This collaborative project seeks to gain a deep mechanistic understanding of the Mediator kinases (CDK8 and CDK19) and their roles in driving hyper-active interferon (IFN) signaling in Down syndrome (DS). A chronic inflammatory state underlies the DS condition and contributes to its co-morbidities; consequently, a means to dampen IFN activation could have broad therapeutic benefits. CDK8 and CDK19 each represent druggable targets and, as potent activators of IFN responses, inhibition of their function could effectively antagonize runaway IFN signaling in DS. CDK8 and its paralog, CDK19, are considered ?Mediator kinases? because of their stable, but reversible, association with the 26-subunit Mediator complex. Mediator is a global regulator of RNA polymerase II (pol II) activity, which transcribes all protein-coding and many non-coding RNAs in the human genome. CDK8 and CDK19 have each been shown to help drive transcriptional responses to IFN cytokines, which are universal regulators of inflammation and immune function. Here, we seek to define their essential? yet poorly understood?cellular and mechanistic roles in the context of DS (Biobank-matched D21 and T21 cells) and IFN signaling. We will combine sophisticated cell-based methods (e.g. transcriptomics and metabolomics) with detailed, cutting-edge biochemical approaches to allow rigorous and reliable assessment of the mechanisms by which CDK8 vs. CDK19 control IFN responses in DS individuals.
In Aim 1, we will probe the role of Mediator kinase activity, whereas in Aim 2 we will focus on the CDK19 protein, which serves key structural/scaffolding roles to enable robust transcriptional responses upon IFN stimulation. Transcriptomic (RNA-seq and PRO-seq) and metabolomics approaches will be applied in Aims 1 and 2, to assess T21-specific signatures (including potential splicing defects) and to identify how CDK8 and/or CDK19 impact cellular responses to IFN. These experiments will also uncover how sequence-specific, DNA-binding transcription factors (TFs) are differentially mobilized in T21 vs. D21 cells, including during IFN stimulation. As part of this, we will establish how CDK8 and CDK19 independently control TF activation or repression, through kinase-dependent or -independent mechanisms.
In Aim 3, we will perform detailed in vitro studies to define the molecular mechanisms by which Mediator kinases regulate pol II transcription initiation, promoter-proximal pausing, or elongation. Mechanistic findings will then be probed further in cells, to better characterize their significance in DS and related IFN signaling cascades. Collectively, these experiments (Aims 1-3) may help establish Mediator kinases (CDK8 & CDK19) as viable therapeutic targets to antagonize hyper-active IFN signaling in DS. Moreover, the balance of targeted/mechanistic studies with discovery-based approaches may yield new and unanticipated therapeutic strategies with relevance for DS and its associated co-morbidities, which also affect the general population.

Public Health Relevance

Individuals with Down syndrome experience chronic inflammation because of hyper-sensitivity to molecules called interferons, which activate immune responses to infections. In this study, we seek to apply diverse and complementary approaches to study two human enzymes that have recently been shown to drive these interferon responses. We anticipate that the mechanistic insights gained will establish the clinical potential of these druggable enzymes as antagonists of inflammatory interferons, which may yield new therapeutic strategies for DS and its co-morbidities, such as auto-immune disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI156739-01
Application #
10114912
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Singleton, Kentner L
Project Start
2020-09-22
Project End
2025-08-31
Budget Start
2020-09-22
Budget End
2021-08-31
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80303