Elucidating the mechanisms of non-canonical CDK8 function in DNA damage signaling

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PROJECT SUMMARY The Mediator of transcription is a multi-subunit protein complex that is a critical component of RNA polymerase II-mediated transcriptional machinery which regulates the transcription of essentially all genes. The core Mediator contains a head, middle and tail module, and an accessory kinase module (CKM) which can reversibly associate with the core Mediator. CKM-bound core Mediator has generally an antagonizing effect on transcription; however, it can selectively to promote the transcription of specific transcripts in certain contexts. In addition to its opposing effects on transcription, CKM influences a variety of cellular processes including stress responses, genome organization and tumorigenesis. However, it is not well-understood how CKM incorporates signals from these divergent pathways, and whether these functions of CKM are dependent on its kinase activity and its interactions with core Mediator. Through a genome wide unbiased interaction screen in yeast to identify novel pathways that contribute to the regulation of DNA damage response, an intricate signaling pathway involved in protecting the integrity of the genome, I discovered an intriguing genetic interaction with the Mediator. Through a focused mutational screen of Mediator-CKM subunits in yeast, I found that all of the four CKM subunits as well as CKM’s kinase activity are essential for cell cycle re-entry following a DNA break. Notably, the CKM mutants did not impair UV damage repair or DNA double-strand break repair through homologous recombination, indicating that CKM specifically impinges on DNA damage response. Furthermore, I demonstrated that CKM contributes to the global downregulation of transcription following DNA damage. To address the crosstalk between CKM and DNA damage signaling and uncover fundamental aspects of CKM regulation, in Aim 1, I will ask if CKM directly interacts with DNA damage factors in yeast and mammalian cells by immunostaining, live cell imaging and immunoprecipitation. I will ask whether the proteins in DNA damage response pathway are direct targets of CKM’s kinase activity by employing phosphoproteomic mass spectrometry analyses. In Aim 2, I will examine how the DNA damage response alters CKM function, focusing on its subunit composition, kinase activity and substrate scope, and genome-wide localization by using a combination of in vivo and in vitro approaches. Lastly, in Aim 3 will focus on the transcriptional aspect of CKM and DNA damage crosstalk in yeast; specifically, the DNA damage- and CKM-dependent downregulation of histone transcripts. I will ask whether this downregulation leads to global nucleosome depletion and increased chromatin mobility to promote homology searching by using internally-calibrated chromatin immunoprecipitation and live cell imaging of DNA break movement. These studies will reveal the molecular mechanisms of previously uncharacterized link between DNA damage and CKM while addressing long-standing questions on CKM modulation.