c-Myc-induced aberrant DNA synthesis and activation of DNA damage response in p300 knockdown cells

Abstract

We previously showed that in quiescent cells, p300/CBP (CREB-binding protein)family coactivators repress c-myc and prevent premature induction of DNA synthesis. p300/CBP-depleted cells exit G(1) early and continue to accumulate in S phase but do not progress into G(2)/M, and eventually they die of apoptosis. Here, we show that the S-phase arrest in these cells is because of an intra-S-phase block. The inappropriate DNA synthesis that occurs as a result of forced expression of c-myc leads to the activation of the DNA damage response as evidenced by the phosphorylation of several checkpoint related proteins and the formation of foci containing gamma-H2AX. The activation of checkpoint response is related to the induction of c-myc, as the phosphorylation of checkpoint proteins can be reversed when cells are treated with a c-Myc inhibitor or when Myc synthesis is blocked by short hairpin RNA. Using the DNA fiber assay, we show that in p300-depleted cells initiation of replication occurs from multiple replication origins. Chromatin loading of the Cdc45 protein also indicates increased origin activity in p300 knockdown cells. Immunofluorescence experiments indicate that c-Myc colocalizes with replication foci, consistent with the recently reported direct role of c-Myc in the initiation of DNA synthesis. Thus, the inappropriate S-phase entry of p300 down-regulated cells is likely to be because of c-Myc-induced deregulated replication origin activity, which results in replicative stress, activation of a DNA damage response, and S-phase arrest. Our results point to an important role for p300 in maintaining genomic integrity by negatively regulating c-myc.

Figures

FIGURE 1.

p300 knockdown proliferating MCF10A cells arrest in S phase (A and B), contain elevated c-Myc levels (C), and show an intra-S-phase block (D and E). A, flow cytometric analysis of proliferating MCF10A cells infected with Ad vectors Adp300sh1 or Adp300shRNA2 targeting two different regions of p300-coding sequences or Adlucsh-targeting luciferase gene(see“Experimental Procedures”). Cells were infected at 25 plaque-forming units/cell with the Ad vectors as shown, and the cells were harvested after 18 h, then their DNA content was measured by flow cytometry as described under “Experimental Procedures.” B, BrdUrd incorporation into DNA of p300-depleted proliferating MCF10A cells. Cells grown on coverslips were infected with Ad vectors as above and were pulse-labeled with 50 μm BrdUrd for 1 h before processing for immunostaining. The cells were then fixed, and BrdUrd was immunoreacted with mouse α-BrdUrd antibody (BD Biosciences). Bound antibodies were visualized using α-mouse antibody conjugated with Alexafluor-592 (red) (BD Biosciences). DNA was detected by staining with DAPI (blue). Immunostaining of cells were visualized at 40× magnification. C, c-Myc protein levels in cells infected with Ad vectors expressing shRNAs described in A. Proliferating MCF10A cells infected with Ad vectors for 18 h were harvested, and cell lysates equivalent to equal amount of proteins were analyzed in Western immunoblots. The antibodies used wereα-p300 (N-15),α-c-Myc (sc-40), and α-actin (sc-1919) from Santa Cruz Biotechnology. D, DNA synthesis assay. Proliferating MCF10A cells were infected with Ad vectors expressing shRNAs and harvested at 0, 6, 12, and 18 h after infection, then harvested, and the number of cells in G0/G1, S, and G2/M fractions were determined by flowcytometry. In the same experiment, in parallel, vector-infected cells were pulse-labeled with [3H]thymidine for 30 min before harvesting at the time points used for cell cycle analysis. Radioactivity on a per cell basis was calculated based on the percent number of cells in S phase as described under “Experimental Procedures.” Average values ± S.D. obtained from two independent experiments, each carried out in triplicate, are shown. Fluorescence-activated cell sorter analysis was also repeated twice, and the distribution of cell numbers in different cell cycle phases is shown for only one experiment. These numbers did not differ by more than 5% between the two experiments. E, chromatin loading of Cdc45. Chromatin-bound proteins, and the proteins present in the nuclear-soluble and the cytoplasmic fractions were prepared as described under “Experimental Procedures.” Cdc45 present in different fractions was determined by Western blots using antibodies specific for Cdc45 (Santa Cruz). Loading controls for each fraction (tubulin for cytoplasmic fraction, a nonspecific band for nuclear fraction, and histones for chromatin fraction) are shown below each panel showing shRNA data. Tubulin levels were determined by reprobing the membrane with an anti-tubulin antibody. Histones were detected by staining the gel by Coomassie Blue. Quantification of Cdc45 band on the autoradiogram was carried out by a densitometer scanning. -Fold increase was based on the values obtained for Adlucsh-infected cells.

