Evidence for the receipt of DNA damage stimuli by PML nuclear domains

Abstract

Promyelocytic leukaemia nuclear domains (PML-NDs) comprise a shell of PML protein and many labile cargo proteins. The nature of their cargo, their juxtaposition to foci of damaged DNA following ionizing radiation (IR), and the altered DNA damage responses in PML null cells all implicate PML-NDs in the DNA damage response. In this work, the propensity of PML-NDs to increase in number and decrease in size following IR has been studied. Serial quantitative studies of endogenous PML-NDs prove that the PML-ND response to IR is not the result of the asymmetry in cell cycle distribution that can follow IR, but reflects more directly the process of DNA damage. The response is swift, sensitive (evident after 1 Gy), and potentially reversible in untransformed fibroblasts. In these cells and in HCT116 colon cancer cells, failure to restore PML-ND number within 24 h correlates with later loss of growth potential--in fibroblasts, through prolonged cell cycle arrest and in HCT116 cells, through apoptosis. Failure to express an intact ATM/CHK2 DNA damage signalling pathway in either cell type leads to a delay in the PML-ND response to IR. Conversely, cell cycle progression following IR in cells that detect damaged DNA accelerates PML-ND reorganization. Collectively, these data show that the increase in PML-ND number seen after irradiation is, in part, triggered by the receipt of the DNA damage stimulus. The senescent cell state is also associated with chronic DNA damage and Hayflick-limited fibroblasts were found to express nuclei with elevated numbers of PML-NDs before IR that remained unresponsive to IR. Though the underlying reasons for damage-induced PML alteration remain obscure, it is noteworthy that significant numbers of PML-NDs juxtapose with ionizing radiation-induced foci after IR. The co-regulation of these structures may necessitate the stereotyped increases in PML-ND number following damage.

Nuclear organization in genome stability: SUMO connections

“…Importantly, the telomeric DNA recruited to PML-NBs for ALT are also linear, presumably containing stretches of ss TG-DNA rather than t-loops [91]. The association and disassociation of repair and checkpoint signaling proteins with PML-NBs both prior to and after irradiation is complex [20,[90][91][92][93][94][95]. Nonetheless two points are noteworthy.…”

“…Nonetheless two points are noteworthy. The DNA damage response marker, γ-H2AX (histone H2AX phosphorylated on serine 139), which results from activation of ATM kinase, extends over large chromatin domains at radiation-induced breaks and colocalizes with PML-NBs hours after exposure to ionizing radiation [90,92]. Secondly, also at late time points radiation-induced MRE11 foci (IRIF), as well as p53, colocalize with PML-NBs.…”

AIRE's CARD Revealed, a New Structure for Central Tolerance Provokes Transcriptional Plasticity

“…Immunofluorescence-Immunostaining of transfectants was as described previously (24). Immunostaining reagents were as follows: anti-PML (clone N19, Santa Cruz Biotechnology), antimurine AIRE (rabbit polyclonal, a kind gift from Professor Peltonen, Biomedicum, Helsinki, Finland), and anti-human AIRE (mouse monoclonal AIRE6.1 as described previously (23)).…”

The Tumor Suppressor PML Specifically Accumulates at RPA/Rad51-Containing DNA Damage Repair Foci but Is Nonessential for DNA Damage-Induced Fibroblast Senescence

“…PML NBs were also found in spatial proximity to DNA single-strand breaks (SSBs) and DSBs (17,26,27). This suggests that PML NBs could serve as DNA damage sensors, DNA repair compartments, and physical sites where DNA repair activities and/or cell cycle checkpoint pathways are coordinated and monitored (17,28). PML protein levels and the number of NBs are elevated when cells encounter stress, e.g., after DNA damage (28)(29)(30) and during senescence induction (31,32).…”

“…This suggests that PML NBs could serve as DNA damage sensors, DNA repair compartments, and physical sites where DNA repair activities and/or cell cycle checkpoint pathways are coordinated and monitored (17,28). PML protein levels and the number of NBs are elevated when cells encounter stress, e.g., after DNA damage (28)(29)(30) and during senescence induction (31,32). Overexpression of PML protein isoform IV induces senescence in primary human and murine fibroblasts, and this process is dependent on p53 and pRb (32)(33)(34).…”