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Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase-negative human cell line.

ABSTRACT: Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism. This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs.

SUBMITTER: Jegou T

PROVIDER: S-EPMC2663922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Live cell dynamics of promyelocytic leukemia nuclear bodies upon entry into and exit from mitosis.

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Functional reorganization of promyelocytic leukemia nuclear bodies during BK virus infection.

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Identifying gene locus associations with promyelocytic leukemia nuclear bodies using immuno-TRAP.

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Project description:The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates consisting of its adapter protein FE65, the histone acetyltransferase TIP60 and the tumour suppressor proteins p53 and PML. APP C-terminal (APP-CT50) complexes co-localize and co-precipitate with p53 and PML. The PML nuclear body generation is induced and fusion occurs over time depending on APP signalling and STED imaging revealed active gene expression within the complex. We further show that the nuclear aggregates of APP-CT50 fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimer's disease brains reveal a highly significant reduction of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT50 signalling to the nucleus takes place in the aged human brain and is involved in the pathophysiology of AD.

| S-EPMC8042982 | biostudies-literature

Identifying chromatin associations with promyelocytic leukemia nuclear bodies using immunoTRAP

Project description:Studies using fluorescence in situ hybridization (FISH) have attempted to determine whether specific gene loci associate with promyelocytic leukemia nuclear bodies (PML NBs). Two drawbacks accompany this approach; the lack of spatial resolution inherent with the technique, and the a priori basis for selecting which genes to probe. To overcome these limitations, we developed a technique, which we call immunoTRAP, which purifies the DNA contacting PML NBs at molecular dimensions. When we combined the immunoTRAP technique with immunoFISH and microarray analysis, not only did we verify a TP53-PML NB association, but were able also to identify novel locus associations such as PML, ABCA7, and TFF1. In addition, we observed that these associations are cell type specific, and induction of significantly higher levels of PML gene expression, as well as other physiological changes brought about by interferon treatment, can lead to a physical association of the PML gene with PML NBs in normal human fibroblasts. Thus, immunoTRAP is a technique capable of identifying the chromatin associations around nuclear subcompartments and is amenable for further downstream applications such as microarray analysis or deep sequencing.

2013-03-28 | GSE40554 | GEO

Identifying chromatin associations with promyelocytic leukemia nuclear bodies using immunoTRAP

2013-03-28 | E-GEOD-40554 | biostudies-arrayexpress

Manipulation of Promyelocytic Leukemia Protein Nuclear Bodies by Marek's Disease Virus Encoded US3 Protein Kinase.

Project description:Promyelocytic leukemia protein nuclear bodies (PML-NBs) are dynamic nuclear structures, shown to be important for herpesvirus replication; however, their role in regulating Marek's disease virus (MDV) infection has not been studied. MDV is an oncogenic alphaherpesvirus that causes lymphoproliferative disease in chickens. MDV encodes a US3 serine/threonine protein kinase that is important for MDV replication and gene expression. In this study, we studied the role of MDV US3 in regulating PML-NBs. Using an immunofluorescence assay, we found that MDV US3 disrupts PML and SP100 in a kinase dependent manner. In addition, treatment with MG-132 (a proteasome inhibitor) could partially restore the levels of PML and SP100, suggesting that a cellular proteasome dependent degradation pathway is involved in MDV US3 induced disruption of PML and SP100. These findings provide the first evidence for the interplay between MDV proteins and PML-NBs.

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Project description:During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

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Promyelocytic leukemia nuclear bodies support a late step in DNA double-strand break repair by homologous recombination.

Project description:The PML protein and PML nuclear bodies (PML-NB) are implicated in multiple cellular functions relevant to tumor suppression, including DNA damage response. In most cases of acute promyelocytic leukemia, the PML and retinoic acid receptor alpha (RARA) genes are translocated, resulting in expression of oncogenic PML-RAR? fusion proteins. PML-NB fail to form normally, and promyelocytes remain in an undifferentiated, abnormally proliferative state. We examined the involvement of PML protein and PML-NB in homologous recombinational repair (HRR) of chromosomal DNA double-strand breaks. Transient overexpression of wild-type PML protein isoforms produced hugely enlarged or aggregated PML-NB and reduced HRR by ~2-fold, suggesting that HRR depends to some extent upon normal PML-NB structure. Knockdown of PML by RNA interference sharply attenuated formation of PML-NB and reduced HRR by up to 20-fold. However, PML-knockdown cells showed apparently normal induction of H2AX phosphorylation and RAD51 foci after DNA damage by ionizing radiation. These findings indicate that early steps in HRR, including recognition of DNA double-strand breaks, initial processing of ends, and assembly of single-stranded DNA/RAD51 nucleoprotein filaments, do not depend upon PML-NB. The HRR deficit in PML-depleted cells thus reflects inhibition of later steps in the repair pathway. Expression of PML-RAR? fusion proteins disrupted PML-NB structure and reduced HRR by up to 10-fold, raising the possibility that defective HRR and resulting genomic instability may figure in the pathogenesis, progression and relapse of acute promyelocytic leukemia.

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