Dynamic PML protein nucleolar associations with persistent DNA damage lesions in response to nucleolar stress and senescence-inducing stimuli

Terezie Imrichova,Zdenek Hodny,Sona Hubackova,Jiri Bartek,Alena Kucerova,Pavla Vasicova,Jan Kosla

doi:10.18632/aging.102248

Terezie Imrichova, Zdenek Hodny + Show 5 more

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https://doi.org/10.18632/aging.102248

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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.

Highlights

One of the biological processes contributing to aging and age-related diseases is cellular senescence – a cell response to various stresses, characterized by protracted halt of cell cycle due to supra-threshold elevation of inhibitors of cyclin-dependent kinases
RPE-1hTERT cells treated with this dose of doxorubicin developed cellular senescence as shown by loss of proliferation, presence of DNA damage detected as γH2A.X foci, characteristic morphological changes, and positivity for senescence-associated β-galactosidase staining (Supplementary Figure 1B–1E)
PML nucleolus-derived structure (PML-NDS) were almost undetected until 48 hours after doxorubicin addition, their presence strongly increased after doxorubicin washout and was associated with the decrease of other PNAs forms as can be observed at 96 hours after doxorubicin removal

Summary

Introduction

One of the biological processes contributing to aging and age-related diseases is cellular senescence – a cell response to various stresses, characterized by protracted halt of cell cycle due to supra-threshold elevation of inhibitors of cyclin-dependent kinases (iCdk). Cellular senescence participates in aging by two main mechanisms: cell cycle arrest of progenitor cells, preventing tissue renewal; and secretion of proinflammatory molecules, leading to chronic inflammation and tissue deterioration DNA damage and persistent DNA damage response The persistent DDR due to irreparable or perpetual DNA damage is thought to be the main mechanism behind most forms of cellular senescence. The nature of this senescence-associated DNA damage seems to be complex and multifactorial though irreparability of telomeres is the factor most frequently cited [6, 7]. The direct evidence that damage of ribosomal DNA (rDNA) loci can cause senescence has been reported [10, 11]

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