Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome
- Department of Biochemistry, University of Colorado, United States
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado, United States
- BioFrontiers Institute, University of Colorado, United States
- Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, United States
- UC-Denver RNA Bioscience Initiative, United States
- Crnic Institute Boulder Branch, United States
- Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, United States
- Metabolon Inc, Durham, United States
- Department of Pharmacology, University of Colorado Anschutz Medical Campus, United States
Figures
Figure 1 with 2 supplements
Experimental overview; elevated cytokines in T21 cells.
(A) Schematic of cell treatment and data collection workflow for metabolomics, cytokine screen (green shading) and RNA-seq (purple shading). (B) Simplified diagram of human metabolic pathways, with …
-
Figure 1—source data 1
RNA-seq data summary.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig1-data1-v1.xlsx
-
Figure 1—source data 2
Metabolomics data.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig1-data2-v1.xlsx
-
Figure 1—source data 3
Cytokine screen data.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig1-data3-v1.xlsx
-
Figure 1—source data 4
Gene set enrichment analysis (GSEA) results.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig1-data4-v1.xlsx
-
Figure 1—source data 5
Ingenuity Pathway Analysis (IPA) results.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig1-data5-v1.xlsx
-
Figure 1—source data 6
GO pathway analysis involving comparisons with Human Trisome Project large cohort study.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig1-data6-v1.xlsx
Figure 1—figure supplement 1
Comparisons between T21 and D21 cells.
(A) Western blots of STAT1 S727 phosphorylation following 45-min treatment with IFNγ with or without 30-min pretreatment with cortistatin A (100 nM) or CCT251545 (250 nM), two structurally distinct …
Figure 1—figure supplement 2
Metabolic differences in T21 vs. D21; data comparison with whole blood cohort clinical studies.
(A) Relative levels of metabolites from core metabolic pathways in untreated T21 cells compared with untreated D21 cells. (B) Differential metabolites summary (ANOVA p-value <0.1) in untreated T21 …
Figure 2 with 5 supplements
Mediator kinase inhibition tempers inflammatory pathways under basal conditions, with T21-specific effects.
(A) Heatmap of genes with differential expression in D21 or T21 cells treated with cortistatin A (CA) (100 nM) compared to DMSO controls. Genes with adjusted p-value <0.01 in one or both cell lines …
-
Figure 2—source data 1
Metabolomics data sibling-matched T21 vs. D21.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig2-data1-v1.xlsx
-
Figure 2—source data 2
Metabolomics data sibling-matched T21 and D21 ±CA.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Cortistatin A (CA) suppresses T21 inflammatory pathways under basal conditions.
Volcano plots showing differentially expressed genes following CA treatment in D21 (A) or T21 (B) cells, with selected genes color-coded based on pro-inflammatory (red), anti-inflammatory (green) …
Figure 2—figure supplement 2
Persistent activation of lipid metabolism and associated transcription factors (TFs) in cortistatin A (CA)-treated T21 cells; reduced expression of RNA processing factors.
(A) Volcano plot showing differentially expressed genes following 24 hr CA treatment (vs. DMSO controls), with selected genes color-coded based on pro-inflammatory (red), anti-inflammatory (green) …
Figure 2—figure supplement 3
Cortistatin A (CA) treatment suppresses oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in T21 cells.
Normalized OCR in D21 (A) or T21 cells (B) treated with 100 nM CA or DMSO for 24 hr. Line plots were generated from biological replicates (n = 3) with error bars representing standard error of the …
Figure 2—figure supplement 4
Elevated oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in T21 vs. D21; cortistatin A (CA) treatment normalizes mitochondrial function and ECAR toward D21 levels.
(A) Bar plots showing quantitation of mitochondrial stress test parameters in vehicle-treated D21 or sibling-matched T21 cells. Lines represent standard error of the mean (n = 3). (B, C) Comparison …
Figure 2—figure supplement 5
Cortistatin A (CA) treatment increases expression of lipid biosynthesis genes and suppresses inflammation and IFN-related genes in a different T21 donor line.
(A) Volcano plot showing differentially expressed genes following 4.5 hr CA treatment (vs. DMSO controls), with selected genes color-coded based on pro-inflammatory (red), anti-inflammatory (green) …
Figure 3 with 2 supplements
Mediator kinase inhibition antagonizes IFNγ transcriptional responses in T21 and D21.
