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 G1 early and continue to accumulate in S phase but do not progress into G2/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 γ-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. 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 G1 early and continue to accumulate in S phase but do not progress into G2/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 γ-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. In eukaryotic cells, initiation of DNA replication takes place from multiple replication origins on each chromosome, providing flexibility for the large eukaryotic DNA to replicate efficiently. However, control mechanisms exist to ensure that DNA replication occurs only once per cell, and when such mechanisms fail, checkpoint responses are activated to monitor the genome integrity and inhibit replication until DNA damage has been repaired (for review, see Ref. 1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1691) Google Scholar). In response to DNA damage, eukaryotic cells activate ATM 3The abbreviations used are: ATM, ataxia telangiectasia-mutated; ATR, ataxia telangiectasia and Rad3-related; Ad, adenovirus; CBP, cAMP-CREB-binding protein; PCNA, proliferating cell nuclear antigen; Chk1, Checkpoint homolog (Schizosaccharomyces pombe) 1; Chk2, checkpoint homolog 2; PARP-1, poly(ADP-ribose) polymerase family member 1; NBS1, Nijmegen Breakage syndrome gene 1; shRNA, short hairpin RNA; AdM4, adenovirus M4 vector; PBS, phosphate-buffered saline; DAPI, 4′,6-diamidino-2-phenylindole; BrdUrd, bromodeoxyuridine; IdUrd, iododeoxyuridine; CldUrd, chlorodeoxyuridine; γ-H2AX, phosphorylated form of histone H2AX./Chk2 and ATR/Chk1 kinase pathways to arrest the cell cycle and allow time to repair DNA. Both ATM and ATR control cell cycle checkpoint signals by phosphorylating a number of checkpoint-related proteins including Chk1 and Chk2 and p53 (for review, see Refs. 2Shiloh Y. Trends Biochem. Sci. 2006; 31: 402-410Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar and 3Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell Biol. 2000; 1: 179-186Crossref PubMed Scopus (667) Google Scholar). p300 and CBP are two highly related and conserved nuclear phosphoproteins that function as transcriptional coactivators, and by interacting with a large number of sequence-specific transcription factors they integrate diverse signal transduction pathways in the nucleus. Because of their intrinsic histone acetyltransferase activity, they also acetylate nucleosomal histones and, thus, with transcription (for review, see Ref. Genes Dev. 2000; PubMed Google Scholar). that p300/CBP an important role in DNA replication and the activation of DNA damage response by interacting with several proteins in DNA replication by proteins in DNA We recently that of p300 or CBP in quiescent cells leads to induction of c-myc that in DNA synthesis in the of Sci. 2001; PubMed Scopus Google Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). DNA proteins such as adenovirus and large also to and p300/CBP and c-myc and S phase in quiescent cells Sci. 2001; PubMed Scopus Google PubMed Scopus Google Scholar). premature DNA synthesis in p300 down-regulated cells to exit they accumulate in S phase but to progress into and die of Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of p300/CBP-depleted cells to S phase is not related to the of because the of to p300/CBP-depleted cells also leads to increased c-Myc synthesis and an S-phase Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). c-myc is to DNA synthesis by in DNA replication (for review, see Ref. Mol. Biol. PubMed Scopus Google Scholar). However, that c-myc can also control the initiation of DNA replication the PubMed Scopus Google Scholar). c-Myc can with DNA replication origin and function as a replication PubMed Scopus Google Scholar). c-Myc can DNA and the deregulated origin activity can DNA damage response by the ATM PubMed Scopus Google Sci. 2006; PubMed Scopus Google Scholar). Because p300/CBP inappropriate DNA synthesis by negatively regulating c-myc and p300/CBP knockdown results in the induction of c-myc in quiescent and proliferating cells Sci. 2001; PubMed Scopus Google Biol. Full Text Full Text PDF PubMed Scopus Google of to the S-phase that occurs in p300/CBP-depleted cells is related to the forced induction of c-myc and the DNA damage response is in these cells. We show that the inappropriate DNA synthesis that occurs in p300 knockdown cells as a result of c-myc induction is likely because of replication origin of DNA replication origin activity replication stress, an intra-S-phase and activation of a DNA damage an important role for p300 in maintaining genomic integrity by negatively regulating c-myc. and for the cells Google Sci. 2001; PubMed Scopus Google Sci. PubMed Scopus Google Scholar). for and cell cycle as Sci. 2001; PubMed Scopus Google Sci. PubMed Scopus Google Scholar). The adenovirus M4 a in in which of the Myc of the gene Sci. PubMed Scopus Google Scholar). Myc and as a result the activity of is Sci. PubMed Scopus Google Scholar). of the two p300 two of p300 and and the shRNA, the in a PubMed Scopus Google Scholar). and Cell in with inhibitor and inhibitor and of protein to as PubMed Scopus Google Scholar). cells. cells per as in the to with with The cells the time in the and the activity from of protein as Sci. 