Volume 38, Issue 1, Pages 54-66 (April 2010) NER Factors Are Recruited to Active Promoters and Facilitate Chromatin Modification for Transcription in the Absence of Exogenous Genotoxic Attack  Nicolas Le May, David Mota-Fernandes, Renier Vélez-Cruz, Izarn Iltis, Denis Biard, Jean Marc Egly  Molecular Cell  Volume 38, Issue 1, Pages 54-66 (April 2010) DOI: 10.1016/j.molcel.2010.03.004 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 NER Factors Form Part of the Initiating and Elongating Transcription Machinery (A–M) Relative mRNA expression of RARβ2 gene from HeLa cells treated with t-RA (1 μM) (A). Error bars represent the standard deviation of three independent experiments. Schematic representation of the RARβ2 gene with the indicated amplicons designed at the upstream (Us), promoter (Pr), and elongation (El, exon 6) regions. ChIP monitoring the t-RA-dependent occupancy of: RAR, RXR, VDR, TIF2, and Med 6 (B, F, and J); RNA pol II, TFIIH subunits (XPD, XPB, and p44) and TFIIF (C, G, and K); XPG, XPA, RPA and XPF (D, H, and L); XPC and CSB (E, I, and M), on the different amplicons of RARβ2 gene. (N–Y) Relative mRNA expression of HMGCS2 gene from HeLa cells treated either with t-RA (N) or with WY14643 (1 μM) and transfected with the pSG5-PPARα (S). Error bars represent the standard deviation of three independent experiments. ChIP monitoring the t-RA- and WY1464 -dependent occupancy of PPAR, RXR, VDR, TIF2, and Med1 (O and T); RNA pol II and TFIIH subunits (XPD, XPB, and p44) (P and U); XPG, XPA, RPA, and XPF (Q and V); XPC and CSB (R and W), on the HMGCS2 promoter (Pr). Each series of ChIP is representative of at least two independent experiments. Values are expressed as percent input, which are the average of at least two qPCR reactions and error is within 10%. Relative RARβ2 mRNA expression (X) and ChIP monitoring the occupancy of RAR, RNA pol II, XPD, XPG, XPA, and CSB (Ya–Yf) on the RARβ2 promoter in t-RA treated HeLa cells in absence (gray histogram) or presence (green histogram) of DRB (100 μM). Molecular Cell 2010 38, 54-66DOI: (10.1016/j.molcel.2010.03.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 NER Factors Are Corecruited with RNA pol II Machinery after UV Irradiation (A–L) Relative mRNA expression of GADD45α (A), RARβ2 (E), and HMGCS2 (I) genes monitored by qPCR from UV-irradiated (20 J/m2) HeLa cells treated with t-RA (1 μM). Relative mRNA expression of RARβ2 gene measured by qPCR from t-RA-treated cells (E, dotted blue line). A dose of 20 J/m2 generates around two photolesions per 10 kb of genomic DNA (van Hoffen et al., 1995). ChIP monitoring the UV- and t-RA-dependent recruitment of p53, RAR, PPAR, and VDR (B, F, and J) as indicated; RNA pol II and TFIIH subunits (XPB, XPD, cdk7) (C, G, and K); XPG, XPA, RPA, CSB and XPC (D, H, and L), on the GADD45α (curves), RARβ2 (histograms), and HMGCS2 (curves) promoters. Each series of ChIPs is representative of two independent experiments. Values are expressed as percent input, as previously. (M–P) For ChIP/ReChIP experiments on GADD45α and RARβ2 promoters, samples were subjected to either (M and O) a first IP against XPA, XPB, or cdk7 and then purified complexes were further subjected to a second IP using antibodies against either pol II or XPA or (N and P) a first IP using an antibody against an unrelated protein (VDR) and then purified complexes were subjected to a second antibody against either XPA, RNA pol II, XPB, or cdk7 or vice versa as indicated. Molecular Cell 2010 38, 54-66DOI: (10.1016/j.molcel.2010.03.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 NER Factors-Containing Transcriptional Complex Can Be Discriminated from a DNA Repair Complex (A and B) Relative GADD45α mRNA expression (blue dotted histograms) and ChIP/ReChIP (colored histograms) monitoring the coimmunoprecipitation of the corresponding promoter using antibodies combinations against RNA pol II/ XPD, XPD/cdk7, RNA pol II/XPG, XPG/XPA, and VDR/pol II from UV-irradiated (20 J/m2) MRC5 fibroblasts in absence (A) or presence (B) of DRB (100 μM), harvested at indicated times. (C and D) Western-blotting analysis of Ab-XPB ChIP samples from chromatin extracts of MRC5 fibroblasts incubated overtime after UV irradiation, in