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Assessing the impact of Ccnc or Cdk8 loss +/- ATR inhibition on mRNA gene transcription using RNA-seq

ABSTRACT: We have found that loss of Ccnc or Cdk8 (members of the RNAPII mediator complex) provide ATRi resistance. As part of invesitgations into the mechanism of this suppression, we probed for specific changes in the transcriptome by performing RNA-seq in WT, Cdk8 KO and Ccnc KO mESCs following DMSO or ATRi (AZD6738) treatment. However, this did not identify any transcripts that were significantly differentially regulated following AZD6738 treatment in both Ccnc and Cdk8 KO, but not WT, cells. Similarly, pathway analyses of proteins whose transcripts were up- or down-regulated in a treatment-independent manner in both Cdk8 and Ccnc KO clones compared to WT mESCs did not provide a clear explanation why the Ccnc/Cdk8 KO cells are ATRi resistant.

ORGANISM(S): Mus musculus

PROVIDER: GSE165359 | GEO | 2021/07/12

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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Assessing the impact of Ccnc or Cdk8 loss +/- ATR inhibition on mRNA gene transcription using RNA-seq

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