PML Nuclear Bodies Research Articles

SUMO Modification of Hepatitis B Virus Core Mediates Nuclear Entry, Promyelocytic Leukemia Nuclear Body Association, and Efficient Formation of Covalently Closed Circular DNA.

Persistence of hepatitis B virus (HBV) infection is due to a nuclear covalently closed circular DNA (cccDNA), generated from the virion-borne relaxed circular DNA (rcDNA) genome in a process likely involving numerous cell factors from the host DNA damage response (DDR). The HBV core protein mediates rcDNA transport to the nucleus and likely affects stability and transcriptional activity of cccDNA. Our study aimed at investigating the role of HBV core protein and its posttranslational modification (PTM) with SUMO (small ubiquitin-like modifiers) during the establishment of cccDNA. HBV core protein SUMO PTM was analyzed in His-SUMO-overexpressing cell lines. The impact of HBV core SUMOylation on association with cellular interaction partners and on the HBV life cycle was determined using SUMOylation-deficient mutants of the HBV core protein. Here, we show that the HBV core protein is posttranslationally modified by the addition of SUMO and that this modification impacts nuclear import of rcDNA. By using SUMOylation-deficient HBV core mutants, we show that SUMO modification is a prerequisite for the association with specific promyelocytic leukemia nuclear bodies (PML-NBs) and regulates the conversion of rcDNA to cccDNA. By in vitro SUMOylation of HBV core, we obtained evidence that SUMOylation triggers nucleocapsid disassembly, providing novel insights into the nuclear import process of rcDNA. HBV core protein SUMOylation and subsequent association with PML bodies in the nucleus constitute a key step in the conversion of HBV rcDNA to cccDNA and therefore a promising target for inhibiting formation of the HBV persistence reservoir. IMPORTANCE HBV cccDNA is formed from the incomplete rcDNA involving several host DDR proteins. The exact process and the site of cccDNA formation are poorly understood. Here, we show that HBV core protein SUMO modification is a novel PTM regulating the function of HBV core. A minor specific fraction of the HBV core protein resides with PML-NBs in the nuclear matrix. SUMO modification of HBV core protein mediates its recruitment to specific PML-NBs within the host cell. Within HBV nucleocapsids, SUMOylation of HBV core induces HBV capsid disassembly and is a prerequisite for nuclear entry of HBV core. SUMO HBV core protein association with PML-NBs is crucial for efficient conversion of rcDNA to cccDNA and for the establishment of the viral persistence reservoir. HBV core protein SUMO modification and the subsequent association with PML-NBs might constitute a potential novel target in the development of drugs targeting the cccDNA.

ATR protects centromere identity by promoting DAXX association with PML nuclear bodies

Centromere protein A (CENP-A) defines centromere identity and nucleates kinetochore formation for mitotic chromosome segregation. Here, we show that ataxia telangiectasia and Rad3-related (ATR) kinase, a master regulator of the DNA damage response, protects CENP-A occupancy at interphase centromeres in a DNA damage-independent manner. In unperturbed cells, ATR localizes to promyelocytic leukemia nuclear bodies (PML NBs), which house the histone H3.3 chaperone DAXX (death domain-associated protein 6). We find that ATR inhibition reduces DAXX association with PML NBs, resulting in the DAXX-dependent loss of CENP-A and an aberrant increase in H3.3 at interphase centromeres. Additionally, we show that ATR-dependent phosphorylation within the C terminus of DAXX regulates CENP-A occupancy at centromeres and DAXX localization. Lastly, we demonstrate that acute ATR inhibition during interphase leads to kinetochore formation defects and an increased rate of lagging chromosomes. These findings highlight a mechanism by which ATR protects centromere identity and genome stability.

LDL Affects the Immunomodulatory Response of Endothelial Cells by Modulation of the Promyelocytic Leukemia Protein (PML) Expression via PKC.

