Cyclin-dependent Kinase 8 Module Expression Profiling Reveals Requirement of Mediator Subunits 12 and 13 for Transcription of Serpent-dependent Innate Immunity Genes in Drosophila

Emilia Kuuluvainen,Heini Hakala,Essi Havula,Michelle Sahal Estimé,Mika Rämet,Ville Hietakangas,Tomi P Mäkelä

doi:10.1074/jbc.m113.541904

Abstract

The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med12 and Med13 link Cdk8 and cyclin C (CycC) to core Mediator. Here we have investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity.

Highlights

Roles of Mediator Cdk8 module subunits in transcriptional regulation are not well defined in metazoans
To compare expression changes following depletion of Cdk8 module components, Drosophila S2 cells were treated with dsRNAs targeting Cdk8, Cyclin C, Med12, or Med13
Distinct Expression Profiles by Cdk8-cyclin C (CycC) and Med12-Med13— Comparison of the expression profiles demonstrated a very significant similarity between Cdk8 and CycC profiles (Pearson’s correlation 0.86; Fig. 1B) with similar changes noted in 97 genes

Summary

Background

Roles of Mediator Cdk module subunits in transcriptional regulation are not well defined in metazoans. The Cdk (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med and Med link Cdk and cyclin C (CycC) to core Mediator. The Cdk module can act as a repressor or activator context dependently through e.g. hindering of RNA polymerase II binding with the core Mediator [2, 3], stimulation of elongation through Cdk9 [4], and interaction with the cohesin loading factor Nipped-B-like [5]. Cdk and Med sometimes cooperate on specific genes [15, 16], but in other contexts Cdk and CycC appear to act independently of Med, suggesting Mediator-independent functions [17, 18]. Med12-Med in Serpent-dependent Transcription and Immunity regulate transcription in concert or independently genomewide

EXPERIMENTAL PROCEDURES

D Increased Decreased

RESULTS

DISCUSSION