The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcriptionblocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)-and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis.antibody microinjection ͉ DNA damage response ͉ RNA polymerase II ͉ nuclear export ͉ phosphorylation T he mechanisms by which DNA lesions are detected and how damage-response pathways are activated in cells are not well understood (1, 2). The stress-response kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) link DNA damage to p53 activation by phosphorylating the Ser-15 site of p53 (3-6). Although the ATM kinase is activated after exposure to ionizing radiation (7), the ATR kinase responds to agents that interfere with replication such as UV light and hydroxyurea (6). The tumor suppressor p53 is induced in response to a variety of different agents in all phases of the cell cycle (8,9). By acting as a transcription factor, p53 can induce the expression of gene products involved in DNA repair, cell cycle arrest, or apoptosis (10, 11). It can also regulate DNA repair and apoptosis by transcription-independent mechanisms (12). Under normal conditions, the p53 protein is rapidly targeted for nuclear export and degradation in a process regulated by the MDM2 protein (13). After cellular stress, the MDM2-mediated negative regulation of p53 is abrogated, and p53 proteins accumulate in the cell nucleus. This nuclear accumulation can be accomplished by (i) DNA damage-induced phosphorylation of p53 and MDM2, leading to the interference of the interaction between MDM2 and p53 (14); (ii) inactivation of proteasomes (15); or (iii) inhibition of the nuclear export machinery (13,16).DNA lesions that block RNA polymerase II induce the recruitment of transcription-coupled repair (TCR) and chromatin remodeling factors to recover RNA synthesis (17)(18)(19). Cells defective in TCR induce p53 and apoptosis at much lower doses than cells with proficient TCR, suggesting that lesions in the transcribed strand of active genes trigger these responses (9,(20)(21)(22). Although these studies have shown a correlation between blockage of ...
Increased levels of EZH2, a critical regulator of cellular memory, signal the presence of metastasis and poor outcome in breast cancer patients. High levels of EZH2 are associated with nuclear pleomorphism, lack of estrogen receptor expression, and decreased nuclear levels of BRCA1 tumor suppressor protein in invasive breast carcinomas. The mechanism by which EZH2 overexpression promotes the growth of poorly differentiated invasive carcinomas remains to be defined. Here, we show that EZH2 controls the intracellular localization of BRCA1 protein. Conditional doxycycline-induced upregulation of EZH2 in benign mammary epithelial cells results in nuclear export of BRCA1 protein, aberrant mitoses with extra centrosomes, and genomic instability. EZH2 inhibition in CAL51 breast cancer cells induces BRCA1 nuclear localization and rescues defects in ploidy and mitosis. Mechanistically, EZH2 overexpression is sufficient for activation of the phosphoinositide 3-kinase/ Akt (PI3K/Akt) pathway specifically through activation of Akt isoform 1. EZH2-induced BRCA1 nuclear export, aneuploidy, and mitotic defects were prevented by treatment with the PI3K inhibitors LY294002 or wortmannin. Targeted inhibition of Akt-1, Akt-2, and Akt-3 isoforms revealed that the EZH2-induced phenotype requires specific activation of Akt-1. The relevance of our studies to human breast cancer is highlighted by the finding that high EZH2 protein levels are associated with upregulated expression of phospho-Akt-1 (Ser473) and decreased nuclear expression of phospho-BRCA1 (Ser1423) in 39% of invasive breast carcinomas. These results enable us to pinpoint one mechanism by which EZH2 regulates BRCA1 expression and genomic stability mediated by the PI3K/Akt-1 pathway. Cancer Res; 71(6); 2360-70. Ó2011 AACR.
Supplementary Tables 1-2 from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer
Supplementary Methods from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer
Supplementary Figures 1-5 from Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer
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