In addition to its function as an intravascular lipid transporter, LDL also triggers signal transduction in endothelial cells (ECs), which, among other things, trigger immunomodulatory cascades, e.g., IL-6 upregulation. However, the molecular mechanisms of how these LDL-triggered immunological responses in ECs are realized are not fully understood. Since promyelocytic leukemia protein (PML) plays a role in promoting inflammatory processes, we examined the relationship between LDL, PML, and IL-6 in human ECs (HUVECs and EA.hy926 cells). RT-qPCR, immunoblotting, and immunofluorescence analyses showed that LDL but not HDL induced higher PML expression and higher numbers of PML-nuclear bodies (PML-NBs). Transfection of the ECs with a PML gene-encoding vector or PML-specific siRNAs demonstrated PML-regulated IL-6 and IL-8 expression and secretion after LDL exposure. Moreover, incubation with the PKC inhibitor sc-3088 or the PKC activator PMA showed that LDL-induced PKC activity leads to the upregulation of PML mRNA and PML protein. In summary, our experimental data suggest that high LDL concentrations trigger PKC activity in ECs to upregulate PML expression, which then increases production and secretion of IL-6 and IL-8. This molecular cascade represents a novel cellular signaling pathway with immunomodulatory effects in ECs in response to LDL exposure.

SOX2 downregulation of PML increases HCMV gene expression and growth of glioma cells.

The presence of human cytomegalovirus (HCMV) in glioblastoma (GBM) and improved outcomes of GBM patients receiving therapies targeting the virus have implicated HCMV in GBM progression. However, a unifying mechanism that accounts for the contribution of HCMV to the malignant phenotype of GBM remains incompletely defined. Here we have identified SOX2, a marker of glioma stem cells (GSCs), as a key determinant of HCMV gene expression in gliomas. Our studies demonstrated that SOX2 downregulated promyelocytic leukemia (PML) and Sp100 and consequently facilitated viral gene expression by decreasing the amount of PML nuclear bodies in HCMV-infected glioma cells. Conversely, the expression of PML antagonized the effects of SOX2 on HCMV gene expression. Furthermore, this regulation of SOX2 on HCMV infection was demonstrated in a neurosphere assay of GSCs and in a murine xenograft model utilizing xenografts from patient-derived glioma tissue. In both cases, SOX2 overexpression facilitated the growth of neurospheres and xenografts implanted in immunodeficient mice. Lastly, the expression of SOX2 and HCMV immediate early 1 (IE1) protein could be correlated in tissues from glioma patients, and interestingly, elevated levels of SOX2 and IE1 were predictive of a worse clinical outcome. These studies argue that HCMV gene expression in gliomas is regulated by SOX2 through its regulation of PML expression and that targeting molecules in this SOX2-PML pathway could identify therapies for glioma treatment.

A stress-induced cilium-to-PML-NB route drives senescence initiation

Cellular senescence contributes to tissue homeostasis and age-related pathologies. However, how senescence is initiated in stressed cells remains vague. Here, we discover that exposure to irradiation, oxidative or inflammatory stressors induces transient biogenesis of primary cilia, which are then used by stressed cells to communicate with the promyelocytic leukemia nuclear bodies (PML-NBs) to initiate senescence responses in human cells. Mechanistically, a ciliary ARL13B-ARL3 GTPase cascade negatively regulates the association of transition fiber protein FBF1 and SUMO-conjugating enzyme UBC9. Irreparable stresses downregulate the ciliary ARLs and release UBC9 to SUMOylate FBF1 at the ciliary base. SUMOylated FBF1 then translocates to PML-NBs to promote PML-NB biogenesis and PML-NB-dependent senescence initiation. Remarkably, Fbf1 ablation effectively subdues global senescence burden and prevents associated health decline in irradiation-treated mice. Collectively, our findings assign the primary cilium a key role in senescence induction in mammalian cells and, also, a promising target in future senotherapy strategies.

Issue Information

The FEBS JournalVolume 290, Issue 7 p. 1667-1669 Issue Cover & InformationFree Access Issue Information First published: 05 April 2023 https://doi.org/10.1111/febs.16490AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Cover Illustration Representative confocal immunofluorescence image showing the colocalization of promyelocytic leukaemia (PML) nuclear bodies with nuclear lipid droplets in a glioblastoma cell line. See the Original Article by Umaru et al. in this issue. Volume290, Issue7April 2023Pages 1667-1669 RelatedInformation

Bioinformatics analyses of combined databases identify shared differentially expressed genes in cancer and autoimmune disease