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Full Text Full Text PDF PubMed Scopus Google Scholar). cells accumulate in S phase but do not progress the of of p300 in cells, we two of proliferating cells with two that two of p300 and for cell cycle in the of cells with an control for showed a cell cycle the two of p300 cells showed an cell cycle of the and cells and in S and the number of cells in the by as with control results by a in which proliferating cells with p300 with to DNA synthesis. The cells in indicate that of the p300-depleted cells for BrdUrd, only of the control cells by cells, see are not that c-Myc protein in p300 cells as with control cells. the results that p300 cells control cells and in S phase but to progress Thus, when p300 is cells do not cycle and are blocked in S p300 in S an for the of the p300 cells to progress S phase is that be DNA replication as a result of an intra-S-phase block. of cells to such as or the of DNA synthesis until the DNA damage is repaired (for review, see Ref. Mol. Biol. 2006; PubMed Scopus Google Scholar). DNA synthesis is in p300-depleted cells, cells with and into DNA on a per cell as In the in a of cells used for per S-phase cell of and a of cells used to cell cycle The results in show that the number of p300-depleted cells in S phase increased by by of cells with p300 with time in the of on a per cell in the p300-depleted cells. the control showed a of as with time In p300 knockdown showed a in results are with cell cycle which showed a of p300-depleted cells in S we do not a for is that replication origins in p300-depleted cells DNA synthesis and S these and as origins. replication for S-phase and activation of the DNA damage Chromatin of Cdc45 in p300 the p300 knockdown cells in S phase of increased origin activity, we the of the loading of Cdc45 Cdc45 has been in initiation of DNA and Cdc45 has been used as an of replication origin activity Mol. Biol. PubMed Scopus Google Sci. PubMed Scopus Google Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the loading of Cdc45 in p300 down-regulated cells, proteins from cells with control and p300 for and as and to time a in S-phase cells in the p300-depleted not in indicate that a in Cdc45 and a in Cdc45 in nuclear results that the origin activity is increased in p300 knockdown cells. The DNA in p300-depleted in S to DNA damage is a of related protein ATR and ATM, which are activated by DNA damage and to the of DNA they a number of including Chk1, Chk2, and the DNA damage response is activated in p300-depleted cells that accumulate in S we the phosphorylation of the proteins of the response to DNA damage R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1691) Google Scholar). cells with the two p300 or the control for cell and to for CBP, and for the phosphorylated checkpoint proteins ATM, Chk1, and in p300 in cells CBP In with Myc protein and p53 Biol. Full Text Full Text PDF PubMed Scopus Google consistent with a role for p300 in of p53 Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). ATM and Chk2 to be phosphorylated showed that p300 in cells not result in Chk1 that ATR/Chk1 is not p300 Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We these results not The results indicate that the DNA damage response activated in p300-depleted cells that accumulate in S cells of c-Myc protein as with control cells in Thus, checkpoint activation in p300 cells with the increased Myc protein early in response to DNA in cells is the phosphorylation of a histone of DNA damage (for review, see Ref. 2Shiloh Y. Trends Biochem. Sci. 2006; 31: 402-410Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). such as replication or DNA can formation of foci containing of of phosphorylated Y. Trends Biochem. Sci. 2006; 31: 402-410Abstract Full Text Full Text PDF PubMed Scopus (468) Google the p300 cells that accumulate in foci, p300 cells on with and for the formation of foci by of the p300 cells to be for foci, a number of control cells showed such results the of replication DNA in p300-depleted cells and Thus, we that p300-depleted cells that accumulate in S phase activate checkpoint of c-myc in p300 with the of DNA the induction of c-myc that occurs in cells with induction of the DNA damage response, we the of p300 activation of c-myc, and phosphorylation of DNA damage response proteins in p300-depleted quiescent cells. cells with p300 and cells the time in Cell from these cells as in indicate that of p300 to the time CBP large in Myc protein by of ATM, Chk2, and not also of cells with p53 when p300 consistent with the role of p300 in maintaining p53 Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). 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We to the of of these cells in S phase in p300/CBP-depleted cells and DNA damage response is activated in these cells and, the activation of the DNA damage response is related to the induction of We used two two of p300 PubMed Scopus Google to the expression of p300 and to the of p300 on The cell cycle we of an intra-S-phase in cells, induction of checkpoint formation of foci, and of Myc in DNA replication two p300 in quiescent and proliferating cells are in that of p300 knockdown is likely related to the of c-myc. Our show that the c-Myc in p300 cells is for the on the induction of DNA damage response in cells that accumulate as a result of of the of of these when Myc by a Myc shRNA, or Myc activity blocked by a Myc We previously showed that the S-phase entry of the p300 or CBP knockdown cells in the of factors be reversed when the induction of c-Myc Sci. 2001; PubMed Scopus Google Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). with we showed that activation of the DNA damage response of checkpoint in p300 knockdown cells can be when Myc are or the Myc activity blocked by a Myc inhibitor and Myc is a and a large number of by to their of the Myc are in the initiation and of DNA replication and and S-phase (for review, see Ref. Mol. Biol. PubMed Scopus Google Scholar). expression of Myc in quiescent cells has been to S phase (for review, see Ref. Dev. PubMed Scopus Google that to be of in which is from as a result of phosphorylation Genes Dev. 2000; PubMed Scopus Google Scholar). However, a that Myc can also the DNA replication origin activity by to proteins PubMed Scopus Google Scholar). Thus, Myc can DNA synthesis by synthesis of proteins in the initiation of DNA synthesis as as by interacting with replication origins. Using DNA fiber we showed that in p300 cells DNA replication is likely from a large number of replication origins as with cells. 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