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Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency. Key words: PML, PML nuclear bodies, homologous recombination, CRISPR/Cas9, homology-directed repair. Les corps nucleaires PML (PML NB) sont des sous-domaines nucleaires qui repondentau stress genotoxiqueen croissant en nombre au moyen de changements de la structure de la chromatine. Toutefois, le role de la proteine PML et des PML NB dans des mecanismes specifiquesde reparation d'ADN n'ont pas ete completement caracterises. Les auteurs ont examine directement le role de PML dans la recombinaison homologue (HR) a l'aide de tests de reparation dirigee par homologie (HDR) extrachromosomique et chromosomique induite par I-SceI et dans la HDR par edition genique mediee par CRISPR/Cas9. Ils ont demontre que la perte de PML peut inhiber la HR lors d'un test de HDR extrachromosomique, mais avait un effet moindre sur la HDR chromosomique mediee par CRISPR/Cas9. La surexpression de PML inhibait aussi la HDR mediee par CRISPR/Cas9 et la HDR induite par I-Sce I sur un rapporteur chromosomique, de maniere specifique a l'isoforme. Toutefois, l'impact de la surexpression de PML sur le rapporteur de HDR chromosomique etait dependant du positionnement chromosomique intranucleaire du rapporteur. Particulierement, la HDR sur le locus genique TAP1, qui est associe aux PML NB, etait reduite comparativement a un locus non associea un PML NB; neanmoins, laHDR pouvait etre reduite sur lelocus non associe a unPML NB par la surexpression de PML. Ainsi, tant la perte que la surexpression des isoformes de PML peuvent inhiber la HDR, et la proximite d'un bris chromosomique a un PML NB peut affecter l'efficacite de la HDR. [Traduit par la Redaction] Mots-cles : PML, corps nucleaires PML, recombinaison homologue, CRISPR/Cas9, reparation dirigee par homologie., Introduction Promyelocytic leukemia nuclear bodies (PML NBs) are proteinaceous nuclear subdomains that are present in most mammalian cell lines and tissues. PML NBs range in size between 0.1-1.0 [micro]m and [...]

Abstract: With the introduction of precision genome editing using CRISPR--Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it [...]

DNA repair by homologous recombination (1) is highly suppressed in G1 cells (2,3) to ensure that mitotic recombination occurs solely between sister chromatids (4). Although many homologous recombination factors are [...]

Promyelocytic leukemia (PML) protein forms the scaffold for PML nuclear bodies (PML NB) that reorganize into Lipid‐Associated PML Structures (LAPS) under fatty acid stress. We determined how the fatty acid oleate alters the interactome of PMLI or PMLII by expressing fusions with the ascorbate peroxidase APEX2 in U2OS cells. The resultant interactome included ESCRT and COPII transport protein nodes. Proximity ligation assay (PLA) revealed that COPII proteins SEC23B, SEC24A and USO1 preferentially associated with PML NBs. Nuclear localization of USO1, but not SEC23B and SEC24A, was reduced in PML knockout cells and restored by PMLII expression. Thus, proximity‐labelling methods identified COPII transport protein interactions with PML NBs that are disrupted by fatty acid stress. [ABSTRACT FROM AUTHOR]

R2137 --> 679.1 --> Lipid droplets (LD) are organelles composed of a phospholipid monolayer surrounding a core of and triacylglycerol and cholesterol ester. LD have a primary role in energy homeostasis and lipotoxic stress through the regulated storage and release of fatty acids. While LD are cytoplasmic, lipoprotein secreting cells harbor nuclear LD (nLD) that associate with PML, the structural protein of PML nuclear bodies (PML‐NB) that regulate DNA repair and stress responses. Knockout studies in U2OS cells indicated that the 50% of nLDs contain PML isoform II (PML‐II) and have a unique lipid and protein composition marked by the lack of canonical PML‐NB‐associated proteins, such as DAXX, SP100 and SUMO. Owing to their unique composition, we term these subnuclear lipid:protein domains, lipid‐associated PML structures (LAPS). To gain insight into LAPS function, we conducted proximity labeling and mass spectrometry (MS) studies using PML‐I and PML‐II modified by fusion to the ascorbate peroxidase APEX2 that uses biotin‐phenol to biotinylate proteins. U2OS and Huh7 cells expressing PML‐APEX2 fusion proteins were treated with and without oleate, and biotinylated proteins were isolated and analyzed by LC‐MS/MS. Using standard filtering techniques (CRAPome, SAINT, Bayesian false discovery rate) we identified several high confidence interacting proteins associated with the COPII coatomer complex, including Sec24A, Sec23IP, Sec24B, and Sec23B. These Sec23/24 proteins were associated with PML‐I and, to a lesser extent, PML‐II under oleate‐treated conditions. Sec23B was observed to associate with both PML‐NBs as well as LAPS by immunofluorescence microscopy in U2OS cells expressing PML‐II‐APEX2. The COPII pathway has previously been implicated in protein delivery to cytoplasmic LD setting a precedence for the interaction we observed with LAPS. Thus, given our preliminary data showing interaction between Sec 23/24 heterodimer, PML‐NBs and LAPS we are now pursuing the hypothesis that lipid membrane deformation and/or cargo transport activity in the nucleus by COPII proteins could play a role in lipid homeostasis. [ABSTRACT FROM AUTHOR]

The p10 fusion-associated small transmembrane protein of avian reovirus induces extensive syncytium formation in transfected cells. Here we show that p10-induced cell-cell fusion is restricted by rapid degradation of the majority of newly synthesized p10. The small ectodomain of p10 targets the protein for degradation following p10 insertion into an early membrane compartment. Paradoxically, conservative amino acid substitutions in the p10 ectodomain hydrophobic patch that eliminate fusion activity also increase p10 stability. The small amount of p10 that escapes intracellular degradation accumulates at the cell surface in a relatively stable form, where it mediates cell-cell fusion as a late-stage event in the virus replication cycle. The unusual relationship between a nonstructural viral membrane fusion protein and the replication cycle of a nonenveloped virus has apparently contributed to the evolution of a novel mechanism for restricting the extent of virus-induced cell-cell fusion. [ABSTRACT FROM AUTHOR]