BackgroundInadequate immunity caused by poor immune surveillance leads to tumorigenesis, while excessive immunity due to breakdown of immune tolerance causes autoimmune genesis. Although the function of immunity during the onset of these two processes appears to be distinct, the underlying mechanism is shared. To date, gene expression data for large bodies of clinical samples are available, but the resemblances of tumorigenesis and autoimmune genesis in terms of immune responses remains to be summed up.MethodsConsidering the high disease prevalence, we chose invasive ductal carcinoma (IDC) and systemic lupus erythematosus (SLE) to study the potential commonalities of immune responses. We obtained gene expression data of IDC/SLE patients and normal controls from five IDC databases (GSE29044, GSE21422, GSE22840, GSE15852, and GSE9309) and five SLE databases (GSE154851, GSE99967, GSE61635, GSE50635, and GSE17755). We intended to identify genes differentially expressed in both IDC and SLE by using three bioinformatics tools including GEO2R, the limma R package, and Weighted Gene Co-expression Network Analysis (WGCNA) to perform function enrichment, protein-protein network, and signaling pathway analyses.ResultsThe mRNA levels of signal transducer and activator of transcription 1 (STAT1), 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase like (OASL), and PML nuclear body scaffold (PML) were found to be differentially expressed in both IDC and SLE by using three different bioinformatics tools of GEO2R, the limma R package and WGCNA. From the combined databases in this study, the mRNA levels of STAT1 and OAS1 were increased in IDC while reduced in SLE. And the mRNA levels of OASL and PML were elevated in both IDC and SLE. Based on Kyoto Encyclopedia of Genes and Genomes pathway analysis and QIAGEN Ingenuity Pathway Analysis, both IDC and SLE were correlated with the changes of multiple components involved in the Interferon (IFN)-Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway.ConclusionThe expression levels of STAT1 and OAS1 manifest the opposite expression tendency across cancer and autoimmune disease. They are components in the IFN-JAK-STAT signaling pathway related to both tumorigenesis and autoimmune genesis. STAT1 and OAS1-associated IFN-JAK-STAT signaling could explain the commonalities during tumorigenesis and autoimmune genesis and render significant information for more precise treatment from the point of immune homeostasis.

Crosstalk between PML and p53 in response to TGF-β1: A new mechanism of cardiac fibroblast activation.

Cardiac fibrosis is a common pathological cardiac remodeling in a variety of heart diseases, characterized by the activation of cardiac fibroblasts. Our previous study uncovered that promyelocytic leukemia protein (PML)-associated SUMO processes is a new regulator of cardiac hypertrophy and heart failure. The present study aimed to explore the role of PML in cardiac fibroblasts activation. Here we found that PML is significantly upregulated in cardiac fibrotic tissue and activated cardiac fibroblasts treated with transforming growth factor-β1 (TGF-β1). Gain- and loss-of-function experiments showed that PML impacted cardiac fibroblasts activation after TGF-β1 treatment. Further study demonstrated that p53 acts as the transcriptional regulator of PML, and participated in TGF-β1 induced the increase of PML expression and PML nuclear bodies (PML-NBs) formation. Knockdown or pharmacological inhibition of p53 produced inhibitory effects on the activation of cardiac fibroblasts. We further found that PML also may stabilize p53 through inhibiting its ubiquitin-mediated proteasomal degradation in cardiac fibroblasts. Collectively, this study suggests that PML crosstalk with p53 regulates cardiac fibroblasts activation, which provides a novel therapeutic strategy for cardiac fibrosis.

Nicotinamide Mononucleotide Administration Prevents Doxorubicin-Induced Cardiotoxicity and Loss in Physical Activity in Mice.

Doxorubicin (Doxo) is a widely used antineoplastic drug with limited clinical application due to its deleterious dose-related side effects. We investigated whether nicotinamide mononucleotide (NMN) could protect against Doxo-induced cardiotoxicity and physical dysfunction in vivo. To assess the short- and long-term toxicity, two Doxo regimens were tested, acute and chronic. In the acute study, C57BL6/J (B6) mice were injected intraperitoneally (i.p.) once with Doxo (20 mg/kg) and NMN (180 mg/kg/day, i.p.) was administered daily for five days before and after the Doxo injection. In the chronic study, B6 mice received a cumulative dose of 20 mg/kg Doxo administered in fractionated doses for five days. NMN (500 mg/kg/day) was supplied in the mice's drinking water beginning five days before the first injection of Doxo and continuing for 60 days after. We found that NMN significantly increased tissue levels of NAD+ and its metabolites and improved survival and bodyweight loss in both experimental models. In addition, NMN protected against Doxo-induced cardiotoxicity and loss of physical function in acute and chronic studies, respectively. In the heart, NMN prevented Doxo-induced transcriptomic changes related to mitochondrial function, apoptosis, oxidative stress, inflammation and p53, and promyelocytic leukemia nuclear body pathways. Overall, our results suggest that NMN could prevent Doxo-induced toxicity in heart and skeletal muscle.