FIGURE 2.

Activation of the DNA damage response in proliferating p300 knockdown cells. A, phosphorylation of DNA damage response proteins Chk2 and ATM in p300-depleted cells. Proliferating MCF10A cells were infected with Ad vectors expressing Luc-sh, p300sh1, or p300sh2 as shown, and the cells were harvested after 18 h. Equal amounts of protein extracts were used for Western immunoblots to detect various proteins as indicated. Sources of antibodies used: α-p300 and α-actin (Santa Cruz),α-CBP (Upstate Biotechnology),α-ATM (Bethyl), α-pATM (Rockland), α-Chk2, α-pChk2, and α-p53 (Cell Signaling). B, formation of γ-H2AX containing foci in p300-depleted cells. Proliferating cells grown on coverslips were infected as above for 18 h, then the cells were immunoreacted with α-γ-H2AX antibody (Upstate). Bound antibodies were detected using α-mouse antibody conjugated with Alexafluor-592 (red). DNA was detected by staining with DAPI (blue). C, the percentage of γ-H2AX positive cells were scored from three different fields, and the average was plotted with ±S.D. At least 100 cells were counted in each field.

FIGURE 3.

A time course study shows depletion of p300, induction of c-Myc, and phosphorylation of Chk2 and ATM in quiescent cells (A) and the formation of γ-H2AX-containing foci (B). A, activation of checkpoint in quiescent cells. MCF10A cells were made quiescent by incubating them in culture media containing 0.2% serum for 48 h, then the cells were infected with Adp300sh1, Adp300sh2 (data not shown; see below), or Adlucsh and maintained in the starvation media as above. Cell extracts were made at the indicated time points as shown in the figure, and equal quantities of proteins were Western-immunoblotted using α-pChk2 and α-pATM antibodies as shown. 0-h time point refers to the time of infection of cells. The blots were then re-probed with α-Chk2, α-ATM, and α-actin antibodies as indicated. Identical data for cells infected with Adp300sh2 were obtained (data not shown). B, formation of γ-H2AX-containing foci. Quiescent MCF10A cells grown on coverslips were infected with two Adp300 shRNAs as indicated above, and the cells were immunoreacted with anti-γ-H2AX antibody at 6, 18, and 24 h post-infection. Bound antibodies were detected using α-mouse antibody conjugated with Alexafluor-488 (green), and the DNA was detected by staining with DAPI (blue). C, the percentage of γ-H2AX-positive cells was determined after scoring cells from three different fields. At least 100 cells were counted in each field. Shown are the average values with ±S.D.

FIGURE 4.

DNA fiber assay showing the activation of multiple replication origins and replication fork terminations in p300-depleted cells. Quiescent MCF10A cells were infected with p300 shRNA viruses, and 18 h later the cells were first labeled with IdUrd for 30 min, washed, and then labeled with CldUrd for 30 min. The cells were lysed, and the DNA fibers were spread on glass slides as described under “Experimental Procedures.” The incorporated IdUrd and CldUrd were immunoreacted with antibodies that recognize IdUrd (α-BrdUrd, BD Biosciences) and CldUrd (α-BrdUrd, Abcam), respectively. The bound antibodies were detected using antibodies tagged with fluorescent dyes (IdUrd, green; CldUrd, red). The images were captured at 40× magnification using a Nikon fluorescent microscope, and data were processed using the Adobe Photoshop software. DNA fibers prepared from quiescent cells treated with serum for 12 h were used as a control. A, a diagrammatic representation of the type of the colored DNA tracks expected in DNA spreads. B, two representative images of labeled DNA tracks from each sample. Arrows represent likely origins. Arrowheads represent likely fork terminations.

FIGURE 5.

