Human mediator kinase subunit CDK11 plays a negative role in viral activator VP16‐dependent transcriptional regulation

Abstract

Mediator is an essential transcriptional cofactor of RNA polymerase II (Pol II) in eukaryotes. This cofactor is a large complex containing up to 30 subunits and consisting of four modules: head, middle, tail, and CDK/Cyclin. Generally, Mediator connects transcriptional regulators, cofactors, chromatin regulators, and chromatin remodellers, with the pre‐initiation complex to provide a platform for the assembly of these factors. Many previous studies have revealed that CDK8, a subunit of the CDK/Cyclin module, is one of the key subunits mediating the pivotal roles of Mediator in transcriptional regulation. In addition to CDK8, CDK11 is conserved among vertebrates as a Mediator subunit and closely resembles CDK8. While the role of CDK8 has been studied extensively, little is known of the role of CDK11 in Mediator. We purified human CDK11 (hCDK11)‐containing protein complexes from an epitope‐tagged hCDK11‐expressing HeLa cell line and found that hCDK11 could independently form Mediator complexes devoid of human CDK8 (hCDK8). To investigate the in vivo transcriptional activity of the complex, we employed a luciferase assay. Although hCDK11 has nearly 80% amino acid sequence identity to hCDK8, siRNA‐knockdown study revealed that hCDK8 and hCDK11 possess opposing functions in viral activator VP16‐dependent transcriptional regulation.

Mediator Complex Recruits Epigenetic Regulators via Its Two Cyclin-dependent Kinase Subunits to Repress Transcription of Immune Response Genes

“…Cell Lines and siRNA-mediated CDK Knockdown-Cell lines stably expressing HA/FLAG-tagged (HF) CDK8 or CDK19 stably expressing cell lines were established previously (25). Normal HeLa S3 and CDK-expressing cells were maintained in DMEM supplemented with 5% calf serum at 37°C in 5% CO 2 .…”

“…These nuclear extracts were prepared from large cultures of previously prepared HA/FLAG-tagged CDK8 (HF-CDK8)-and CDK19 (HF-CDK19)-expressing stable cell lines previously established by our group (19,25). Each CDK-containing Mediator complex was purified using anti-FLAG M2 agarose beads (Fig.…”

“…Human CDK19 shares a high degree of amino acid sequence identity with CDK8. In previous studies, we demonstrated that CDK19 forms a CDK8-independent Mediator complex (25). Furthermore, we observed that CDK19 is expressed in a tissue-specific manner, whereas CDK8 is ubiquitously expressed (26).…”

“…DNA microarray analysis of the target genes of each CDK complex revealed extensive overlap in their target gene preferences (26). Therefore, we decided to explore the idea that Mediators play a pivotal role in transcriptional regulation (19,25).…”

The Response of Secondary Genes to Lipopolysaccharides in Macrophages Depends on Histone Deacetylase and Phosphorylation of C/EBPβ

“…In fact, the binding of TFIIB to the preinitiation complex depends on the Mediator complex (35). Given that, TFIIB was not incorporated into the preinitiation complex of the nos2 promoter after HDAC inhibition, we hypothesized that CDK8 acts as a transcriptional repressor, as described in other genes (36,37). CDK8 can regulate transcription by targeting the cdk7/cyclin H subunits of the general transcription initiation factor IIH.…”