Recruitment of TRIM33 to cell-context specific PML nuclear bodies regulates nodal signaling in mESCs.

TRIM33 is a chromatin reader required for mammalian mesendoderm differentiation after activation of Nodal signaling, while its role in mESCs is still elusive. Here, we report that TRIM33 co-localizes with promyelocytic leukemia nuclear bodies (PML-NBs) specifically in mESCs, to mediate Nodal signaling-directed transcription of Lefty1/2. We show that TRIM33 puncta formation in mESCs depends on PML and on specific assembly of PML-NBs. Moreover, TRIM33 and PML co-regulate Lefty1/2 expression in mESCs, with both PML protein and formation of mESCs-specific PML-NBs being required for TRIM33 recruitment to these loci, and PML-NBs directly associating with the Lefty1/2 loci. Finally, a TurboID proximity-labeling experiment confirmed that TRIM33 is highly enriched only in mESCs-specific PML-NBs. Thus, our study supports a model in which TRIM33 condensates regulate Nodal signaling-directed transcription in mESCs and shows that PML-NBs can recruit distinct sets of client proteins in a cell-context-dependent manner.

Preprint Highlight: ATR protects centromere identity by promoting DAXX association with PML nuclear bodies.

ATR ( Ataxia telangiectasia and Rad3-related) is an essential kinase in the DNA damage response, but it also displays some DNA damage-independent roles in mitosis. By using an ATR inhibitor and genetic manipulations, this work shows that ATR phosphorylates the histone H3.3 chaperone DAXX to maintain its localization to PML (promyelocytic leukemia) nuclear bodies in interphase cells and thus ensures proper localization of centromere protein A (CENP-A) to centromeres. Reduced CENP-A localization on centromeres after ATR inhibition lasted through mitosis, resulting in chromosome segregation errors. This work adds to the understanding of ATR's role in genome stability independent of DNA damage response and may shed new light on how to reduce toxicity of ATR inhibitor-based cancer therapeutics.

Oleic acid-bound FABP7 drives glioma cell proliferation through regulation of nuclear lipid droplet formation.

Fatty acid-binding protein 7 (FABP7), one of the fatty acid (FA) chaperones involved in the modulation of intracellular FA metabolism, is highly expressed in glioblastoma, and its expression is associated with decreased patients' prognosis. Previously, we demonstrated that FABP7 requires its binding partner to exert its function and that a mutation in the FA-binding site of FABP7 affects tumour biology. Here, we explored the role of FA ligand binding for FABP7 function in tumour proliferation and examined the mechanism of FABP7 and ligand interaction in tumour biology. We discovered that among several FA treatment, oleic acid (OA) boosted cell proliferation of FABP7-expressing cells. In turn, OA increased FABP7 nuclear localization, and the accumulation of FABP7-OA complex in the nucleus induced the formation of nuclear lipid droplet (nLD), as well as an increase in colocalization of nLD with promyelocytic leukaemia (PML) nuclear bodies. Furthermore, OA increased mRNA levels of proliferation-related genes in FABP7-expressing cells through histone acetylation. Interestingly, these OA-boosted functions were abrogated in FABP7-knockout cells and mutant FABP7-overexpressing cells. Thus, our findings suggest that FABP7-OA intracellular interaction may modulate nLD formation and the epigenetic status thereby enhancing transcriptionof proliferation-regulating genes, ultimately driving tumour cell proliferation.

Targeting Interferon Pathway in TBLR1-RARa-Driven Promyelocytic Leukemia in the Context of ATRA and ATO Unresponsiveness

