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Gene expression profiling of the Arabidopsis Mediator MED5 mutants ref4-1 and ref4-3, CDK8 mutant cdk8-1 and ref4-3 cdk8-1

ABSTRACT: Purpose: To identify changes in gene expression in ref4-3 mutant that contribute to the mutant’s altered growth and metabolite accumulation by comparing differential gene expression in ref4-3 and ref4-3 cdk8-1. Methods: Gene expression profiles were generated from 20-day-old, pooled Arabidopsis rosettes in biological triplicate, by paried-end sequencing of 100 bp fragments using Illumina HiSeq 2500. The sequence reads that passed quality filters were aligned to the TAIR10 reference genome using TopHat. Gene counts were determined using HTseq-count. Differential expression analysis was performed using edgeR. Results: We identified 7,355 genes that were differentially expressed in ref4-3 compared to wild type, part of which were significantly rescued by loss of CDK8 (FDR <0.05).

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE111290 | GEO | 2019/02/22

REPOSITORIES: GEO

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Project description:Gene regulation by cytokine-activated STAT transcription factors requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation was reported to occur upon promoter binding by an unknown kinase. Here we show that the Mediator CDK8 module phosphorylates S727 of the STAT1 TAD in the interferon (IFN) signaling pathway as well as the TADs of other STATs. Microarray analysis reveals that CDK8-mediated STAT1 TAD phosphorylation positively or negatively regulates over 40% of IFN-gamma-responsive genes, and RNA polymerase II occupancy correlates with gene expression changes. This selective regulation occurs despite CDK8 occupancy and STAT1 S727 phosphorylation at both S727 phosphorylation-dependent and -independent IFN-gamma target genes. Independently of its role as STAT1 S727 kinase CDK8 acts as a positive regulator of IFN-gamma responses. These data reveal a dual input of CDK8 in STAT1-controlled transcription and propose a key role for CDK8 in TAD phosphorylation of other STATs during cytokine responses. STAT1 WT and STAT1 S727A mouse fibroblasts were treated with siRNA to CDK8 (siCdk8 smart pool, On Target Plus, Dharmacon) and control siRNA (siCtrl) and stimulated with IFN-gamma for 4 h or left untreated. Total RNA from three independent experiments for each treatment and each genotype was isolated from cells using Trizol reagent (Invitrogen) following the manufactures protocol and used for expression analysis using Agilent Whole Mouse Genome Microarrays, 8x60K. Standard protocols for labeling and hybridization were followed. In brief, fluorescent cRNA was generated using Low Input Quick Amp Labeling Kit (Agilent). The amplified cyanine 3-labeled cRNA samples were then purified using SV Total RNA Isolation System (Promega) and hybridized to microarray slides. Microarray slides were washed and scanned with an Agilent Scanner. Note: The outlier array #10 [SA.CDK.gamma.R3] was removed from subsequent analysis and its processed data was not provided. However, its raw data file has been linked as a supplementary file at the foot of the Series record.

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Gene expression profiling of the Arabidopsis Mediator MED5 mutants ref4-1 and ref4-3, CDK8 mutant cdk8-1 and ref4-3 cdk8-1

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