CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization.

Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.

Alternative lengthening of telomeres is a self-perpetuating process in ALT-associated PML bodies.

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.

Genomic Profiling by ALaP-Seq Reveals Transcriptional Regulation by PML Bodies through DNMT3A Exclusion.

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.

The Nuclear SUMO-Targeted Ubiquitin Quality Control Network Regulates the Dynamics of Cytoplasmic Stress Granules.

Exposure of cells to heat or oxidative stress causes misfolding of proteins. To avoid toxic protein aggregation, cells have evolved nuclear and cytosolic protein quality control (PQC) systems. In response to proteotoxic stress, cells also limit protein synthesis by triggering transient storage of mRNAs and RNA-binding proteins (RBPs) in cytosolic stress granules (SGs). We demonstrate that the SUMO-targeted ubiquitin ligase (StUbL) pathway, which is part of the nuclear proteostasis network, regulates SG dynamics. We provide evidence that inactivation of SUMO deconjugases under proteotoxic stress initiates SUMO-primed, RNF4-dependent ubiquitylation of RBPs that typically condense into SGs. Impairment of SUMO-primed ubiquitylation drastically delays SG resolution upon stress release. Importantly, the StUbL system regulates compartmentalization of an amyotrophic lateral sclerosis (ALS)-associated FUS mutant in SGs. We propose that the StUbL system functions as surveillance pathway for aggregation-prone RBPs in the nucleus, thereby linking the nuclear and cytosolic axis of proteotoxic stress response.

Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

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.

TSPYL5 Depletion Induces Specific Death of ALT Cells through USP7-Dependent Proteasomal Degradation of POT1.

A significant fraction (∼10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). There are no known molecular, ALT-specific, therapeutic targets. We have identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1-a shelterin component-and protects POT1 from proteasomal degradation exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies and identify TSPYL5 as an ALT+ cancer-specific therapeutic target.

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells.

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

PML isoforms: a molecular basis for PML pleiotropic functions.

PML is a stress-responsive protein that coordinates assembly of phase-separated nuclear aggregates, known as PML nuclear bodies (PML-NBs), where a large number of protein interactors and chromatin processes are finely regulated. Tampering with the PML gene produces a variety of phenotypic consequences that include promoting or interfering with tumor progression but the molecular underpinnings of PML pleiotropy are still elusive. In this review, we explore the contribution of PML splicing isoforms to PML-NB assorted activities. We describe recent literature indicating that distinct PML isoforms drive formation of specialized PML-NBs and perform unique functions and we suggest that future research efforts should delve into the contribution of isoform specificity to help elucidate the complex functionality of the PML gene.

A Systems-Level Study Reveals Regulators of Membrane-less Organelles in Human Cells.

Membrane-less organelles (MLOs) are liquid-like subcellular compartments that form through phase separation of proteins and RNA. While their biophysical properties are increasingly understood, their regulation and the consequences of perturbed MLO states for cell physiology are less clear. To study the regulatory networks, we targeted 1,354 human genes and screened for morphological changes of nucleoli, Cajal bodies, splicing speckles, PML nuclear bodies (PML-NBs), cytoplasmic processing bodies, and stress granules. By multivariate analysis of MLO features we identified hundreds of genes that control MLO homeostasis. We discovered regulatory crosstalk between MLOs, and mapped hierarchical interactions between aberrant MLO states and cellular properties. We provide evidence that perturbation of pre-mRNA splicing results in stress granule formation and reveal that PML-NB abundance influences DNA replication rates and that PML-NBs are in turn controlled by HIP kinases. Together, our comprehensive dataset is an unprecedented resource for deciphering the regulation and biological functions of MLOs.

DAXX safeguards heterochromatin formation in embryonic stem cells.

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the three-dimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in de-repression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability.

Exploring JC polyomavirus sequences and human gene expression in brain tissue of patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a rare neurological condition associated with reactivation of dormant JC polyomavirus (JCPyV). In this study, we characterized gene expression and JCPyV rearrangements in PML brain tissue. Infection of white matter astrocytes and oligodendrocytes as well as occasional brain cortex neurons was shown. PML brain harbored exclusively rearranged JCPyV variants. Viral transcripts covered the whole genome on both strands. Strong differential expression of human genes associated with neuroinflammation, blood-brain-barrier permeability and neurodegenerative diseases was shown. Pathway analysis revealed wide immune activation in PML brain. The study provides novel insights into the pathogenesis of PML.

