Identification and characterization of Brd4 interacting proteins relevant to oncogenic DNA viruses and human cancers

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The Bromodomain-containing protein Brd4 has been implicated in a variety of human cancers. On the one hand, Brd4 is targeted by a number of viral proteins encoded by oncogenic DNA tumor viruses including human papillomaviruses (HPV), Epstein-Barr-virus (EBV), human herpesvirus 8 (HHV8) as well as Merkel cell polyomavirus (MCPV). For these viruses the interaction with Brd4 has been demonstrated to have roles in the maintenance of viral genomes as well as transcriptional regulation of both viral and cellular genes. On the other hand, a genomic translocation (t15;19), that results in a gene fusion of Brd4 with the NUT (nuclear protein in testis) gene leads to an aggressive form of squamous cell carcinoma called NUT-midline carcinoma (NMC). In two thirds of NMC cases the NUT gene is fused with Brd4, resulting in the expression of a Brd4-NUT fusion protein. While it can be assumed that some Brd4 functions may be retained after viral infections or fusion with NUT, it is likely that other functions might be altered, lost or even created due to the modified cellular physiological conditions. I hypothesize that the alterations in Brd4 functions found under these pathological conditions will also be reflected by a change in cellular proteins that bind to Brd4. I thus performed a proteomic approach to identify cellular Brd4 interaction partners in the presence and absence of different viral proteins by mass-spectrometry and subsequent CompPASS software analysis. My preliminary data identified several new Brd4 interaction partners. Interestingly, these proteins include proteins that only bind to Brd4 in the presence of a specific viral protein as well as proteins that lost their binding ability towards Brd4 in the presence of a viral protein. These proteins are interesting candidates for further analysis as deregulation of Brd4 interactions by viral protein expression potentially contributes to pathogenesis. I will therefore validate these newly identified interactions and study their physiological relevance during specific viral infections. Additionally, I will expand my mass-spectrometry based interaction studies and use a similar approach to identify proteins that show altered binding behavior towards Brd4 upon the fusion of Brd4 to NUT in NMC. Both studies will provide new insights into specific Brd4 functions and dysfunctions in cancer and after infection by specific oncogenic viruses.