1. Volume 68, Issue 6, Pages 1054-1066.e6 (December 2017)
Cockayne’s Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation 
Alexey Epanchintsev, Federico Costanzo, Marc-Alexander Rauschendorf, Manuela Caputo, Tao Ye, Lise-Marie Donnio, Luca Proietti-de- Santis, Frederic Coin, Vincent Laugel, Jean-Marc Egly 
Molecular Cell 
Volume 68, Issue 6, Pages e6 (December 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 68, 1054-1066.e6DOI: (10.1016/j.molcel.2017.11.009)
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3. Figure 1
Gene Expression in CSA, CSB, and Rescued Cells after Genotoxic Stress
(A) Venn diagram of RNA-seq showing the number of downregulated genes upon UV irradiation in CSA- (CS3BE) and CSB- (CS1AN) deficient cells.
(B) Amount of ATF3-dependent genes in common between CSA and CSB cells that were downregulated.
(C) Pie chart showing genome-wide distribution of ATF3-binding events among the downregulated genes.
(D) seqMINER heatmaps from ChIP-seq data showing site-specific ATF3 (lanes 1–4) and Pol II (lanes 5–8) binding events within 0, 8, and 24 hr after UV in CS1AN+CSBWT (WT) and CS1AN (Mut) cell lines. Data aligned to 8 hr ATF3 peak position.
(E) RT-PCR of ATF3 in CS3BE, CS3BE+CSAWT, CS1AN, and CS1AN+CSBWT cells 4 hr after UV.
(F) RT-PCR of DHFR, CDK5RAP2, NRG1, ID1, NIPBL, DYRK1A, and RAD50 genes in CS3BE, CS3BE+CSAWT, CS3BE+siATF3, and CS3BE+siCtrl cells, 24 hr after UV.
Gene symbols are indicated at the right of each figure. Values are presented as fold expression in relation to the internal expression control GAPDH and to expression level of each gene at time point t = 0 (without UV irradiation).
(G) Western blot of extracts from CS3BE cells transiently transfected with either siCtrl or siATF3; cells were collected 24 hr post-UV.
Error bars represent SD from at least three independent experiments.
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4. Figure 2
CSA and CSB Overcome ATF3-Dependent Transcriptional Repression
(A1–D1) Western blot of ATF3 accumulation in (A1) CS3BE, (B1) CS3BE+CSAWT, (C1) CS1AN/CSBQ678E, and (D1) CS1AN/CSBQ678E+CSB WT cells over time (as indicated at the top of each panel) after UV irradiation; in the rescued cells, CSAWT and CSBWT were identified as well as α-tubulin as a control.
(A2–D2) ChIP assay determining the presence of Pol II at the DHFR core promoter, and of ATF3, CSB, or CSBQ678E at the ATF/CRE site.
(A3–D3) ChIP assay determining the presence of CSA, DDB1, and CUL4A and ChIP/reChIP assay for CSB/CSA.
(A4–D4) ChIP/reChIP assay determining the presence of ATF3/CSB and CSB/Pol II at the CRE/ATF and core promoter of DHFR.
(A5–D5) ChIP assay determining the presence of the acetylated H3K9, tri-methylated H3K4, or di-methylated H3K9 and tri-methylated H3K27 24 hr after UV. All results are presented as fold enrichment (ratio of the value obtained at each time point relative to time t = 0 in comparison with chromatin input). Each point represents the average of three RT-PCR reactions of three independent experiments.
(E and F) Immunoprecipitation assays showing interaction between (E) ATF3 and CSB and CSA proteins in CS1AN+CSBWT whole-cell extract and (F) ATF3, CSA, and CUL4A in CS3BE+CSAWT whole-cell extract.
(G and H) Reciprocal pull-down assays showing interactions between recombinant ATF3 and CSB proteins. Molecular weight is shown in kDa.
Error bars represent SD from at least three independent experiments.
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5. Figure 3 CSA and CSB Are Promoting ATF3 Ubiquitination
(A–D) ChIP/reChIP of Ubi/ATF3, Ubi/CSB, and CSB/MDM2 showing the presence of ATF3 and CSB in a ubiquitinated protein complex as well as the partnership between CSB and MDM2 at CRE/ATF site of DHFR.
(E) Scheme of the setup and feature of the experiment.
(F) Streptavidin immunoprecipitation assay from CS3BE+CSAWT, CS3BE, CS1AN+CSBWT, and CS1AN cells showing the ubiquitination of endogenous ATF3. Cells were treated or not with MG132 proteasomal inhibitor. Blot was performed against ATF3.
(G) Streptavidin immunoprecipitation assay showing kinetic of endogenous ubiquitinated ATF3 in response to UV stress in CS3BE+CSAWT and CS3BE cells. Blot was performed against ATF3 (upper panel; middle panel represents a longer blot exposure) or against ubiquitin (lower panel). Time points, 0, 8, 16, and 24 hr; MG132 at 22 hr; cells were treated 2 hr with MG132; control (Co), no pEBB-BT-ubiquitin transfection.
(H) In vitro ubiquitination of ATF3 in the presence of recombinant MDM2, CSA, and CSB as indicated at the top of the panel. Relative percentage of recombinant ATF3 ubiquitination is represented at the bottom of the panel.
Error bars represent SD from at least three independent experiments.
