PML Regulates the Epidermal Differentiation Complex and Skin Morphogenesis during Mouse Embryogenesis.

The promyelocytic leukemia (PML) protein is an essential component of nuclear compartments called PML bodies. This protein participates in several cellular processes, including growth control, senescence, apoptosis, and differentiation. Previous studies have suggested that PML regulates gene expression at a subset of loci through a function in chromatin remodeling. Here we have studied global gene expression patterns in mouse embryonic skin derived from Pml depleted and wild type mouse embryos. Differential gene expression analysis at different developmental stages revealed a key role of PML in regulating genes involved in epidermal stratification. In particular, we observed dysregulation of the late cornified envelope gene cluster, which is a sub-region of the epidermal differentiation complex. In agreement with these data, PML body numbers are elevated in basal keratinocytes during embryogenesis, and we observed reduced epidermal thickness and defective hair follicle development in PML depleted mouse embryos.

Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1.

Nuclear lipid droplets (nLDs) form on the inner nuclear membrane by a mechanism involving promyelocytic leukemia (PML), the protein scaffold of PML nuclear bodies. We report that PML structures on nLDs in oleate-treated U2OS cells, referred to as lipid-associated PML structures (LAPS), differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100, and DAXX. These nLDs were also enriched in CTP:phosphocholine cytidylyltransferase α (CCTα), the phosphatidic acid phosphatase Lipin1, and DAG. Translocation of CCTα onto nLDs was mediated by its α-helical M-domain but was not correlated with its activator DAG. High-resolution imaging revealed that CCTα and LAPS occupied distinct polarized regions on nLDs. PML knockout U2OS (PML KO) cells lacking LAPS had a 40-50% reduction in nLDs with associated CCTα, and residual nLDs were almost devoid of Lipin1 and DAG. As a result, phosphatidylcholine and triacylglycerol synthesis was inhibited in PML KO cells. We conclude that in response to excess exogenous fatty acids, LAPS are required to assemble nLDs that are competent to recruit CCTα and Lipin1.

[PML isoforms and TGF-ß response]. / Les isoformes de PML et la réponse au TGF-ß.

PML/TRIM19 is the organizer of PML nuclear bodies (NB), a multiprotein complex associated to the nuclear matrix, which recruit a large number of proteins involved in various cellular processes. Alternative splicing from a single PML gene generates 6 nuclear PML isoforms (PMLI to PMLVI) and one cytoplasmic isoform, PMLVII. Murine PML-null primary cells are resistant to TGF-ß-induced apoptosis. Cytoplasmic PML is an essential activator of TGF-ß signaling by increasing the phosphorylation of transcription factors SMAD2/3 while nuclear PML plays a role in TGF-ß-induced caspase 8 activation and apoptosis. TGF-ß targets nuclear PML by inducing its conjugation to SUMO. In the nucleus, PML is mainly expressed in the nucleoplasm with a small fraction in the nuclear matrix. In response to TGF-ß, PML and caspase 8 shift to the nuclear matrix, where both PML and caspase 8 colocalise within PML NBs. Here, we review the implication of cytoplasmic and nuclear PML isoforms in TGF-ß response.

TITLE: Les isoformes de PML et la réponse au TGF-ß. ABSTRACT: PML (promyelocytic leukemia) est la protéine organisatrice des corps nucléaires, une structure multiprotéique associée à la matrice nucléaire, impliquée dans différents processus cellulaires. Sept isoformes principales de PML, dont six nucléaires (PMLI à VI) et une cytoplasmique (PMLVII), sont générées par épissage alternatif d'un gène unique. D'une part, PML dans le cytoplasme régule positivement le signal de transduction donné par le TGF-ß, en augmentant la phosphorylation des facteurs de transcription SMAD2/3 et, d'autre part, PML augmente dans le noyau l'activation de la caspase 8 et l'apoptose en réponse au TGF-ß. L'absence de PML rend les cellules résistantes à l'apoptose induite par le TGF-ß. Dans le noyau, PML est localisée majoritairement dans le nucléoplasme, une petite fraction étant cependant retrouvée dans la matrice nucléaire. Le TGF-ß cible PML dans le noyau en induisant sa conjugaison à SUMO (small ubiquitin modifier), son transfert et celui de la caspase 8 vers la matrice nucléaire où les deux protéines se localisent au sein des corps nucléaires PML. Cette revue rend compte des implications de PML dans le cytoplasme et le noyau dans la réponse au TGF-ß.

PML nuclear body-residing proteins sequentially associate with HPV genome after infectious nuclear delivery.