Successful BK virus-specific T cell therapy in a kidney transplant recipient with progressive multifocal leukoencephalopathy.

The strategy for progressive multifocal leukoencephalopathy (PML) in solid organ transplant recipients primarily focuses on reducing immunosuppressive therapy. However, this approach offers limited efficacy and carries a high risk of graft loss. Here, we present the case of a 64-year-old male kidney transplant recipient with a high degree of immunosuppression who developed PML in October 2022. Despite the standard reduction of immunosuppressive therapy, the patient's condition continued to deteriorate, as evidenced by worsening neurological symptoms and increasing JC virus (JCV) DNA levels in cerebrospinal fluid. This prompted the innovative use of BKPyV-virus-specific T cell (BKPyV-VST) therapy, given the genetic similarities between BK and JCVs. Infusion of third-party donor BKPyV-VST resulted in clinical stabilization, a significant reduction in JCV-DNA levels, and the emergence of a JCV-specific T cell response, as observed in enzyme-linked immunospot assays and TCRß sequencing. This represents the first case report of successful third-party BKPyV-VST therapy in a kidney recipient presenting PML, without graft-versus-host disease or graft dysfunction.

LINE-1: an emerging initiator of cGAS-STING signalling and inflammation that is dysregulated in disease.

The cGAS-STING (cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)) axis integrates DNA damage and cellular stress with type I interferon (IFN) signalling to facilitate transcriptional changes underlying inflammatory stress responses. The cGAS-STING pathway responds to cytosolic DNA in the form of double-stranded DNA, micronuclei, and long interspersed nuclear element 1 (L1) retroelements. L1 retroelements are a class of self-propagating non-long terminal repeat transposons that have remained highly active in mammalian genomes. L1 retroelements are emerging as important inducers of cGAS-STING and IFN signalling, which are often dysregulated in several diseases, including cancer. A key repressor of cGAS-STING and L1 activity is the exonuclease three prime repair exonuclease 1 (TREX1), and loss of TREX1 promotes the accumulation of L1. In addition, L1 dysregulation is a common theme among diseases with chronic induction of type I IFN signalling through cGAS-STING, such as Aicardi-Goutières syndrome, Fanconi anemia, and dermatomyositis. Although TREX1 is highly conserved in tetrapod species, other suppressor proteins exist that inhibit L1 retrotransposition. These suppressor genes when mutated are often associated with diseases characterized by unchecked inflammation that is associated with high cGAS-STING activity and elevated levels of L1 expression. In this review, we discuss these interconnected pathways of L1 suppression and their role in the regulation of cGAS-STING and inflammation in disease.

Nuclear lipid droplets - how are they different from their cytoplasmic siblings?

Lipid droplets (LDs) in the cytoplasm are formed in the endoplasmic reticulum (ER) and are connected with various organelles, both structurally and functionally. This is in contrast to LDs in the nucleus, which are separated from organelles in the cytoplasm. How nuclear lipid droplets form and what function they have were not known for many years. Recent results have revealed that nuclear LDs in hepatocytes are derived from lipoprotein precursors in the ER lumen, whereas those in non-hepatocytes and budding yeast newly form in the inner nuclear membrane. Although nuclear LDs are far fewer in number than cytoplasmic LDs, the unique location appears to bestow upon them specific functions, which are potentially linked to nuclear biology. This Review will provide an overview of our current understanding of nuclear LDs, discuss how they are different from cytoplasmic LDs and highlight knowledge gaps that need to be filled in future studies.

SLF2 Interacts with the SMC5/6 Complex to Direct Hepatitis B Virus Episomal DNA to Promyelocytic Leukemia Bodies for Transcriptional Repression.

