Research Paper Volume 11, Issue 17 pp 7206—7235
Dynamic PML protein nucleolar associations with persistent DNA damage lesions in response to nucleolar stress and senescence-inducing stimuli
- 1 Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic
- 2 Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark
- 3 Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
- 4 Present address: Institute of Biotechnology, Czech Academy of Sciences, Prague-West, Czech Republic
Received: July 19, 2019 Accepted: August 22, 2019 Published: September 7, 2019
https://doi.org/10.18632/aging.102248How to Cite
Copyright © 2019 Imrichova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Diverse stress insults trigger interactions of PML with nucleolus, however, the function of these PML nucleolar associations (PNAs) remains unclear. Here we show that during induction of DNA damage-induced senescence in human non-cancerous cells, PML accumulates at the nucleolar periphery simultaneously with inactivation of RNA polymerase I (RNAP I) and nucleolar segregation. Using time-lapse and high-resolution microscopy, we followed the genesis, structural transitions and destiny of PNAs to show that: 1) the dynamic structural changes of the PML-nucleolar interaction are tightly associated with inactivation and reactivation of RNAP I-mediated transcription, respectively; 2) the PML-nucleolar compartment develops sequentially under stress and, upon stress termination, it culminates in either of two fates: disappearance or persistence; 3) all PNAs stages can associate with DNA damage markers; 4) the persistent, commonly long-lasting PML multi-protein nucleolar structures (PML-NDS) associate with markers of DNA damage, indicating a role of PNAs in persistent DNA damage response characteristic for senescent cells. Given the emerging evidence implicating PML in homologous recombination-directed DNA repair, we propose that PNAs contribute to sequestration and faithful repair of the highly unstable ribosomal DNA repeats, a fundamental process to maintain a precise balance between DNA repair mechanisms, with implications for genomic integrity and aging.
Abbreviations
5-FUrd: 5-fluorouridine; AMD: actinomycin D; B23: nucleophosmin; DAPI: 4',6-diamidino-2-phenylindole; DDR: DNA damage response; DHX9: DExH-box helicase 9/nuclear DNA helicase II; doxo: doxorubicin hydrochloride; EdU: 5-ethynyl-2′-deoxyuridin; EGFP: enhanced green fluorescent protein; γH2AX: histone H2AX phosphorylated on serine 139; hMSC: human mesenchymal stem cells; HR: homologous recombination; iCdk: inhibitors of cyclin-dependent kinases; INK4: inhibitors of CDK4; IR: ionizing radiation; Kip: kinase inhibitory protein; NHEJ: non-homologous end joining: PAF49: RNA polymerase I-associated factor 49; PBS: phosphate buffered saline; PML: promyelocytic leukemia; PML NBs: PML nuclear bodies; PML-KO: PML knock-out; PML-NDS: PML nucleolus-derived structure; PML-WT: PML wild-type; PNAs: PML nucleolar associations; rDNA: ribosomal DNA; RFP: red fluorescent protein; RNAP I: RNA polymerase I; RPE-1hTERT: retinal pigment epithelial cells immortalized with human telomerase reverse transcriptase; SIM: structured illumination microscopy; SiR-DNA: silicon rhodamine DNA dye; STED: stimulated emission depletion; WO: doxorubicin washout.