Molecular Cell, Volume 51 Supplemental Information Enhanced Chromatin Dynamics by FACT Promotes Transcriptional Restart after UV-Induced DNA Damage Christoffel Dinant, Giannis Ampatziadis-Michailidis, Hannes Lans, Maria Tresini, Anna Lagarou, Malgorzata Grosbart, Arjan Theil, Wiggert A. van Cappellen, Hiroshi Kimura, Jiri Bartek, Maria Fousteri, Adriaan B. Houtsmuller, Wim Vermeulen, and Jurgen A. Marteijn Inventory of Supplemental Information: Figure S1, related to Figure 1 Immunofluorescence images of different core histones and different histone exchange assay. Figure S2, related to Figure 2 Effect of NAP1L1 on histone exchange Efficiencies of sirna knockdown Effect of PARP and CK2 inhibitors on histone exchange Figure S3, related to Figure 3 Control blot of SSRP1-GFP and SPT16-GFP Immunofluorescence of SPT16 at UV damage in time Figure S4, related to Figure 4 Function of SPT16 in transcriptional restart and presence near TC-NER complexes Supplemental References
2
Figure S1, related to Figure 1 A. HeLa cells stably expressing the indicated GFP-tagged histones were locally UV irradiated with 100 J/m 2 by irradiation through a micro-porous filter. The local UV-irradiated area was visualized by anti-cpd immunofluorescent counterstaining. The general distribution and steady-state level of histones was not significantly affected at LUD. B. Local UV-C laser (266nm) DNA damage (LUD) infliction (arrow) in HeLa cells stably expressing H2A-GFP, showing that also with this procedure the total amount of core histones is not changed at LUD. C. HeLa cells were subjected to local UV-C laser (266nm) DNA damage infliction (arrow) 6-8 hrs after transient transfection of H2A-GFP. At this timepoint the expression of the transfected H2A-GFP starts, enabling us to follow de novo histone incorporation in the chromatin in time. D. HeLa cells expressing H2A-GFP (green) and HeLa cells expressing H2B-RFP (red) were PEG-mediated fused and local UV-irradiated 30 min after fusion. Images were taken 1 hr after local UV-C laser irradiation (arrow). E. Enhanced H2A-GFP exchange at LUD in S-phase and non S-phase cells, identified by the absence of the typical S- phase-specific PCNA foci using expression of PCNA-Cherry. PCNA also localizes to LUD. 3
4
Figure S2, related to Figure 2 A. H2A-GFP exchange at LUD in HeLa cells transfected with control sirna or sirna targeting NAP1L1 B. NAP1L1 expression levels in cells transfected with non-targeting control sirna (sictrl) or sirna directed against NAP1L1 (right lane, sinap1l1). A nonspecific cross-reacting band is used as loading control. C. Western blots for the knockdown efficiencies of sissrp1, sispt16 and sifact (both sissrp1 and sispt16 simultaneously). Tubulin is used as loading control. Graphs show quantification of SSRP1 (left) and SPT16 (right) proteins levels normalized to tubulin levels. D. Quantification of histone exchange in unperturbed or locally UV-C exposed regions of the bleached part of the nucleus transfected with the indicated sirna s. Fluorescence recovery directly after LUD is plotted against time in sec. for H2A-GFP, (n > 8 cells, mean ± s.e.m.). E. H2A-GFP exchange at LUD in HeLa cells, in the presence of PARP or CK2 inhibitors (1hr). Top panel images of half nucleus bleached immediately captured after local UV-C damage infliction, lower panel 15 minutes after UV damage. F. Quantification of histone exchange in unperturbed or locally UV-C exposed regions of the bleached part of the nucleus untreated or pre-incubated for 1 hour with the indicated inhibitors (n > 8 cells, mean ± s.e.m.). G. The activity of PARP inhibitor (AZD2281, 10 µm) is shown by the decrease of H 2 0 2 -induced (5 min treatment with 500 µm) PAR staining using immunofluorescence (Eltze et al., 2008). H. The efficacy of CK2 inhibition by 10 µm DMAT is illustrated by the strong redistribution of the CK2 substrate NPM/B23 (Louvet et al., 2006; Negi and Olson, 2006). 5
