Chromatin2 3 4 TSAMedChemExpress TSA Z-score ChromatinBUbi. vs. Ubi. (MG132)TMPO FANCD2 LDLRAP1 RPLFANCI RPS10 PCNA RPACDTYBX1/2 DDB2 NAP1L1 RPL7 ERCCCUEDC-0 5 Z-score Ubiquitylation4 6 8 Z-score UbiquitylationUV Phosphoproteome We also recorded the UV-induced phosphoproteome (Figure 3C; Table S6A). USP32 Serine, threonine, and tyrosine phosphorylation sites were detected. Of these, 543 serines, 91 threonines, and 1 tyrosine MDC1 (MAPK9 Y185) were markedly more phosHTATSF1 phorylated in Actinomycin IV price response to UV irradiation. SMC3 As expected, damage-induced phosphorylation of H2AX (H2AFX) at serine -5 0 5 140 was detected (gH2AX). 0 2 4 6 8 10 -10 -5 0 5 10 Z-score Phosphorylation By analyzing the sequence motifs that Z-score Phosphorylation increase in phosphorylation status after UV irradiation, we found that the ATM/ UV Ubiquitylome ATR consensus motif S/T-Q was generally enriched. In total, We next used SILAC proteomics in combination with affinity we detected 396 S/TQ phosphorylation sites. Forty-five of these purification of ubiquitin remnants to identify >10,000 ubiquityla- were not described in the phosphosite plus reference database tion sites, proteome-wide. Of these, 900 were affected by (http://www.phosphosite.org) and 14 of these increased in phosDNA damage. As a positive control, and consistent with prior phorylation in response to UV irradiation. Lists of these sites can work by others (Povlsen et al., 2012; Elia et al., 2015b), we de- be found in Tables S6B and S6C. tected markedly increased levels of RPA1- (K163, K167, K331), Intriguingly, the Cohesin complex was also phosphorylated at PCNAK164-, FANCIK523-, and FANCD2K561 ubiquitylation upon several sites in a UV-induced manner. Nipped-B-like (NIPBL), an UV treatment (Figure 3B; Table S5). essential part of the Cohesin loading-complex, had a UVUbiquitylation of Cohesin subunits changed markedly upon induced ATM/ATR phosphorylation site as well. Moreover, a DNA damage: RAD21K573 became ubiquitylated, while SMC1A wide variety of other proteins, which have primarily been impliappears to become de-ubiquitylated at several sites, further rein- cated in the DNA double strand break response were also phosforcing the connection suggested by the RNAPII interactome. The phorylated after UV irradiation. These included ATRIP, BRCA1, YBX proteins, involved in both transcriptional and translational CHEK1, CHEK2, CLSPN, FANCD2, MDC1, NBN, RAD50, TIPIN, control (Matsumoto and Bay, 2005), became heavily ubiquitylated TP53BP1, and XRCC4, BCLAF1, and THRAP3.Z-socre Phos (MG132) Z-socre Phos (MG132)BCLAF1 NBN BRCA1 FOXO3 THRAP3 MARK2 RAD50 NBN TP53BP1 SMC1A-CPhos. vs. Phos. (MG132)after UV irradiation as well. A connection between YBX proteins and the DNA damage response has not previously been reported, but the fact that elevated levels of these proteins occur in a number of human malignancies and is associated with poor prognosis and disease recurrence (Kosnopfel et al., 2014), is potentially significant in this connection. Interestingly, however, the group of proteins that appeared to have the most marked increase in sitespecific ubiquitylation comprised ribosome proteins and included RPS10, RPL7, and RPL12 (Figure 3B; Table S5).Z-score Ubiq. (MG132) -5 0Z-score Ubiq. (MG132)Cell Reports 15, 1597?610, May 17, 2016A Transfection of a 21,120 poolsiRNA library into MRC5VA cells 48 hrs UV-irradiation 18 hrs Labelling of nascent RNA by 5’ethyl uridine (EU) pulse 2 hrs Covalent attachment of flurophore