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) mostly driven by the t(15;17) translocation that yields the PML-RARα (PR) fusion protein. Apart from PML, other partner genes have been uncovered to fuse with RARα forming rare X-RARα fusion genes. Among them, TBLR1-RARα (TR) is a novel oncogene to induce APL identified in our previous studies, which elicited differed response and worse prognosis in contrast to the canonical PR fusion gene. Despite the textbook rationale of all-trans retinoid acid (ATRA) and arsenic trioxide (ATO) turning most APL patients with PR fusion gene from highly fatal to highly curable, TR-driven APL did not yield long-term remission both in murine models and patients, highlighting the necessity to dissect the mechanism of drug unresponsiveness and explore novel targeted therapies. In this study, we first established two doxycycline-inducible leukemia cell lines to directly initiate the expression of TR and PR fusion protein in U937 cells, respectively. Transcriptome sequencing analysis showed that interferon (IFN) pathways were significantly suppressed in TR rather than PR-induced leukemia cells. Then we administered IFNs as well as the standard regimen of ATRA and ATO in TR- and PR-induced APL, comparing therapy responses and biological phenotypes with in vitro and in vivo studies. Main results are summarized as follows concerning TR- in comparison to PR-driven APL. Firstly, ATO was unresponsive in TR-induced leukemia cells, which failed to elicit oncoprotein degradation, apoptosis and loss of self-renewal. Secondly, lower doses of ATRA were inadequate. Although a lower dose of ATRA was sufficient to trigger differentiation, increasing doses of ATRA further promoted the degradation of oncoprotein and reduced the colony formation capacity of TR leukemic cells, which ultimately conferred survival benefits in TR mice. Thirdly, type I IFNs was promising, which induced apoptosis, cooperated with ATRA to boost differentiation, and exhibited potential to reduce self-renewal, reflecting anti-leukemia efficacy. Finally, type I IFNs combining with ATRA displayed survival advantage to TR mice, and murine STING agonist DMXAA, which acted upstream to produce type I IFNs, significantly endowed TR mice with extended survival time. Based on our results, we further propose mechanistic hypotheses of TR-APL in comparison with the classic PR-APL. In TR-driven APL, despite the unresponsiveness of ATO, we may utilize STING-IFN pathway agonist to upregulate PML expression, which may obliterate self-renewal independent of ATO response triggered by initial PML nuclear body disruption observed in PR-driven APL, providing therapeutic implications for APL driven by rare RARα fusion genes. Our study aims to gain further understanding of TR-driven APL and integrate insights into principles underlying leukemogenesis mediated by rare RARα fusion genes, which may benefit such a therapy-resistant population and enable APL to be a bona fide curable leukemia.

The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers.

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.

Promyelocytic Leukemia Protein Potently Restricts Human Cytomegalovirus Infection in Endothelial Cells.

PML nuclear bodies (PML-NBs) are dynamic macromolecular complexes that mediate intrinsic immunity against viruses of different families, including human cytomegalovirus (HCMV). Upon HCMV infection, PML-NBs target viral genomes entering the nucleus and restrict viral immediate–early gene expression by epigenetic silencing. Studies from several groups performed in human fibroblast cells have shown that the major PML-NB components PML, Daxx, Sp100 and ATRX contribute to this repression in a cooperative manner. Their role for HCMV restriction in endothelial cells, however, has not yet been characterized although infected endothelium is thought to play a crucial role for HCMV dissemination and development of vascular disease in vivo. Here, we use conditionally immortalized umbilical vein endothelial cells (HEC-LTT) as a cell culture model to elucidate the impact of PML-NB proteins on lytic HCMV infection. Depletion of individual PML-NB proteins by lentiviral transduction showed a particularly strong antiviral effect of PML in HEC-LTT, compared to human fibroblasts. A closer characterization of this antiviral function revealed that PML may not only effectively inhibit HCMV immediate-early gene expression but also act at later steps of the viral replication cycle. At contrast, we surprisingly noted an antiviral behavior of Daxx in complementary approaches: Depletion of Daxx resulted in decreased viral gene expression, while overexpression of Daxx promoted HCMV infection. In summary, our data demonstrate a cell type-specific effect of PML-NB components on lytic HCMV infection and suggest an important role of PML in the inhibition of HCMV dissemination through infected endothelial cells.

Exploration of nuclear body-enhanced sumoylation reveals that PML represses 2-cell features of embryonic stem cells

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation. PML NBs recruit many partner proteins, but the actual biochemical mechanism underlying their pleiotropic functions remains elusive. Similarly, PML role in embryonic stem cell (ESC) and retro-element biology is unsettled. Here we demonstrate that PML is essential for oxidative stress-driven partner SUMO2/3 conjugation in mouse ESCs (mESCs) or leukemia, a process often followed by their poly-ubiquitination and degradation. Functionally, PML is required for stress responses in mESCs. Differential proteomics unravel the KAP1 complex as a PML NB-dependent SUMO2-target in arsenic-treated APL mice or mESCs. PML-driven KAP1 sumoylation enables activation of this key epigenetic repressor implicated in retro-element silencing. Accordingly, Pml−/− mESCs re-express transposable elements and display 2-Cell-Like features, the latter enforced by PML-controlled SUMO2-conjugation of DPPA2. Thus, PML orchestrates mESC state by coordinating SUMO2-conjugation of different transcriptional regulators, raising new hypotheses about PML roles in cancer.

