Volume 55, Issue 1, Pages 123-137 (July 2014) NCOA4 Transcriptional Coactivator Inhibits Activation of DNA Replication Origins  Roberto Bellelli, Maria Domenica Castellone, Teresa Guida, Roberto Limongello, Nina Alayne Dathan, Francesco Merolla, Anna Maria Cirafici, Andrea Affuso, Hisao Masai, Vincenzo Costanzo, Domenico Grieco, Alfredo Fusco, Massimo Santoro, Francesca Carlomagno  Molecular Cell  Volume 55, Issue 1, Pages 123-137 (July 2014) DOI: 10.1016/j.molcel.2014.04.031 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 NCOA4 Interacts with the MCM2–7 Complex (A and B) HeLa cell protein extracts, total (A) or fractionated (B), were subjected to immunoprecipitation-western blotting with the indicated antibodies. Lysis buffer alone (−lysate) was immunoprecipitated as a control. (C) HeLa cells released from nocodazole arrest were harvested at the indicated time points; chromatin-bound, soluble, or total protein extracts were immunoblotted with the indicated antibodies. (D) Chromatin immunoprecipitation of human Lamin B2 (LB2) (upper) and c-Myc (lower) DNA replication origins, performed using the indicated antibodies. Upper panel: map of LB2 and Ppv1 genes depicting specific exons (closed boxes) and position of PCR fragments. Lower panel: map of Myc gene locus indicating specific exons (closed boxes), alternative promoters and transcription starts (P0, P1, P2, and P3), and position of PCR fragments. Ethidium bromide staining of PCR products is shown (see also Figure S1). Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Immunodepletion of XNCOA4 from Xenopus laevis Egg Extracts Increases DNA Replication (A and B) Xenopus laevis egg extracts were subjected to immunoprecipitation-western blotting with the indicated antibodies (see also Figure S2B). The antibody alone (no extract) served as control. (C) Chromatin-bound XNCOA4, MCM7, and PCNA proteins analyzed by western blotting at the indicated time points. Histone H3 western blotting was used as a loading control. (D) Upper: western blot of Xenopus laevis egg extracts that had been mock (preimmune serum) or XNCOA4 (antibody against XNCOA4) immunodepleted. Antibody against XNCOA4 alone (no extract) was used as control. (E) Mock-depleted, XNCOA4-immunodepleted, or XNCOA4-immunodepleted extracts supplemented with 30 nM NUS-NCOA4 recombinant protein extracts were harvested at the indicated time points, and DNA replication was measured by Cerenkov counting. Percentage of replicated sperm DNA was calculated according to Blow and Laskey (1986) (average results ± SD) (see also Figure S2). Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Immunodepletion of XNCOA4 Increases the Number of Active Replication Forks in Xenopus laevis Egg Extracts (A) Chromatin was incubated in mock- or XNCOA4-depleted extracts containing 15 μM aphidicolin and transferred to fresh extract supplemented with 0.5 mM roscovitine after 45 min. Samples were harvested at the indicated time points, and DNA replication was monitored by Cerenkov counting (average results ± SD). (B) Scheme of the DNA fiber stretching experiment and visualization of DNA fibers by immunofluorescence. (C) Distribution of interorigin distance in mock- and XNCOA4-depleted extracts in samples in which biotin-dUTP was added at 60 min (left) and 90 min (right) (p < 0.001). Representative images of DNA fibers are shown. Results are from three independent experiments where mean percentage of replicated sperm DNA after 120 min, calculated according to Blow and Laskey (1986), was 78.5% and 84% in mock- and XNCOA4-depleted extracts, respectively. (D) Left: nascent strand alkaline gel electrophoresis of mock- and XNCOA4-depleted extracts released from Ara-C (0.2 mM) arrest. dCTP (1 mM) was added at 40 min, and aliquots were sampled at 2, 4, 8, 12, and 16 min. Right: relative amount of radioactivity in alkaline electrophoresis lanes of mock- and XNCOA4-depleted extracts sampled at 4 and 12 min, plotted as a function of molecular size. Percentage of replicated sperm DNA was >70% in both mock- and XNCOA4-depleted extracts (see also Figure S3). Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 An Excess of NCOA4 Inhibits DNA Replication in Xenopus laevis Egg Extracts (A) Upper: scheme of NCOA4 full-length and N- and C-terminal fragments. Lower: Xenopus laevis egg extracts supplemented with the indicated recombinant proteins (150 nM) were harvested at the indicated time points, and DNA replication was monitored by Cerenkov counting. Percentage of replicated sperm nuclei is shown (average results ± SD). (B) Upper: scheme of MCM7 full-length and C-terminal protein fragment (MCM7[C]). Middle: Xenopus laevis egg extracts were subjected to an in vitro pull-down assay using the indicated recombinant proteins. Bound proteins were immunoblotted with antibody against MCM7. Lower: agarose gel electrophoresis autoradiography of DNA synthesized in Xenopus laevis egg extracts in the presence or absence of 300 nM MCM7(C) recombinant protein. (C) Upper: agarose gel electrophoresis autoradiography of DNA synthesized in Xenopus laevis egg extracts using the indicated concentrations of NUS-NCOA4 alone or with 300 nM MCM7(C). Lower: percentage of DNA synthesized in each lane was measured by phosphorimaging (average results ± SD). Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 An Excess of NCOA4 Inhibits Origin Activation in Xenopus laevis Egg Extracts (A) Chromatin binding of the indicated replication factors monitored at the indicated time points upon incubation with NUS-NCOA4 or buffer alone by western blotting with the indicated antibodies. (B) Upper: loading of RPA and MCM7 on chromatin in the presence of buffer, 150 nM NUS-NCOA4, and/or aphidicolin (10 μM). Lower: DNA synthesis of each sample was monitored by agarose gel electrophoresis and autoradiography. (C) Chromatin-bound DNA polα (polα), DNA polε (polε), MCM7, and NUS-NCOA4 in the presence or absence of NUS-NCOA4 (150 nM) at the indicated time points was monitored by western blotting (see also Figure S4). Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 NCOA4−/− Cells Feature Reduced Interorigin Distance and Replication Stress (A) Reduced interorigin distance in NCOA4−/− cells. Upper: scheme of two adjacent replicons observed on individual DNA fibers. CldU is detected in red and IdU in green. Middle: representative images of newly firing replication origins in NCOA4+/+ and NCOA4−/− cells. Lower: average interorigin distance values in NCOA4+/+ and NCOA4−/− MEFs ± SD (∗∗∗p < 0.001). (B) Reduced fork speed in NCOA4−/− cells. Upper: scheme of a replicon observed on a stained DNA fiber after consecutive pulse labeling with CldU and IdU. Middle: representative images of replication tracks of NCOA4+/+ and NCOA4−/− MEFs. Lower: replication fork speed distribution in NCOA4−/− and NCOA4+/+ cells (p < 0.001). (C) Fork asymmetry (ratio between the two strands < 0.6) in NCOA4−/− cells. Upper: scheme of newly firing replication origins upon pulse labeling with CldU and IdU. Middle: representative images of newly firing replication origins in NCOA4+/+ and NCOA4−/− cells. Lower: percentages of asymmetric replication forks in NCOA4+/+ and NCOA4−/− MEFs ± SD (∗∗∗p < 0.001) (see also Figures S5–S7). Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 NCOA4−/− MEFs Undergo Premature Senescence (A) Upper: representative immunofluorescence staining for pATM S1981 of NCOA4−/− and NCOA4+/+ cells. Cell nuclei were counterstained with DRAQ5. Lower: percentage of pATM S1981-positive +/+ and −/− cells ± SD (∗∗p < 0.01). (B) Western blot analysis of cell lysates from NCOA4−/− and NCOA4+/+ MEFs using the indicated antibodies. Tubulin and H3 histone served as loading controls. (C) Upper: representative immunofluorescence staining of NCOA4−/− and NCOA4+/+ cells for pS/TQ ATM/ATR substrates. Lower: percentage of pS/TQ ATM/ATR substrates-positive +/+ and −/− cells ± SD (∗∗p < 0.01). (D) Left: representative immunofluorescence staining for γH2AX of NCOA4−/− and NCOA4+/+ cells. Right: percentage of γH2AX-positive NCOA4+/+ and NCOA4−/− cells ± SD (∗∗∗p < 0.001). (E) Left: representative immunofluorescence staining of NCOA4−/− and NCOA4+/+ cells for 53BP1. Right: percentage of 53BP1-positive +/+ and −/− cells ± SD (∗∗∗p < 0.001). (F) Proliferation of NCOA4+/+, NCOA4+/−, and NCOA4−/− MEFs measured as the accumulation of population doubling level (PDL) (∗∗∗p < 0.001). (G) Bar graphs showing the percentage of senescent cells (SA-β-galactosidase positive) ± SD in NCOA4+/+, NCOA4+/−, and NCOA4−/− cells at the indicated passages (∗∗∗p < 0.001). A representative picture of passage 4 NCOA4+/+ and NCOA4−/− cells is shown (see also Figure S7). Molecular Cell 2014 55, 123-137DOI: (10.1016/j.molcel.2014.04.031) Copyright © 2014 Elsevier Inc. Terms and Conditions