to EU-labelled RNA S.Chromatin2 3 4 Z-score ChromatinBUbi. vs. Ubi. (MG132)TMPO FANCD2 LDLRAP1 RPLFANCI RPS10 PCNA RPACDTYBX1/2 DDB2 NAP1L1 RPL7 ERCCCUEDC-0 5 Z-score Ubiquitylation4 6 8 Z-score UbiquitylationUV Phosphoproteome We also recorded the UV-induced phosphoproteome (Figure 3C; Table S6A). USP32 Serine, threonine, and tyrosine phosphorylation sites were detected. Of these, 543 serines, 91 threonines, and 1 tyrosine MDC1 (MAPK9 Y185) were markedly more phosHTATSF1 phorylated in response to UV irradiation. SMC3 As expected, damage-induced phosphorylation of H2AX (H2AFX) at serine -5 0 5 140 was detected (gH2AX). 0 2 4 6 8 10 -10 -5 0 5 10 Z-score Phosphorylation By analyzing the sequence motifs that Z-score Phosphorylation increase in phosphorylation status after UV irradiation, we found that the ATM/ UV Ubiquitylome ATR consensus motif S/T-Q was generally enriched. In total, We next used SILAC proteomics in combination with affinity we detected 396 S/TQ phosphorylation sites. Forty-five of these purification of ubiquitin remnants to identify >10,000 ubiquityla- were not described in the phosphosite plus reference database tion sites, proteome-wide. Of these, 900 were affected by (http://www.phosphosite.org) and 14 of these increased in phosDNA damage. As a positive control, and consistent with prior phorylation in response to UV irradiation. Lists of these sites can work by others (Povlsen et al., 2012; Elia et al., 2015b), we de- be found in Tables S6B and S6C. tected markedly increased levels of RPA1- (K163, K167, K331), Intriguingly, the Cohesin complex was also phosphorylated at PCNAK164-, FANCIK523-, and FANCD2K561 ubiquitylation upon several sites in a UV-induced manner. Nipped-B-like (NIPBL), an UV treatment (Figure 3B; Table S5). essential part of the Cohesin loading-complex, had a UVUbiquitylation of Cohesin subunits changed markedly upon induced ATM/ATR phosphorylation site as well. Moreover, a DNA damage: RAD21K573 became ubiquitylated, while SMC1A wide variety of other proteins, which have primarily been impliappears to become de-ubiquitylated at several sites, further rein- cated in the DNA double strand break response were also phosforcing the connection suggested by the RNAPII interactome. The phorylated after UV irradiation. These included ATRIP, BRCA1, YBX proteins, involved in both transcriptional and translational CHEK1, CHEK2, CLSPN, FANCD2, MDC1, NBN, RAD50, TIPIN, control (Matsumoto and Bay, 2005), became heavily ubiquitylated TP53BP1, and XRCC4, BCLAF1, and THRAP3.Z-socre Phos (MG132) Z-socre Phos (MG132)BCLAF1 NBN BRCA1 FOXO3 THRAP3 MARK2 RAD50 NBN TP53BP1 SMC1A-CPhos. vs. Phos. (MG132)after UV irradiation as well. A connection between YBX proteins and the DNA damage response has not previously been reported, but the fact that elevated levels of these proteins occur in a number of human malignancies and is associated with poor prognosis and disease recurrence (Kosnopfel et al., 2014), is potentially significant in this connection. Interestingly, however, the group of proteins that appeared to have the most marked increase in sitespecific ubiquitylation comprised ribosome proteins and included RPS10, RPL7, and RPL12 (Figure 3B; Table S5).Z-score Ubiq. (MG132) -5 0Z-score Ubiq. (MG132)Cell Reports 15, 1597?610, May 17, 2016A Transfection of a 21,120 poolsiRNA library into MRC5VA cells 48 hrs UV-irradiation 18 hrs Labelling of nascent RNA by 5’ethyl uridine (EU) pulse 2 hrs Covalent attachment of flurophore to EU-labelled RNA S.