DNA-binding domain of STAT1 was identified. This conserved signal is critical for the IFNγ-induced nuclear import of phosphorylated STAT1 dimers. Mutations of two leucines within this NLS-sequence inhibits nuclear entry of tyrosinephosphorylated STAT1, which in turn prevents induction of IFNγ-inducible target genes. Interestingly, the nuclear import of unphosphorylated STAT1 continues and the STAT1-dependent constitutive expression of caspases and the TNFα-mediated induction of apoptosis proceed unaltered. Thus, tyrosine-phosphorylated and unphosporylated STAT1 proteins shuttle via independent pathways to distinct sets of target genes. Furthermore, it was demonstrated that STAT1 proteins C-terminal fused with a CAAX-motif functioning as a membrane anchor are fully competent to induce TNFα-mediated apoptosis. In contrast, membrane-bound STAT1 lost the ability to activate IFNγ-dependent target genes. These results indicate that STAT1 dimers require nuclear localisation for the activation of IFNγ-dependent target genes and that nuclear accumulation is not necessary for the induction of the STAT1-dependent activation of caspase genes and TNFα\- mediated apoptosis

SUMO conjugation of STAT1 protects cells from hyperresponsiveness to IFNγ

The biologic effects of IFNγ are mediated by the transcription factor STAT1. The activity of STAT1 is inhibited by small ubiquitin-like modifier (SUMO) conjugation. This occurs both directly through decreasing STAT1 tyrosine phosphorylation and indirectly by facilitating STAT1 dephosphorylation consequential to increased STAT1 solubility because of suppressed paracrystal assembly. However, the physiologic implications of SUMO conjugation have remained unclear. Here, we used fibroblasts and bone marrow–derived macrophages (BMMs) from knockin mice expressing SUMO-free STAT1 to explore the consequences of STAT1 sumoylation for IFNγ signaling. Our experiments demonstrated buffer property of paracrystals for activated STAT1, such that SUMO-mediated paracrystal dispersal profoundly reduced phosphorylation of STAT1, which affected both the activating tyrosine 701 and the transcription-enhancing serine 727. Accordingly, the curtailed STAT1 activity in the nucleus caused by SUMO conjugation resulted in diminished transcription of IFNγ-responsive genes; and increased the IFNγ concentration more than 100-fold required to trigger lipopolysaccharide-induced cytotoxicity in bone marrow–derived macrophages. These experiments identify SUMO conjugation of STAT1 as a mechanism to permanently attenuate the IFNγ sensitivity of cells, which prevents hyperresponsiveness to this cytokine and its potentially self-destructive consequences. This sets the mode of SUMO-mediated inhibition apart from the other negative STAT regulators known to date

Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain

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

A family-wide assessment of latent STAT transcription factor interactions reveals divergent dimer repertoires

The conversion of STAT proteins from latent to active transcription factors is central to cytokine signalling. Triggered by their signal-induced tyrosine phosphorylation, it is the assembly of a range of cytokine-specific STAT homo- and heterodimers that marks a key step in the transition of hitherto latent proteins to transcription activators. In contrast, the constitutive self-assembly of latent STATs and how it relates to the functioning of activated STATs, is understood less well. To provide a more complete picture, we developed a co-localization-based assay and tested all 28 possible combinations of the seven unphosphorylated STAT (U-STAT) proteins in living cells. We identified five U-STAT homodimers ―STAT1, STAT3, STAT4, STAT5A and STAT5B― and two heterodimers ―STAT1:STAT2 and STAT5A:STAT5B― and performed semi-quantitative assessments of the forces and characterizations of binding interfaces that support them. One STAT protein ―STAT6― was found to be monomeric. This comprehensive analysis of latent STAT self-assembly lays bare considerable structural and functional diversity in the ways that link STAT dimerization before and after activation