Blocking c-Myc activity using a Myc inhibitor or reducing c-Myc protein levels using a Myc shRNA reverses the DNA damage response. A, inhibition of transcriptional activity induced in p300-depleted proliferating cells by the Myc inhibitor. Proliferating MCF10A cells were infected with Ad vectors expressing various shRNAs as described in the legend to Fig. 2A and infected 2 h later with an Ad vector containing an Myc-responsive promoter-luciferase reporter construct and a mutant version of AdM4 in which the Myc binding sites are mutated (AdM4 and AdM4mut, respectively, Ref. 12)). Cells were then treated with an Myc inhibitor as described under “Experimental Procedures” for 18 h and harvested, and the luciferase activity in the cell lysates was quantified (12). Values from two independent experiments each carried out in triplicate with ±S.D. are shown. B, inhibition of Chk2 phosphorylation in the presence of the Myc inhibitor in proliferating MCF10A cells depleted for p300. Cells were infected with Adp300sh1 or -2 or Adlucsh for 18 h, then the cells were treated with Myc inhibitor (60 μm) or DMSO (control) for 16 h. Cells were then harvested, and Myc and the phosphorylated and non-phosphorylated Chk2 levels were assayed by Western immunoblotting using antibodies as described in legend to Fig. 2A. C, inhibition of transcriptional activity in quiescent MCF10A cells infected with Ad vectors expressing shRNA. Cells were infected with two Ad vectors targeting p300 followed by the AdM4 and AdM4mut vectors and treated with the Myc inhibitor, and luciferase activity in the cell lysates was quantified exactly as described in A. The experiment was carried out in triplicate, and the values from two different experiments are shown with ±S.D. D, inhibition of checkpoint activation by the Myc inhibitor in p300-depleted quiescent cells. Serum-starved MCF10A cells were infected with various Ad vectors for 18 h and treated with the Myc inhibitor as described in A, then harvested18 h later. Myc, pChk2, and Chk2 levels were determined by Western immunoblotting. Details of the antibodies are as described in Fig. 2A. E, down-regulation of Myc protein levels by Myc shRNA prevents the phosphorylation of Chk2. Proliferating MCF10A cells were infected with an Ad vector expressing Myc shRNA, and after 2 h the cells were again infected with Ad vectors expressing p300 or luciferase specific shRNAs. The cells were harvested 18 h later, then the protein extracts were subjected to Western immunoblotting to detect Myc and phosphorylated and unphosphorylated Chk2 levels. F, inhibition of the formation of the γ-H2AX-containing foci in p300-depleted quiescent cells by the Myc inhibitor. Serum-starved cells grown on coverslips were infected with Adp300shRNA1 or -2 for 18 h then treated with the Myc inhibitor as described above. Cells were then immunoreacted with α-γH2AX antibody. Detection of γ-H2AX was as detailed in Fig. 2C. The percentage of γ-H2AX positive cells is shown with ±S.D.

FIGURE 6.

c-Myc localizes to replication centers during DNA replication. A, c-Myc and BrdUrd co-localization was carried out in quiescent cells infected with Ad vectors expressing different shRNAs for the time periods as indicated and pulse-labeled with BrdUrd. Further details of this experiment are as described in legend to Fig. 1B. The control cells (quiescent cells expressing lucshRNAs) did not show any BrdUrd incorporation (not shown). B, proliferating MCF10A cells grown on coverslips were infected with Ad vectors expressing shRNAs targeting p300 or luciferase mRNAs for 18 h and were pulse-labeled for 1 h with 50 μm BrdUrd. Cells were then fixed and processed. BrdUrd and c-Myc were immunoreacted with rat α-BrdUrd antibody and mouse α-c-Myc antibodies as appropriate. The bound antibodies were detected using α-rat antibody conjugated with Alexafluor-592 (red) and α-mouse antibody conjugated with Alexaflour-488 (green). DNA content was visualized by staining with DAPI (blue). C, quantification of the number of cells (percentage) shown in B in which BrdUrd and c-Myc staining merged. D, distribution of cells in different cell cycle fractions (shown next to the cell cycle profiles) after synchronization and quantification of c-Myc. Serum-starved Adlucsh-infected cells were serum-stimulated for 16 h then harvested (control). Serum-starved cells infected with Adp300sh1 were maintained in serum-free media for 16 h then harvested (experimental). The distribution of cells in different cell cycle fractions was determined by fluorescence-activated cell sorter, and Myc protein levels were determined by Western immunoblotting. E, confocal images showing co-localization of BrdUrd and Myc staining in p300-depleted and control cells present in S phase. In parallel with the experiment described in D, using identical growth conditions, cells were grown on coverslips and infected with Ad vectors expressing shRNAs for 16 h using conditions as described above. They were then pulse-labeled for 1 h with 50 μm BrdUrd, then immunoreacted with α-BrdUrd and α-c-Myc antibodies as described earlier. The immunostained cells were examined using Nikon EZ-C1 confocal microscope at 100× magnification, and the images were captured and processed by the Adobe Photoshop software. Representative images are shown from each group.

FIGURE 7.

A, colocalization of c-Myc or PCNA with DNA replication foci in p300-depleted quiescent cells and the effect of Myc inhibitor on the c-Myc or PCNA colocalization. Cells grown on coverslips were made quiescent by serum starvation, infected with Ad vectors for 16 h, then pulse-labeled with BrdUrd for 30 min. Immunofluorescence details are described in Fig. 6A. For localization of PCNA, an α-PCNA antibody was used (sc-56; Santa Cruz). B, confocal images showing colocalization of BrdUrd staining with Myc or PCNA staining. Samples used in A were examined under a confocal microscope as described in Fig. 6E.