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Multiomic Analysis of the UV-Induced DNA Damage Response

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Multiomic Analysis of the UV-Induced DNA Damage Response Resource Multiomic Analysis of the UV-Induced DNA Damage Response Graphical Abstract Authors Stefan Boeing, Laura Williamson, Vesela Encheva, ..., Michael Howell, Ambrosius P. Snijders, Jesper Q. Svejstrup Correspondence [email protected] In Brief Boeing et al. investigate the UV-induced DNA damage response by combining a range of proteomic and genomic screens. A function in this response for the melanoma driver STK19 as well as a number of other factors are uncovered. Highlights d A multiomic screening approach examines the UV-induced DNA damage response d Multiple factors are connected to the transcription-related DNA damage response d Melanoma gene STK19 is required for a normal DNA damage response Boeing et al., 2016, Cell Reports 15, 1597–1610 May 17, 2016 ª 2016 The Author(s) http://dx.doi.org/10.1016/j.celrep.2016.04.047 Cell Reports Resource Multiomic Analysis of the UV-Induced DNA Damage Response Stefan Boeing,1,5 Laura Williamson,1 Vesela Encheva,2 Ilaria Gori,3 Rebecca E. Saunders,3 Rachael Instrell,3 Ozan Aygun,€ 1,7 Marta Rodriguez-Martinez,1 Juston C. Weems,4 Gavin P. Kelly,5 Joan W. Conaway,4,6 Ronald C. Conaway,4,6 Aengus Stewart,5 Michael Howell,3 Ambrosius P. Snijders,2 and Jesper Q. Svejstrup1,* 1Mechanisms of Transcription Laboratory, the Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK 2Protein Analysis and Proteomics Laboratory, the Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK 3High Throughput Screening Laboratory, the Francis Crick Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK 4Stowers Institute for Medical Research, Kansas City, MO 64110, USA 5Bioinformatics and Biostatistics Laboratory, the Francis Crick Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK 6Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA 7Present address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2016.04.047 SUMMARY sures that RNAPII is ubiquitylated and degraded by the protea- some, enabling repair by other mechanisms (Wilson et al., 2013). In order to facilitate the identification of factors and Importantly, bulky DNA lesions not only block RNAPII prog- pathways in the cellular response to UV-induced ress, but also affect transcription genome-wide so that even DNA damage, several descriptive proteomic screens un-damaged genes temporarily cease to be expressed (Mayne and a functional genomics screen were performed in and Lehmann, 1982; Rockx et al., 2000; Proietti-De-Santis parallel. Numerous factors could be identified with et al., 2006). The mechanisms and factors that underlie TC- high confidence when the screen results were super- NER and the more general DNA-damage-induced repression of gene expression are still poorly understood. imposed and interpreted together, incorporating bio- Cockayne syndrome B protein (CSB, also named ERCC6) logical knowledge. A searchable database, bioLOGIC, plays a key role in both TC-NER and the global transcription which provides access to relevant information about a response to DNA damage (Vermeulen and Fousteri, 2013). protein or process of interest, was established to host CSB is recruited to damage-stalled RNAPII, allowing assembly the results and facilitate data mining. Besides uncov- of the core NER machinery around it (Fousteri et al., 2006). ering roles in the DNA damage response for numerous CSB is also required for the subsequent DNA incisions, permit- proteins and complexes, including Integrator, Cohe- ting lesion removal (Anindya et al., 2010). Importantly, CSB addi- sin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we tionally helps regulate global RNAPII-mediated transcription. uncovered a role for the poorly studied, melanoma- Indeed, CSB is crucial for the general recovery of transcription associated serine/threonine kinase 19 (STK19). Be- after DNA damage (Mayne and Lehmann, 1982), in a process sides effectively uncovering relevant factors, the that is partly independent of its role in repair (Rockx et al., 2000; Proietti-De-Santis et al., 2006). CSB contains a function- multiomic approach also provides a systems-wide ally important ubiquitin-binding domain (Anindya et al., 2010) overview of the diverse cellular processes connected and is itself both ubiquitylated (Groisman et al., 2003; Groisman to the transcription-related DNA damage response. et al., 2006) and phosphorylated (Christiansen et al., 2003), sup- porting the idea that post-translational modifications (PTMs) are important in the DNA damage response. Some CSB ubiquityla- INTRODUCTION tion is carried out by a ubiquitin ligase complex containing CSA (Groisman et al., 2006), a TC-NER factor that transfers to The cellular response to bulky DNA lesions, such as those chromatin only after DNA damage (Kamiuchi et al., 2002). induced by UV irradiation is multi-faceted. The effect of such With these factors and mechanisms in mind, we set out to damage on transcription is particularly complex. Bulky DNA le- chart the transcription-related DNA damage response. In mod- sions in the transcribed strand cause stalling of RNA polymerase ern ‘‘global screens,’’ the characteristics of thousands of pro- II (RNAPII), resulting in a block to transcript elongation. Damage- teins or genes can be mapped concomitantly, but it is often stalled RNAPII then functions as a molecular beacon that triggers problematic to recognize the important candidates in a list of transcription-coupled nucleotide excision repair (TC-NER), the hundreds of scoring proteins. In the hope of addressing this dif- process whereby DNA damage in the transcribed strand of ficulty, we developed a multiomic approach. In this approach, active genes is preferentially removed (Gaillard and Aguilera, distinct global screens were performed under the same condi- 2013). On the other hand, if the DNA lesion for some reason tions and the results then overlapped and integrated. Specif- cannot be removed by TC-NER, a mechanism of last resort en- ically, we used quantitative proteomics to determine the impact Cell Reports 15, 1597–1610, May 17, 2016 ª 2016 The Author(s) 1597 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). UV-irradiation Local Global Remodeler UVSSAUVSSA CSB RNAPII XPF-RPACSA-Cul XPG ERCC1 DNA RNA ! TFIIH Transcription 0 6 12 24 48 hrs Interactomes PTMs Chromatin Proteome SiRNA screen Upon UV, interaction with UV-induced Chromatin translocation Nascent transcription Ubiquitylation ?? upon UV-irradiation ?? RNAPII CSB Phosphorylation - + after UV Light Heavy Light Heavy UV siRNAs untreated UV-treated untreated UV-treated Light Heavy Chromatin extraction Tryptic digest untreated UV-treated Measure nascent FLAG-IP & control IP Enrich K-Gly-Gly peptides, transcription or Chromatin extraction Peptide Elution Enrich Phos-peptides Mass Spectrometry Mass Spectrometry Mass Spectrometry low normal high Data integration, bioLOGIC Network analysis, identification of individual ‘hits’ Figure 1. Graphical Overview of the Multiomic Approach to Charting the Transcription-Related DNA Damage Response UV-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 inter- tracted from HEK293 cells at 3 hr after UV-induced DNA dam- actome, (3) chromatin association dynamics, (4) the protein age. We hoped this would uncover factors across the whole ubiquitylome, and (5) the phosphoproteome. This was comple- DNA damage response, from early events, such as DNA damage mented with (6) a functional RNAi screen. Candidates were signaling and gene expression shutdown, to late events, such as then ranked based on their performance in the screens overall post-incision repair factors and transcription-recovery proteins and further filtered for biological relevance and technical robust- (see Figure 1). The proteomic screens were performed under ness and are searchable using a newly established database, identical conditions and all made use of quantitative stable named bioLOGIC. The multiomic approach not only confirmed isotope labeling by amino acids in cell culture (SILAC) prote- the involvement of well-known TC-NER factors, but also uncov- omics (Ong et al., 2002), enabling us to distinguish between ered numerous new factors and cellular pathways that have not ‘‘constitutive’’ and UV-induced interactors and modifications. previously been connected to the transcription-related DNA Moreover, proteasome inhibition has previously been shown to damage response. One example is the poorly studied melanoma prevent dissociation of certain DNA-damage-induced protein in- gene STK19. teractions (Groisman et al., 2006). We therefore also carried out all proteomic experiments in the presence of proteasome inhib- RESULTS itor MG132. To uncover factors with a role in the transcription-related DNA CSB Interactome damage response, we carried out a combination of proteomic CSB is the central transcription-repair coupling factor and is spe- and genomic screens. The UV-induced DNA damage response cifically recruited to damage-stalled RNAPII. The UV-induced has typically been studied at early time-points (30 min to 1 hr af- CSB
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