Subnuclear promyelocytic leukemia (PML) nuclear bodies (NBs) are targeted by many DNA viruses after nuclear delivery. PML protein is essential for formation of PML NBs. Sp100 and Small Ubiquitin-Like Modifier (SUMO) are also permanently residing within PML NBs. Often, large DNA viruses disassemble and reorganize PML NBs to counteract their intrinsic antiviral activity and support establishment of infection. However, human papillomavirus (HPV) requires PML protein to retain incoming viral DNA in the nucleus for subsequent efficient transcription. In contrast, Sp100 was identified as a restriction factor for HPV. These findings suggested that PML NBs are important regulators of early stages of the HPV life cycle. Nuclear delivery of incoming HPV DNA requires mitosis. Viral particles are retained within membrane-bound transport vesicles throughout mitosis. The viral genome is released from transport vesicles by an unknown mechanism several hours after nuclear envelope reformation. The minor capsid protein L2 mediates intracellular transport by becoming transmembranous in the endocytic compartment. Herein, we tested our hypothesis that PML protein is recruited to incoming viral genome prior to egress from transport vesicles. High-resolution microscopy revealed that PML protein, SUMO-1, and Sp100 are recruited to incoming viral genomes, rather than viral genomes being targeted to preformed PML NBs. Differential immunofluorescent staining suggested that PML protein and SUMO-1 associated with transport vesicles containing viral particles prior to egress, implying that recruitment is likely mediated by L2 protein. In contrast, Sp100 recruitment to HPV-harboring PML NBs occurred after release of viral genomes from transport vesicles. The delayed recruitment of Sp100 is specific for HPV-associated PML NBs. These data suggest that the virus continuously resides within a protective environment until the transport vesicle breaks down in late G1 phase and imply that HPV might modulate PML NB assembly to achieve establishment of infection and the shift to viral maintenance.

PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers.

High-grade serous ovarian cancer (HGSOC) remains an unmet medical challenge. Here, we unravel an unanticipated metabolic heterogeneity in HGSOC. By combining proteomic, metabolomic, and bioergenetic analyses, we identify two molecular subgroups, low- and high-OXPHOS. While low-OXPHOS exhibit a glycolytic metabolism, high-OXPHOS HGSOCs rely on oxidative phosphorylation, supported by glutamine and fatty acid oxidation, and show chronic oxidative stress. We identify an important role for the PML-PGC-1α axis in the metabolic features of high-OXPHOS HGSOC. In high-OXPHOS tumors, chronic oxidative stress promotes aggregation of PML-nuclear bodies, resulting in activation of the transcriptional co-activator PGC-1α. Active PGC-1α increases synthesis of electron transport chain complexes, thereby promoting mitochondrial respiration. Importantly, high-OXPHOS HGSOCs exhibit increased response to conventional chemotherapies, in which increased oxidative stress, PML, and potentially ferroptosis play key functions. Collectively, our data establish a stress-mediated PML-PGC-1α-dependent mechanism that promotes OXPHOS metabolism and chemosensitivity in ovarian cancer.

Pml nuclear body disruption cooperates in APL pathogenesis and impairs DNA damage repair pathways in mice.

A hallmark of acute promyelocytic leukemia (APL) is altered nuclear architecture, with disruption of promyelocytic leukemia (PML) nuclear bodies (NBs) mediated by the PML-retinoic acid receptor α (RARα) oncoprotein. To address whether this phenomenon plays a role in disease pathogenesis, we generated a knock-in mouse model with NB disruption mediated by 2 point mutations (C62A/C65A) in the Pml RING domain. Although no leukemias developed in PmlC62A/C65A mice, these transgenic mice also expressing RARα linked to a dimerization domain (p50-RARα model) exhibited a doubling in the rate of leukemia, with a reduced latency period. Additionally, we found that response to targeted therapy with all-trans retinoic acid in vivo was dependent on NB integrity. PML-RARα is recognized to be insufficient for development of APL, requiring acquisition of cooperating mutations. We therefore investigated whether NB disruption might be mutagenic. Compared with wild-type cells, primary PmlC62A/C65A cells exhibited increased sister-chromatid exchange and chromosome abnormalities. Moreover, functional assays showed impaired homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways, with defective localization of Brca1 and Rad51 to sites of DNA damage. These data directly demonstrate that Pml NBs are critical for DNA damage responses, and suggest that Pml NB disruption is a central contributor to APL pathogenesis.

C-terminal motifs in promyelocytic leukemia protein isoforms critically regulate PML nuclear body formation.

Promyelocytic leukemia protein (PML) nuclear bodies (NBs), which are sub-nuclear protein structures, are involved in a variety of important cellular functions. PML-NBs are assembled by PML isoforms, and contact between small ubiquitin-like modifiers (SUMOs) with the SUMO interaction motif (SIM) are critically involved in this process. PML isoforms contain a common N-terminal region and a variable C-terminus. However, the contribution of the C-terminal regions to PML-NB formation remains poorly defined. Here, using high-resolution microscopy, we show that mutation of the SIM distinctively influences the structure of NBs formed by each individual PML isoform, with that of PML-III and PML-V minimally changed, and PML-I and PML-IV dramatically impaired. We further identify several C-terminal elements that are important in regulating NB structure and provide strong evidence to suggest that the 8b element in PML-IV possesses a strong ability to interact with SUMO-1 and SUMO-2, and critically participates in NB formation. Our findings highlight the importance of PML C-termini in NB assembly and function, and provide molecular insight into the PML-NB assembly of each distinctive isoform.