(A) Heatmap comparing gene expression patterns (RNA-seq) in IFNγ-treated D21 or T21 cells ±CA. This comparison shows cortistatin A (CA)-specific effects during IFNγ stimulation, which broadly …
Figure 3—figure supplement 1
The response to exogenous IFNγ is suppressed in T21 vs. D21 cells.
(A) Heatmap of genes with differential expression in D21 or T21 cells treated with IFNγ compared to controls. Genes with adjusted p-value <0.01 in one or both cell lines are shown. (B) Average …
Figure 3—figure supplement 2
Mediator kinase inhibition antagonizes IFNγ transcriptional responses.
Volcano plots of differentially expressed genes in IFNγ-treated cells ±CA in D21 (A) or T21 (B) cells. Names of selected genes are color-coded based on pro-inflammatory (red) or anti-inflammatory …
Figure 4 with 1 supplement
Mediator kinase inhibition reverses pro-inflammatory metabolic and cytokine changes triggered by IFNγ.
Effect of IFNγ treatment on select classes of lipid metabolites is shown (IFNγ vs. Ctrl) alongside the effect of cortistatin A (CA) treatment in IFNγ-treated cells, in D21 (A) and T21 (B) cells. The …
-
Figure 4—source data 1
Metabolomics data sibling-matched T21 and D21 ±CA ±IFNγ.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Mediator kinases transcriptionally enable cytokine responses to IFNγ.
(A) Heatmap of average relative cytokine levels in cells treated with IFNγ + CA compared to IFNγ alone. Only cytokines with relative levels of ≥1.5 (log2FC; red shading) or ≤0.75 (blue shading) in …
Figure 5 with 2 supplements
Mediator kinases regulate splicing in pathway-specific ways.
(A) Table of alternative exon usage from untreated T21 cells compared to D21. Inclusion criteria were assessed at FDR <0.05, |InclusionLevelDifference| >0.2, and ≥2 reads/replicate. (B) Sashimi …
-
Figure 5—source data 1
Splicing data (rMATS); sibling-matched T21 and D21.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig5-data1-v1.xlsx
-
Figure 5—source data 2
Splicing data (rMATS); non-sibling T21 and sibling T21 t = 24 hr.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig5-data2-v1.xlsx
-
Figure 5—source data 3
Ingenuity Pathway Analysis (IPA) results from splicing data.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig5-data3-v1.xlsx
Figure 5—figure supplement 1
Additional information about splicing changes.
(A) Table of alternative splicing events (RNA-seq data) from untreated T21 cells compared to D21. Inclusion criteria were an FDR <0.05, |InclusionLevelDifference| >0.2, and ≥2 reads/replicate. (B) …
Figure 5—figure supplement 2
Summary of splicing changes ±CA in non-sibling T21 (t = 4.5 hr) and sibling-matched T21 at 24 hr.
(A) Table of alternative exon usage from non-sibling T21 cells ±CA at t = 4.5 hr. Inclusion criteria were assessed at FDR <0.05, |InclusionLevelDifference| >0.2, and ≥2 reads/replicate. (B) Table of …
Figure 6
Mediator kinase inhibition rapidly suppresses pro-inflammatory transcription factors (TFs) in T21 cells.
(A) MA plot of Transcription Factor Enrichment Analysis (TFEA) results comparing control (DMSO) vs. cortistatin A (CA)-treated T21 cells under basal conditions, from PRO-seq experiments completed …
-
Figure 6—source data 1
Transcription Factor Enrichment Analysis (TFEA) results from PRO-seq data, T21 ±CA.
- https://cdn.elifesciences.org/articles/100197/elife-100197-fig6-data1-v1.xlsx
Figure 7
Working model for Mediator kinase-dependent regulation of IFNγ signaling.
Under conditions of IFN stimulation, Mediator kinase inhibition suppresses pro-inflammatory transcriptional responses, including expression of cytokine genes and receptors. Mechanistically, this …
Author response image 1
Information about cortistatin A.
(A) KiNativ kinome screen from HEK293 lysates. CA blocked capture of only CDK8/CDK19 in this MSbased assay, among over 200 kinases detected. (B) Equilibrium binding constants and kinetics for CA. …