Reliable assessment of telomere maintenance mechanisms in neuroblastoma

BackgroundTelomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved.MethodsWhole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15).ResultsTumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations.ConclusionsWe here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients.

Regulating the p53 Tumor Suppressor Network at PML Biomolecular Condensates.

Simple SummaryThe cell nucleus is organized into different sub-nuclear compartments to control specific cellular processes, including PML nuclear bodies (NBs), also termed PML biomolecular condensates. PML-NBs form multi-protein complexes that are highly responsive to cellular stress, and regulate cell fate decisions in response to genome damage. By concentrating proteins that directly control the activity of the tumor suppressor p53, PML biocondensates guide the cellular response either towards induction of cell death or senescence through altering p53 modifications. In this review, we discuss the molecular mechanisms controlling PML condensate formation, and how they impact the regulation of p53 activity, and propose a list of promising candidate proteins that may contribute to the regulation of p53 at PML biomolecular condensates.By forming specific functional entities, nuclear biomolecular condensates play an important function in guiding biological processes. PML biomolecular condensates, also known as PML nuclear bodies (NBs), are macro-molecular sub-nuclear organelles involved in central biological processes, including anti-viral response and cell fate control upon genotoxic stress. PML condensate formation is stimulated upon cellular stress, and relies on protein–protein interactions establishing a PML protein meshwork capable of recruiting the tumor suppressor p53, along with numerous modifiers of p53, thus balancing p53 posttranslational modifications and activity. This stress-regulated process appears to be controlled by liquid–liquid phase separation (LLPS), which may facilitate regulated protein-unmixing of p53 and its regulators into PML nuclear condensates. In this review, we summarize and discuss the molecular mechanisms underlying PML nuclear condensate formation, and how these impact the biological function of p53 in driving the cell death and senescence responses. In addition, by using an in silico approach, we identify 299 proteins which share PML and p53 as binding partners, thus representing novel candidate proteins controlling p53 function and cell fate decision-making at the level of PML nuclear biocondensates.

Smc5/6 silences episomal transcription by a three-step function.

In addition to its role in chromosome maintenance, the six-membered Smc5/6 complex functions as a restriction factor that binds to and transcriptionally silences viral and other episomal DNA. However, the underlying mechanism is unknown. Here, we show that transcriptional silencing by the human Smc5/6 complex is a three-step process. The first step is entrapment of the episomal DNA by a mechanism dependent on Smc5/6 ATPase activity and a function of its Nse4a subunit for which the Nse4b paralog cannot substitute. The second step results in Smc5/6 recruitment to promyelocytic leukemia nuclear bodies by SLF2 (the human ortholog of Nse6). The third step promotes silencing through a mechanism requiring Nse2 but not its SUMO ligase activity. By contrast, the related cohesin and condensin complexes fail to bind to or silence episomal DNA, indicating a property unique to Smc5/6.

The chromatin remodeling protein ATRX positively regulates IRF3-dependent type I interferon production and interferon-induced gene expression.

The chromatin remodeling protein alpha thalassemia/mental retardation syndrome X-linked (ATRX) is a component of promyelocytic leukemia nuclear bodies (PML-NBs) and thereby mediates intrinsic immunity against several viruses including human cytomegalovirus (HCMV). As a consequence, viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation. Here, we show that depletion of ATRX results in an overall impaired antiviral state by decreasing transcription and subsequent secretion of type I IFNs, which is followed by reduced expression of interferon-stimulated genes (ISGs). ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-β promoter to facilitate transcription. Furthermore, whole transcriptome sequencing revealed that ATRX is required for efficient IFN-induced expression of a distinct set of ISGs. Mechanistically, we found that ATRX positively modulates chromatin accessibility specifically upon IFN signaling, thereby affecting promoter regions with recognition motifs for AP-1 family transcription factors. In summary, our study uncovers a novel co-activating function of the chromatin remodeling factor ATRX in innate immunity that regulates chromatin accessibility and subsequent transcription of interferons and ISGs. Consequently, ATRX antagonization by viral proteins and ATRX mutations in tumors represent important strategies to broadly compromise both intrinsic and innate immune responses.