Hepatitis B virus (HBV) chronically infects approximately 300 million people worldwide, and permanently repressing transcription of covalently closed circular DNA (cccDNA), the episomal viral DNA reservoir, is an attractive approach toward curing HBV. However, the mechanism underlying cccDNA transcription is only partially understood. In this study, by illuminating cccDNA of wild-type HBV (HBV-WT) and transcriptionally inactive HBV that bears a deficient HBV X gene (HBV-ΔX), we found that the HBV-ΔX cccDNA more frequently colocalizes with promyelocytic leukemia (PML) bodies than that of HBV-WT cccDNA. A small interfering RNA (siRNA) screen targeting 91 PML body-related proteins identified SMC5-SMC6 localization factor 2 (SLF2) as a host restriction factor of cccDNA transcription, and subsequent studies showed that SLF2 mediates HBV cccDNA entrapment in PML bodies by interacting with the SMC5/6 complex. We further showed that the region of SLF2 comprising residues 590 to 710 interacts with and recruits the SMC5/6 complex to PML bodies, and the C-terminal domain of SLF2 containing this region is necessary for repression of cccDNA transcription. Our findings shed new light on cellular mechanisms that inhibit HBV infection and lend further support for targeting the HBx pathway to repress HBV activity. IMPORTANCE Chronic HBV infection remains a major public health problem worldwide. Current antiviral treatments rarely cure the infection, as they cannot clear the viral reservoir, cccDNA, in the nucleus. Therefore, permanently silencing HBV cccDNA transcription represents a promising approach for a cure of HBV infection. Our study provides new insights into the cellular mechanisms that restrict HBV infection, revealing the role of SLF2 in directing HBV cccDNA to PML bodies for transcriptional repression. These findings have important implications for the development of antiviral therapies against HBV.

Spinal cord involvement in progressive multifocal leukoencephalopathy and immune reconstitution inflammatory syndrome.

Progressive multifocal leukoencephalopathy (PML) is an opportunistic infectious demyelinating disease of the central nervous system caused by JC polyomavirus predominantly affecting immunocompromised individuals. Nowadays, HIV, hematological malignancies and iatrogenic immune suppression account for most PML cases. For unknown reasons, spinal cord is classically protected from PML lesions. Here, we report the course of a patient harboring spinal cord lesions in the context of PML with immune reconstitution inflammatory syndrome and review the eight other cases reported in the literature so far. Then, we discuss the evolving spectrum of PML over recent years, potentially making its diagnosis more challenging.

Phase separation is required for PML nuclear body biogenesis and function.

PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid-liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild-type (WT) NBs, PML A216V derived from arsenic-resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled-coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS-crippled and uncrippled NBs showed aggregation- and ring-like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS-driven NB formation was the prerequisite for partner recruitment, post-translational modifications (PTMs), and PML-driven cellular regulations, such as ROS stress control, mitochondria production, and PML-p53-mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis.

Identification of a novel fusion gene, RARA::ANKRD34C, in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia that is distinguished by the chromosomal translocation t(15;17)(q24;q21), which leads to the fusion of the promyelocytic leukemia (PML) gene with the retinoic acid receptor alpha (RARA). Recently, we identified a novel fusion gene in APL, RARA::ankyrin repeat domain 34C (ANKRD34C), identified its functions by morphological, cytogenetic, molecular biological and multiplex fluorescence in situ hybridization analyses, and demonstrated the potential therapeutic effect clinically and experimentally of all-trans retinoic acid (ATRA); the findings have important implications for the diagnosis and treatment of atypical APL.

Progressive multifocal leukoencephalopathy in a patient with B-cell chronic lymphocytic leukemia after COVID-19 vaccination, complicated with COVID-19 and mucormycosis: a case report.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a rare and fatal opportunistic viral demyelinating infectious disease of the central nervous system (CNS). There are various clinical presenting symptoms for the disease. CASE PRESENTATION: This paper presents a clinical case of PML in a patient with B-Chronic lymphocytic leukemia (B-CLL), previously treated with Chlorambucil, later complicated later with COVID-19 and mucormycosis. CONCLUSION: PML can develop in the setting of cellular immune dysfunction. Late diagnosis of this disease based on nonspecific symptoms is common, therefore when we face a neurological complication in a CLL or immunocompromised patient, we should consider PML infection. A remarkable feature of this case is the possible triggering effect of COVID-19 vaccination for emergence of PML as the disease can be asymptomatic or sub-clinical before diagnosis.