ABSTRACT
Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.
Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Fibroblasts/drug effects , Interferon-gamma/pharmacology , Transcription, Genetic/drug effects , Animals , Cyclin-Dependent Kinase 8/genetics , Cyclin-Dependent Kinases/genetics , Fibroblasts/enzymology , Fibroblasts/virology , HCT116 Cells , Host-Pathogen Interactions , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , RNA Polymerase II/metabolism , STAT1 Transcription Factor/metabolism , Signal Transduction , Vesiculovirus/pathogenicityABSTRACT
Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation-associated mRNA decay remains to be established. Using time-resolved high-resolution RNA binding analysis of the mRNA-destabilizing protein tristetraprolin (TTP), an inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non-destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP-controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA-stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co-regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome-wide atlas (http://ttp-atlas.univie.ac.at) of cis-acting elements controlling mRNA decay in inflammation.
Subject(s)
Lipopolysaccharides/pharmacology , Macrophages/immunology , RNA, Messenger/chemistry , Tristetraprolin/metabolism , Animals , Binding Sites , Cells, Cultured , Gene Expression Profiling/methods , Gene Expression Regulation , HEK293 Cells , Humans , Macrophages/drug effects , Mice , RNA Stability , RNA, Messenger/metabolism , Sequence Analysis, RNAABSTRACT
The bioavailability of the major pro-inflammatory cytokines IL-1α and IL-1ß is tightly controlled by transcription and post-translational processing to prevent hyperinflammation. The role of mRNA decay in maintenance of physiological IL-1 amounts remained unknown. Here we show that the down-regulation of Il1a and Il1b mRNA by the mRNA-destabilizing protein TTP (gene Zfp36) is required for immune homeostasis. The TTP deficiency syndrome, a multi organ inflammation in TTP-/- mice, was significantly ameliorated upon deletion of the IL-1 receptor. Il1a and Il1b played non-redundant roles in triggering the pathological IL-1 signaling in TTP-/- mice. Accordingly, tissues from TTP-/- animals contained increased amounts of Il1b mRNA. Unexpectedly, TTP destabilized Il1b mRNA in cell type-specific ways as evident from RNA-Seq and mRNA stability assays. These results demonstrate that TTP-driven mRNA destabilization depends on the cellular context. Moreover, such context-defined mRNA decay is essential for keeping steady state IL-1 levels in the physiological range.