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6. Figure 4
CSA and CSB Promote Recruitment of the Proteasome for ATF3 Degradation
(A1–D1) ChIP showing the recruitment of PSMD1 and PSMB5 subunits of proteasome to the ATF/CRE site of DHFR.
(A2–D2) ChIP/reChIP showing the co-localization of CSB with either PSMD1 or PSMB5 at CRE/ATF site.
(E) Heatmaps of ATF3, CSA, CSB, MDM2, and PSMB5 within 0 and 8 hr after UV irradiation; data aligned to 8 hr ATF3 peak position.
(F) Site-specific peak distribution around ATF3 (intensity corresponds to the right axis) binding for CSA, CSB, and MDM2 (intensity corresponds to the left axis).
(G and H) Immunoprecipitation assay showing the interaction between (G) CSA, PSMD1, and PSMB5 in CS3BE+CSAWT cells and (H) CSB, PSMD1, and PSMB5 in CS1AN+CSBWT cells. Both (G) and (H) are the parts of the original immunoprecipitations mentioned in Figures 2F and 2E, respectively.
(I) In vitro degradation of ATF3 using whole-cell extracts from CS3BE, CS3BE + CSAWT, CS1AN, and CS1AN + CSBWT cells treated or not with UV and/or MG132 as indicated at the top of the panel. Incubation time was 6 hr. For each western blot, the resolution of the molecular weight is shown in kDa.
Error bars represent SD from at least three independent experiments.
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7. Figure 5 UBD of CSB Facilitates Proteasomal Degradation ATF3
(A) Scheme of CSB indicating the helicase-like motifs, the UBD (1,400–1,428 aa), and the Q678E mutation.
(B) Western blot of ATF3 accumulation in CS1AN+CSBΔUBD cells upon UV irradiation.
(C1–C5) ChIP of (C1) CSB/ΔUBD, Pol II, and ATF3, and (C2) DDB1, CUL4A, and CSA; ChIP/reChIP of (C2) CSB/CSA, (C3) ATF3/CSB and CSB/Pol II, (C4) Ubi/ATF3, Ubi/CSB, and CSB/MDM2; and ChIP of (C5) PSMD1 and PSMB5 proteins at CRE/ATF site and core promoter of DHFR.
(D) In vitro recombinant ATF3 degradation assay in the presence of whole-cell extracts prepared from CS1AN cells, expressing either CSBWT treated with UV and MG132 or just CSB/ΔUBD as indicated at the top of the panel. Incubation time was 0 and 6 hr. (D) and Figure 4I are parts of the same original experiment.
Error bars represent SD from at least three independent experiments.
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8. Figure 6 ATF3 Ubiquitination Is Crucial for RNA Synthesis Recovery
(A) Fluorescence-activated cell sorting (FACS) analysis showing the rate of cell death upon UV irradiation in CS1AN cells transfected with siCtrl and siATF3. We assume the total apoptosis rate by counting events in upper left and right corners of histograms.
(B) Western blot showing the levels of ATF3 in CS3BE and CS3BE KO ATF3 cells upon UV treatment as well as the efficiency of CUL4A and MDM2 protein silencing after respective siRNA treatment.
(C1) ChIP assay determining the presence of CSB and Pol II at the DHFR core promoter as well as CSB/Pol II through ChIP/reChIP assay in CS3BE/KO ATF3 +CSAWT cells.
(C2) ChIP assay determining the presence of CSA and CUL4A and ChIP/reChIP assay for CSB/CSA and CSB/MDM2 on CRE/ATF site.
(C3) ChIP assay determining the presence of PSMD1 and PSMB5 proteasomal subunits on CRE/ATF site.
(C4) ChIP/reChIP assay determining the presence of CSB/PSMD1 and CSB/PSMB5 interaction on CRE/ATF site of DHFR.
(D) Reciprocal immunoprecipitation experiment showing the ubiquitination levels of WT, m1, and m2 mutant constructs of ATF3 co-transfected together with pEBB-BT-ubiquitin plasmid into 293T cells. Streptavidin precipitation (left panel) or vice versa anti-flag immunoprecipitation (right panel) revealed with anti-ATF3 antibody or with anti-biotin antibody, detecting the biotin-ubiquitinated fraction. Control represents cells transfected only with pEBB-BT-ubiquitin construct. The pattern shows the ubiquitinated form of ATF3.
(E) FACS analysis showing the rate of cell death upon UV irradiation in CS1AN cells transfected with WT, m1, and m2 ATF3 constructs. The percentage from three independent experiments was presented in the histogram in Figure S1E.
(F) ChIP assay showing the recruitment of WT ATF3, m1, and m2 ATF3 mutants to the ATF/CRE site as well as Pol II occupation of the core promoter 24 hr after UV.
(G) Immunoprecipitation assay carried out in 293T cells showing the interaction between WT and m1 ATF3 proteins, CSB, and PSMD1 subunit of the proteasome.
(H) ChIP assay showing the recruitment of ATF3 to the ATF/CRE site as well as Pol II occupation on the core promoter upon siRNA treatment 24 hr after UV irradiation.
(I) ChIP assay showing the level of ATF3 ubiquitination on the ATF/CRE site upon siRNA treatment 24 hr after UV irradiation.
Error bars represent SD from at least three independent experiments.
Molecular Cell  , e6DOI: ( /j.molcel )
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