Damage (Vermeulen and Fousteri, 2013). CSB is recruited to damage-stalled RNAPII, allowing assembly of the core NER machinery around it (Fousteri et al., 2006). CSB is also required for the subsequent DNA incisions, permitting lesion removal (Anindya et al., 2010). Importantly, CSB additionally helps regulate EPZ-5676 site global RNAPII-mediated transcription. Indeed, CSB is crucial for the general recovery of transcription after DNA damage (Mayne and Lehmann, 1982), in a process that is partly independent of its role in repair (Rockx et al., 2000; Proietti-De-Santis et al., 2006). CSB contains a functionally important ubiquitin-binding domain (Anindya et al., 2010) and is itself both ubiquitylated (Groisman et al., 2003; Groisman et al., 2006) and phosphorylated (Christiansen et al., 2003), supporting the idea that post-translational modifications (PTMs) are important in the DNA damage response. Some CSB ubiquitylation is carried out by a ubiquitin ligase complex containing CSA (Groisman et al., 2006), a TC-NER factor that transfers to chromatin only after DNA damage (Kamiuchi et al., 2002). With these factors and mechanisms in mind, we set out to chart the transcription-related DNA damage response. In modern “global screens,” the characteristics of thousands of proteins or genes can be mapped concomitantly, but it is often problematic to recognize the important candidates in a list of hundreds of scoring proteins. In the hope of addressing this difficulty, we developed a multiomic approach. In this approach, distinct global screens were performed under the same conditions and the results then overlapped and integrated. Specifically, we used quantitative proteomics to determine the impactCell Reports 15, 1597?610, May 17, 2016 ?2016 The Author(s) 1597 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).UV-irradiationLocalUVSSA UVSSAGlobalRemodelerRNAPII CSB XPFRPACSA-Cul XPG ERCCRNA!TFIIHDNATranscription0 648 hrsInteractomes Upon UV, interaction with ?? RNAPII CSB ??Light HeavyPTMs UV-inducedUbiquitylation Phosphorylation Heavy Light-Chromatin Proteome Chromatin translocation upon UV-irradiationSiRNA screen Nascent transcription after UVsiRNAs UV+Light Heavyuntreated UV-treated Chromatin extraction FLAG-IP control IP Peptide Elution Mass Spectrometryuntreated UV-treated Tryptic digest Enrich K-Gly-Gly peptides, or Enrich Phos-peptides Mass Spectrometryuntreated UV-treated Chromatin extraction Mass Spectrometry Measure nascent transcriptionlownormalhighData integration, bioLOGIC Network analysis, identification of individual `hits’Figure 1. Graphical Overview of the Multiomic Approach to Charting the Transcription-Related DNA Damage ResponseUV-induced DNA damage has effects both at the local (“repairosome”) and the global level. The proteomic screens and the siRNA screen used to investigate the damage response are outlined. UV irradiation (30 J/m2) was used for all proteomic analysis, while 15 J/m2 was used in the RNAi screen.of DNA damage on (1) the RNAPII interactome, (2) the CSB interactome, (3) chromatin association dynamics, (4) the protein ubiquitylome, and (5) the phosphoproteome. This was complemented with (6) a ACY241 site functional RNAi screen. Candidates were then ranked based on their performance in the screens overall and further filtered for biological relevance and technical robustness and are searchable using a newly established database, named bioLOGIC. The multiomic approach not only conf.Damage (Vermeulen and Fousteri, 2013). CSB is recruited to damage-stalled RNAPII, allowing assembly of the core NER machinery around it (Fousteri et al., 2006). CSB is also required for the subsequent DNA incisions, permitting lesion removal (Anindya et al., 2010). Importantly, CSB additionally helps regulate global RNAPII-mediated transcription. Indeed, CSB is crucial for the general recovery of transcription after DNA damage (Mayne and Lehmann, 1982), in a process that is partly independent of its role in repair (Rockx et al., 2000; Proietti-De-Santis et al., 2006). CSB contains a functionally important ubiquitin-binding domain (Anindya et al., 2010) and is itself both ubiquitylated (Groisman et al., 2003; Groisman et al., 2006) and phosphorylated (Christiansen et al., 2003), supporting the idea that post-translational modifications (PTMs) are important in the DNA damage response. Some CSB ubiquitylation is carried out by a ubiquitin ligase complex containing CSA (Groisman et al., 2006), a TC-NER factor that transfers to chromatin only after DNA damage (Kamiuchi et al., 2002). With these factors and mechanisms in mind, we set out to chart the transcription-related DNA damage response. In modern “global screens,” the characteristics of thousands of proteins or genes can be mapped concomitantly, but it is often problematic to recognize the important candidates in a list of hundreds of scoring proteins. In the hope of addressing this difficulty, we developed a multiomic approach. In this approach, distinct global screens were performed under the same conditions and the results then overlapped and integrated. Specifically, we used quantitative proteomics to determine the impactCell Reports 15, 1597?610, May 17, 2016 ?2016 The Author(s) 1597 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).UV-irradiationLocalUVSSA UVSSAGlobalRemodelerRNAPII CSB XPFRPACSA-Cul XPG ERCCRNA!TFIIHDNATranscription0 648 hrsInteractomes Upon UV, interaction with ?? RNAPII CSB ??Light HeavyPTMs UV-inducedUbiquitylation Phosphorylation Heavy Light-Chromatin Proteome Chromatin translocation upon UV-irradiationSiRNA screen Nascent transcription after UVsiRNAs UV+Light Heavyuntreated UV-treated Chromatin extraction FLAG-IP control IP Peptide Elution Mass Spectrometryuntreated UV-treated Tryptic digest Enrich K-Gly-Gly peptides, or Enrich Phos-peptides Mass Spectrometryuntreated UV-treated Chromatin extraction Mass Spectrometry Measure nascent transcriptionlownormalhighData integration, bioLOGIC Network analysis, identification of individual `hits’Figure 1. Graphical Overview of the Multiomic Approach to Charting the Transcription-Related DNA Damage ResponseUV-induced DNA damage has effects both at the local (“repairosome”) and the global level. The proteomic screens and the siRNA screen used to investigate the damage response are outlined. UV irradiation (30 J/m2) was used for all proteomic analysis, while 15 J/m2 was used in the RNAi screen.of DNA damage on (1) the RNAPII interactome, (2) the CSB interactome, (3) chromatin association dynamics, (4) the protein ubiquitylome, and (5) the phosphoproteome. This was complemented with (6) a functional RNAi screen. Candidates were then ranked based on their performance in the screens overall and further filtered for biological relevance and technical robustness and are searchable using a newly established database, named bioLOGIC. The multiomic approach not only conf.
