Regulation of STAT1 and Cytokine Responses by CDK8

Cytokines are signaling polypeptides that are fundamental for activation of immune responses. Cytokines act by stimulating the JAK-STAT signaling pathway which in turn causes profound changes in gene expression. These gene expression changes are brought about by the STAT transcription factors. Although the basic principles of STAT-dependent transcription are well described, the mechanisms that determine the amplitude and magnitude of transcription of STAT target genes are poorly understood. These quantitative features, which are critically involved in health and disease, are likely to be regulated by transcriptional co-factors by means that remain to be determined. The proposed project will address the role for the CDK8 kinase in regulation of STAT-dependent transcription and cytokine responses, with major focus on STAT1 and interferon signaling. CDK8 is transcriptional co-factor known to act in chromatin-associated manner by binding and controlling the transcription activator function of the Mediator complex. Both CDK8 and the Mediator complex play an essential role in mammalian gene transcription although the precise functions are still not fully understood. The few so far reported studies suggest that CDK8 can act in the context of the Mediator complex as a molecular switch controlling the frequency of initiation or re-initiation of transcription and elongation efficiency. Kinase-dependent and independent effects have been described, which was unexpected. Surprisingly, CDK8 can increase or decrease the transcription output. The reasons for this ambiguity are not known. We recently described CDK8 to phosphorylate in chromatin-associated manner transactivation domains of STATs in response to cytokines. Furthermore, we found that CDK8 regulates STAT1 transcriptional activity also independently of TAD phosphorylation and in gene-specific ways. The proposed project will investigate mechanisms of CDK8-dependent effects on STAT1-dependent transcription in responses to IFNs. As one of the major advances, we will employ tools allowing transcriptome-wide determination of kinase-dependent and -independent CDK8 functions in transcription initiation, re-initiation and elongation as well as in chromatin recruitment of key transcription regulators. We will establish a genetically tractable mammalian cell-based system suitable for genetic screens for CDK8 effectors. The conclusions derived from the proposed studies on the model IFN-STAT1 pathway will be tested in other cytokine-induced JAK-STAT pathways. Thus, the study will generate a comprehensive knowledge about CDK8 function in cytokine responses with important implications for understanding of the pathway-specific and the recently reported oncogenic CDK8 effects.

Immune reactions are controlled by small secreted proteins, so called cytokines, such that a robust but not destructively strong immune response against infection is launched. The effects of cytokines are caused by their ability to precisely stimulate transcription (i.e., the reading of the gene sequence and mRNA synthesis) of dozens of genes that perform important tasks in the immune response. However, different genes are activated differently strong and for different periods of time. How this important gene-specific activation comes about is essentially unexplained. Transcriptional gene activation requires the interaction of specific transcription factors with RNA polymerase II (RNAPII), an enzyme that synthesizes mRNA. The communication between transcription factors and RNAPII is mediated by the so called Mediator, a complex of many different proteins, which together form a hub of transcriptional control. An important part of the mediator are the Mediator kinases and control switches CDK8 and CDK19. These kinases are very similar in sequence and have long been believed to perform similar functions as well. Accordingly, these functions should be predominantly based on the phosphorylation (i.e., modification) of other proteins by CDK8 or CDK19. However, the results of our study that was supported by the Research Fund (FWF) clearly indicate that CDK8 and CDK19 regulate different genes and that they also employ different regulatory mechanisms in transcriptional responses to the important cytokine interferon. These results imply that CDK8 and CDK19 play a central role in gene-specific transcriptional activation. The exact mode of operation of the Mediator kinases CDK8 and CDK19 will be investigated in a follow-up project. The Mediator kinases CDK8 and CDK19 can be pharmacologically regulated; however, a therapeutic use of the CDK8 or CDK19 inhibition is not possible without knowledge of the exact function of these enzymes. Our proposed study will therefore also help to define suitable applications for therapeutic inhibition of CDK8 and CDK19 in immune disorders.