absence (C) or pretreated with DRB (100 μM) during 6 hr (D). The WB signals for XPF, XPB, p62, cdk7, cyc H, and RPA were quantified using Genetool and plotted on the graphs. For each single lane, XPB was used as reference. HC indicates the heavy chain of the antibody. (E and F) MRC5 cells were treated or not by DRB (100 μM), as indicated (E and F), during 6 hr and UV irradiated with 70 J/m2 through a 6 mm pore filter and fixed 30 min later. Immunofluorescent labeling was performed using rabbit polyclonal anti-XPC, anti-XPB, anti-XPA, and mouse monoclonal anti-cyclobutane pyrimidine dimers (CPD) antibodies. Nuclei were counterstained with DAPI and slides were merged. Molecular Cell 2010 38, 54-66DOI: (10.1016/j.molcel.2010.03.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Sequential Recruitment of NER Factors on RARβ2 Promoter Necessary for Histones PTMs and Active DNA Demethylation (A–Y) Relative RARβ2 mRNA expression monitored by qPCR from stable SiControl, SiXPC, SiXPA, and SiERCC1 HeLa cell lines treated with t-RA (1 μM). ChIP monitoring the t-RA-dependent occupancy of RAR, RXR, VDR (B–E), RNA pol II, XPB, cdk7 (F–I), XPC, CSB, XPA (J–M), XPG, XPF, Gadd45α (N–Q) and di-/trimethylated histones H3K9, H3K4, acetylated H3K9/14, and DDB1 (R–U) on RARβ2 promoter from stable SiControl, SiXPC, SiXPA, and SiERCC1 HeLa chromatin extracts as indicated. Schematic representation of the RARβ2 promoter with the indicated CpG islands, restriction enzymes, and primers used to evaluate the methylation status (lower panel). Methylation of CpG islands of RARβ2 promoter was determined by measuring the ratio of PCR products obtained after digestion by BamHI/Hpa II, BamHI/ DpnI, or just BamHI of genomic DNA of t-RA treated stable SiControl (V), SiXPC (W), SiXPA (X), and SiERCC1 (Y) HeLa cells. Molecular Cell 2010 38, 54-66DOI: (10.1016/j.molcel.2010.03.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 RARβ2 Induction, NER Factors Recruitment, Histones PTMs, and DNA Demethylation on Active Promoter in Fibroblasts Derived from XP and CS Patients (A–E) Relative RARβ2 mRNA expression monitored by qPCR from GM14867 (XPC/R579st) (Aa), XP12RO (XPA/R207st) (Ba), and CS1AN (CSB/Q336st) (Da) fibroblasts treated with t-RA (10 μM) and compared to the corresponding rescued fibroblasts as indicated. Error bars represent the standard deviation of three independent experiments. ChIP monitoring the t-RA-dependent occupancy of RAR, VDR (Ab, Bb, Cb, Db, and Eb), RNA pol II, XPB, cdk7 (Ac, Bc, Cc, Dc, and Ec), XPC, CSB, XPA (Ad, Bd, Cd, Dd, and Ed), XPG, XPF, Gadd45α (Ae, Be, Ce, De, and Ee) and di-/trimethylated histones H3K9, H3K4 (Af, Bf, Cf, Df, and Ef) on RARβ2 promoter from GM14867, XP12RO, XP12RO rescued, CS1AN and CS1AN rescued chromatin extracts as indicated. Methylation of CpG islands of RARβ2 promoter determined by the ratio of PCR products obtained after digestion by HpaII, BamHI/ DpnI or just BamHI of genomic DNA from t-RA treated, GM14867 (Ag), XP12RO (Bg), XP12RO rescued (Cg) CS1AN (Dg), and CS1AN rescued (Eg) fibroblasts. Error bars represent the standard deviation of three independent experiments. Molecular Cell 2010 38, 54-66DOI: (10.1016/j.molcel.2010.03.004) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Sequential Recruitment of the NER Factors at the Promoter of Activated Genes Facilitates Chromatin Remodeling Upon the t-RA ligand induction, the transactivation complex is formed once RAR/RXR has targeted its responsive element; coactivators and mediator are assembled at RARβ2 promoter together with the transcriptional machinery. XPC is next recruited and allows the sequential arrival of the others NER factors in the following order CSB, XPA/RPA, XPG/XPF/Gadd45α. This association of NER factors and Gadd45α with transcription machinery leads to a cascade of histones PTMs. Concomitantly, an active demethylation of 5′CpG islands of RARβ2 gene occurs. Altogether, these remodeling chromatin events are crucial for accurate RNA synthesis. We can mention that XPG has, at least, a double action of stabilization of TFIIH and chromatin remodeling in transcription process. Molecular Cell 2010 38, 54-66DOI: (10.1016/j.molcel.2010.03.004) Copyright © 2010 Elsevier Inc. Terms and Conditions