Rabbit Anti-Cdk8 monoclonal antibody, clone TO3141

The Mediator complex (Mediator) plays pivotal roles in activating transcription by RNA polymerase II, but relatively little is known about its roles in repression. Here, we identified the histone arginine methyltransferase PRMT5 and WD repeat protein 77/methylosome protein 50 (WDR77/MEP50) as Mediator cyclin-dependent kinase (CDK)-interacting proteins and studied the roles of PRMT5 in the transcriptional regulation of CCAAT enhancer-binding protein (C/EBP) beta target genes. First, we purified CDK8- and CDK19-containing complexes from HeLa nuclear extracts and subjected these purified complexes to mass spectrometric analyses. These experiments revealed that two Mediator CDKs, CDK8 and CDK19, individually interact with PRMT5 and WDR77, and their interactions with PRMT5 cause transcriptional repression of C/EBP beta target genes by regulating symmetric dimethylation of histone H4 arginine 3 (H4R3me2s) in the promoter regions of those genes. Furthermore, the recruitment of the DNA methyltransferase DNMT3A correlated with H4R3 dimethylation potentially leading to DNA methylation at the promoter proximal region and tight inhibition of preinitiation complex formation. In vertebrates, C/EBP beta regulates many genes involved in immune responses and cell differentiation. These findings shed light on the molecular mechanisms of the repressive roles of Mediator CDKs in transcription of C/EBP beta target genes and might provide clues that enable future studies of the functional associations between Mediators and epigenetic regulation.

Background: Varicella-zoster virus (VZV) is the causative agent of varicella and zoster. The immediate-early protein, IE62 is the predominant VZ virion tegument protein, transactivating the expression of all kinetic classes of VZV genes. IE62 is localized to punctae that form DNA replication compartments in the nuclei of VZV infected cells. The morphological changes and the increase in the size of replication compartments that express IE62 are correlated with production of VZ virions. Mammalian Mediator serves as a coactivator of IE62 and functions by bridging DNA-binding transcription factors, RNA polymerase II (RNAP II) and their target DNAs for VZV replication. While VZV is highly sensitive to type I interferons (IFNs), how IFN-alpha inhibits early events during VZV replication is poorly understood. Results: In this study, we performed in situ analysis to investigate the effects of IFN-alpha on the dynamic interactions of IE62 with the Mediator MED25 subunit and the RNAP II negative regulator cycle-dependent kinase 8 (CDK8) in VZV infected cells by confocal immunofluorescence. We found that in addition to dose-dependent inhibition of the yields of infectious virus by IFN treatment, IFN-alpha prominently impeded the development of large IE62(+) nuclear compartments and significantly decreased transcription of VZV genes. Both the expression level and stable recruitment of MED25 to IE62(+) replication compartments were inhibited by IFN-alpha. While IFN-alpha treatment upregulated CDK8 expression, redistribution and recruitment of CDK8 to IE62(+) replication compartments in infected cells was not affected by VZV. Conclusion: IFN-alpha exerts multiple inhibitory activities against virus infections. In this study, we provide visionary demonstration that continuous translocation of MED25 into VZV replication compartments ensures production of virions. IFN-alpha greatly impedes the formation of a stable complex between IE62 and the Mediator complex thereby suppresses VZV gene transcription. Our demonstration that IFN-alpha-induced antiviral effect against VZV infection is through inhibiting the reorganization of nuclear components uncovers a novel function of IFN-alpha. Targeting the interaction between IE62 and MED25 may offer a novel approach to the development of antiviral agents against VZV infection.