The number of PML nuclear bodies increases in early S phase by a fission mechanism

The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) at telomeres. Here, we dissected the role of APBs in a human ALT cell line by performing an RNA interference screen using an automated 3D fluorescence microscopy platform and advanced 3D image analysis. We identified 29 proteins that affected APB formation, which included proteins involved in telomere and chromatin organization, protein sumoylation and DNA repair. By integrating and extending these findings, we found that APB formation induced clustering of telomere repeats, telomere compaction and concomitant depletion of the shelterin protein TRF2 (also known as TERF2). These APB-dependent changes correlated with the induction of a DNA damage response at telomeres in APBs as evident by a strong enrichment of the phosphorylated form of the ataxia telangiectasia mutated (ATM) kinase. Accordingly, we propose that APBs promote telomere maintenance by inducing a DNA damage response in ALT-positive tumor cells through changing the telomeric chromatin state to trigger ATM phosphorylation.

Section: An Rnai Screen With Automated 3d Image Analysis Identifies 2supporting

confidence: 78%

PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening

Osterwald

Deeg

Chung

et al. 2015

“…After digestion with HindIII and BamHI, the cDNA was inserted between the HindIII and BamHI sites of the pdiHcRed-C1 vector [a modification of pHcRed1-C1 from Clontech Laboratories kindly supplied by David Bazett-Jones, The Hospital for Sick Children, Toronto, Canada (Dellaire et al, 2006)] just after the cDNA encoding a dimeric version of HcRed. The cDNA encoding the fusion protein was then excised with NheI and BamHI and inserted between the NheI and BamHI sites of pcDNA3.1/Hygro(-) (Invitrogen).…”

Section: Plasmid Constructsmentioning

confidence: 99%

Mitotic chromosome interactions of Epstein-Barr nuclear antigen 1 (EBNA1) and human EBNA1-binding protein 2 (EBP2)

Nayyar

Shire

Frappier

2009

by tethering the episomes to the cellular chromosomes in mitosis. A host nucleolar protein, EBNA1-binding protein 2 (EBP2), has been shown to be important for interactions between EBNA1 and chromosomes in metaphase and to associate with metaphase chromosomes. Here, we examine the timing of the chromosome associations of EBNA1 and EBP2 through mitosis and the regions of EBNA1 that mediate the chromosome interactions at each stage of mitosis. We show that EBP2 is localized to the nucleolus until late prophase, after which it relocalizes to the chromosome periphery, where it remains throughout telophase. EBNA1 is associated with chromosomes early in prophase through to telophase and partially colocalizes with chromosomal EBP2 in metaphase through to telophase. Using EBNA1 deletion mutants, the chromosome association of EBNA1 at each stage of mitosis was found to be mediated mainly by a central glycinearginine region, and to a lesser degree by N-terminal sequences. These sequence requirements for chromosome interaction mirrored those for EBP2 binding. Our results suggest that interactions between EBNA1 and chromosomes involve at least two stages, and that the contribution of EBP2 to these interactions occurs in the second half of mitosis.

“…The level of expression of GFP-PML-I was modest, in the order of 10-20% of total PML (data not shown). In three independent observations, classical PML nuclear bodies were found to be highly mobile structures that divide or fuse with themselves (Dellaire et al, 2006), whereas SANB fuse with incoming classical nuclear bodies, but were never observed to divide or to bud from the nucleolus, suggesting that these domains are relatively stable (supplementary material Movie 1).…”

Section: Dynamic Analysis Of Sanbmentioning

confidence: 99%

A nucleolar targeting signal in PML-I addresses PML to nucleolar caps in stressed or senescent cells

Condemine

Takahashi

Bras

et al. 2007

The promyelocytic leukemia (PML) tumour suppressor is the organiser of PML nuclear bodies, which are domains the precise functions of which are still disputed. We show that upon several types of stress, endogenous PML proteins form nucleolar caps and eventually engulf nucleolar components. Only two specific PML splice variants (PML-I and PML-IV) are efficiently targeted to the nucleolus and the abundant PML-I isoform is required for the targeting of endogenous PML proteins to this organelle. We identified a nucleolar targeting domain within the evolutionarily conserved C-terminus of PML-I. This domain contains a predicted exonuclease III fold essential for the targeting of the PML-I C-terminus to nucleolar fibrillar centres. Furthermore, spontaneous or oncogene retrieval-induced senescence is associated with the formation of very large PML nuclear bodies that initially contain nucleolar components. Later, poly-ubiquitin conjugates are found on the outer shell or within most of these senescence-associated PML bodies. Thus, unexpectedly, the scarcely studied PML-I isoform links PML bodies, nucleolus, senescence and proteolysis.