SummaryGene regulation by cytokine-activated transcription factors of the signal transducer and activator of transcription (STAT) family requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation occurs upon promoter binding by an unknown kinase. Here, we show that the cyclin-dependent kinase 8 (CDK8) module of the Mediator complex phosphorylated regulatory sites within the TADs of STAT1, STAT3, and STAT5, including S727 within the STAT1 TAD in the interferon (IFN) signaling pathway. We also observed a CDK8 requirement for IFN-γ-inducible antiviral responses. Microarray analyses revealed that CDK8-mediated STAT1 phosphorylation positively or negatively regulated over 40% of IFN-γ-responsive genes, and RNA polymerase II occupancy correlated with gene expression changes. This divergent regulation occurred despite similar CDK8 occupancy at both S727 phosphorylation-dependent and -independent genes. These data identify CDK8 as a key regulator of STAT1 and antiviral responses and suggest a general role for CDK8 in STAT-mediated transcription. As such, CDK8 represents a promising target for therapeutic manipulation of cytokine responses.
Highlights d Mediator kinases CDK8 and CDK19 are distinct transcription regulators in the IFN-g pathway d CDK8 acts as a kinase while CDK19 functions as a scaffold in driving IFN-g gene profiles d CDK8 and CDK19 activate distinct gene sets d Mediator kinase CDK8 promotes polymerase II pause release during the IFN-g response
Interferons regulate immunity by inducing DNA binding of the transcription factor STAT1 through Y701 phosphorylation. Transcription by STAT1 needs to be restricted to minimize the adverse effects of prolonged immune responses. It remains unclear how STAT1 inactivation is regulated such that the transcription output is adequate. Here we show that efficient STAT1 inactivation in macrophages is coupled with processive transcription. Ongoing transcription feeds back to reduce the promoter occupancy of STAT1 and, consequently, the transcriptional output. Once released from the promoter, STAT1 is ultimately inactivated by Y701 dephosphorylation. We observe similar regulation for STAT2 and STAT3, suggesting a conserved inactivation mechanism among STATs. These findings reveal that STAT1 promoter occupancy in macrophages is regulated such that it decreases only after initiation of the transcription cycle. This feedback control ensures the fidelity of cytokine responses and provides options for pharmacological intervention. Cytokines perform their functions as key regulators of immune responses through activation of the JAK-STAT signaling pathway (1). Inadequate (either low or exacerbated) cytokine signaling may result in diseases such as immunodeficiency, autoimmunity, or cancer (2, 3). The strength of cytokine responses is regulated by various positive and negative feedback mechanisms that act at all steps of the signaling pathway: the cytokine receptors, JAKs, and STAT transcription factors. Yet it remains unclear how the process of cytokine-induced transcription is controlled once the transcription machinery has been turned on by activated STAT.STAT1 is indispensable for the biological function of interferons (IFNs), which are crucial cytokines for antiviral and antibacterial immunity. STAT1 nuclear translocation and DNA binding are activated by JAK-mediated phosphorylation of Y701. Other modifications that tune STAT1 function in IFN signaling include CDK8-mediated S727 phosphorylation, IB kinase ε (IKKε)-mediated S708 phosphorylation, and K703 sumoylation (4-6). Y701-phosphorylated STAT1 binds to target gene promoters in two major forms: (i) STAT1 homodimers induced by type I, II, and III IFNs bind to gamma interferon-activated sequence (GAS) elements, and (ii) the trimeric interferon-stimulated gene factor 3 (ISGF3; composed of STAT1, STAT2, and IRF9) induced by type I and III IFNs binds to interferon-stimulated response elements (ISRE) (1, 7). The principal mechanism of STAT1 inactivation is Y701 dephosphorylation, which causes both STAT1 homodimers and ISGF3 to lose their DNA-binding activity and to relocate to the cytoplasm. The nuclear T-cell protein tyrosine phosphatase (TC-PTP) is the major Y701-directed phosphatase (8). STAT1 acetylation was reported to facilitate dephosphorylation by TC-PTP (9), but this issue has been controversial (10). The access of phosphatase to phosphorylated Y701 appears to be restricted, since DNA-bound STAT1 is protected from Y701 dephosphorylation (11,12). For type II IFN (IFN-␥) ...
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