Ur screens, while PCF11 and Integrator subunits also previously scored in

Ur screens, while PCF11 and Integrator subunits also previously scored in an siRNA screen for increased damage signaling (Paulsen et al., 2009), all in all providing a solid starting point for further detailed functional analysis of their physiological role. In general, an involvement in the DNA damage response of these basal mRNA processing/termination factors might potentially help explain the dramatic downregulation of transcription occurring upon DNA damage. However, the role in the DNA damageCell Reports 15, 1597?610, May 17, 2016response of other high-scoring proteins with an often poorly understood role in transcription, such as HTATSF1, TCERG1, RPRD1A/B, RPRD2, and SND1 certainly also merits further study. Obviously, the ultimate goal of any screening approach is to uncover new factors without prior connections to the process of interest. We believe that the multiomic approach achieved this goal; numerous proteins of unknown function, including several enzymes, were uncovered that are connected to the transcription-related DNA damage response. As an example, we followed up on two proteins, ASCC3 and STK19. Our data on ASCC3 will be described elsewhere (L.W., A.S., J.S., S.B., G.P.K., M.H., M. Saponaro, P. East, R. Mitter, A. Lobley, J. Walker, and B. Spencer-Dene, unpublished data). In the case of STK19, there is some evidence that it is a kinase (GomezEscobar et al., 1998), but no cellular function had been assigned to the protein. Our data now place this extremely poorly studied protein in the DNA-damage response. Indeed, the multiomic analysis showed that STK19 interaction with CSB increases dramatically after DNA damage in the presence of MG132. Moreover, in the absence of STK19, transcription fails to recover upon UV irradiation. Crucially, our follow-up experiments showed that STK19 deficiency also results in UV sensitivity, and the protein is recruited to sites of DNA damage. STK19 was previously identified in two cancer genomics studies of genes that are frequently mutated in melanoma patients (Hodis et al., 2012; Lawrence et al., 2014), making its role in the transcription-related DNA damage response particularly exciting. Uncovering the precise role of STK19 in the DNA damage response is an important future goal. In conclusion, by investigating a complex cellular process from a number of distinct angles, the data presented here PXD101 web provides an unprecedented systems level view at the transcription-related DNA damage and at the same time uncovers numerous factors involved in it. The detailed study of the many connections revealed will be a major undertaking.EXPERIMENTAL PROCEDURESRNAi Screen The siRNA screen was performed in MRC5VA cells with the Dharmacon Human siGENOME library. Plates were exposed to short-wavelength UV (UVC) light and then incubated for a further 18 hr before labeling nascent RNA with 50 -ethynyl uridine. Automated image acquisition was performed (Cellomics Array Scan VTI) using a 103 objective. Image analysis was performed using HCS Studio (Thermo Scientific). For further details, please see the PX-478 molecular weight Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and twelve tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.04.047. AUTHOR CONTRIBUTIONS S.B. performed proteomic experiments (except for the C7ORF26 interactome, which was analyzed by O.A.). V.E. and B.S. were responsible f.Ur screens, while PCF11 and Integrator subunits also previously scored in an siRNA screen for increased damage signaling (Paulsen et al., 2009), all in all providing a solid starting point for further detailed functional analysis of their physiological role. In general, an involvement in the DNA damage response of these basal mRNA processing/termination factors might potentially help explain the dramatic downregulation of transcription occurring upon DNA damage. However, the role in the DNA damageCell Reports 15, 1597?610, May 17, 2016response of other high-scoring proteins with an often poorly understood role in transcription, such as HTATSF1, TCERG1, RPRD1A/B, RPRD2, and SND1 certainly also merits further study. Obviously, the ultimate goal of any screening approach is to uncover new factors without prior connections to the process of interest. We believe that the multiomic approach achieved this goal; numerous proteins of unknown function, including several enzymes, were uncovered that are connected to the transcription-related DNA damage response. As an example, we followed up on two proteins, ASCC3 and STK19. Our data on ASCC3 will be described elsewhere (L.W., A.S., J.S., S.B., G.P.K., M.H., M. Saponaro, P. East, R. Mitter, A. Lobley, J. Walker, and B. Spencer-Dene, unpublished data). In the case of STK19, there is some evidence that it is a kinase (GomezEscobar et al., 1998), but no cellular function had been assigned to the protein. Our data now place this extremely poorly studied protein in the DNA-damage response. Indeed, the multiomic analysis showed that STK19 interaction with CSB increases dramatically after DNA damage in the presence of MG132. Moreover, in the absence of STK19, transcription fails to recover upon UV irradiation. Crucially, our follow-up experiments showed that STK19 deficiency also results in UV sensitivity, and the protein is recruited to sites of DNA damage. STK19 was previously identified in two cancer genomics studies of genes that are frequently mutated in melanoma patients (Hodis et al., 2012; Lawrence et al., 2014), making its role in the transcription-related DNA damage response particularly exciting. Uncovering the precise role of STK19 in the DNA damage response is an important future goal. In conclusion, by investigating a complex cellular process from a number of distinct angles, the data presented here provides an unprecedented systems level view at the transcription-related DNA damage and at the same time uncovers numerous factors involved in it. The detailed study of the many connections revealed will be a major undertaking.EXPERIMENTAL PROCEDURESRNAi Screen The siRNA screen was performed in MRC5VA cells with the Dharmacon Human siGENOME library. Plates were exposed to short-wavelength UV (UVC) light and then incubated for a further 18 hr before labeling nascent RNA with 50 -ethynyl uridine. Automated image acquisition was performed (Cellomics Array Scan VTI) using a 103 objective. Image analysis was performed using HCS Studio (Thermo Scientific). For further details, please see the Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and twelve tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.04.047. AUTHOR CONTRIBUTIONS S.B. performed proteomic experiments (except for the C7ORF26 interactome, which was analyzed by O.A.). V.E. and B.S. were responsible f.