6
Figure S3, related to Figure 3 A. Comparison of expression levels of SSRP1-GFP, SPT16-GFP and their endogenous counterparts in U20S cells. B. Immuno-fluorescence, showing SPT16 co-localization at LUD as indicated by CPD staining 2, 6 and 24 hrs after local UV-C damage. C. Accelerated H2A- GFP exchange at LUD occurs in the absence of SSRP1 but not of SPT16. D. Immunofluorescence, showing SPT16 co-localization at LUD as indicated by CPD staining after transfection with control sirna or sirna targeting SSRP1. E. Quantification of the SPT16- GFP or SSRP1-GFP accumulation kinetics at LUD after the indicated sirna mediated knockdown. (n > 12 cells, mean ± s.e.m.). 7
8
Figure S4, related to Figure 4 A. Recruitment kinetics at LUD of GFP tagged version of the early NER factors DDB2, XPC and CSB upon RNAi-mediated knockdown of FACT is normal. (n > 10 cells, mean ± s.e.m.) B. Mock and UV-irradiated (1 hr. after 20 J/m 2 ) VH10 and CS1AN (CS-B) cells (both sv40 immortalized), were subject to SPT16 ChIP and stained for XPA. C. Mock and UV-irradiated (1 hr after 20 J/m 2 ) CS1AN (CS-B) and XP21RO (XP-C) cells (both htert immortalised) were in vivo crosslinked and ChIP-ed with p89 (XPB) antibodies for ChIP-on-western analysis. XPB interacts with RNApolII and TC-NER factors CSA, CSB, as well as SPT16 in response to UV-damage only in TC-NER proficient cells. C and D: longer exposures of the blots of the indicated proteins are depicted in the lower panel. D. Mock and UV-irradiated (1 hr after 20 J/m 2 ) CS1AN (CS-B) cells (htert immortalised) were in vivo crosslinked and ChIP-ed with antibodies against the late elongating form of RNAPII. An increased association between SPT16 and RNApolII is observed in CS-B cells. For figures B, C and D: samples were normalized to equal levels of the ChIP-ed protein. Immunoblot analysis of the coimmunoprecipitated proteins was performed with antibodies as indicated. E. Transcription levels as determined by 2-hour pulse-labelling with EU in U2OS cells. After fixation, EU is fluorescently labelled with Alexa594. Transcription is inhibited upon treatment with 25 μg/ml α-amanitin or 100 μm DRB. F. FRAP graph in which the relative fluorescence recovery after bleaching is plotted against time (s) of SPT16-GFP expressing cells untreated or treated with 25 μg/ml α-amanitin for the indicated times. (n > 15 cells, mean ± s.e.m.). G. FRAP graph in which the relative fluorescence recovery after bleaching is plotted against time (s) of SPT16- GFP expressing cells untreated or treated 100 μm DRB for 1 hour. (n > 12 cells, mean ± s.e.m.). 9
Supplemental References Eltze, T., Boer, R., Wagner, T., Weinbrenner, S., McDonald, M.C., Thiemermann, C., Burkle, A., and Klein, T. (2008). Imidazoquinolinone, imidazopyridine, and isoquinolindione derivatives as novel and potent inhibitors of the poly(adp-ribose) polymerase (PARP): a comparison with standard PARP inhibitors. Mol Pharmacol 74, 1587-1598. Louvet, E., Junera, H.R., Berthuy, I., and Hernandez-Verdun, D. (2006). Compartmentation of the nucleolar processing proteins in the granular component is a CK2-driven process. Mol Biol Cell 17, 2537-2546. Negi, S.S., and Olson, M.O. (2006). Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23. J Cell Sci 119, 3676-3685. 10