Skeletal muscle regeneration is driven by muscle stem cells (MuSCs), which proliferate, differentiate, and fuse to reform myofibers and restore muscle function. This myogenesis process is driven both by intrinsic MuSC properties and extrinsic cues. While coordinated inflammatory signals are necessary for healthy regeneration, chronic inflammation participates in various pathologies affecting the skeletal muscle. In the idiopathic inflammatory myopathy dermatomyositis (DM), MuSCs exhibit impaired myogenesis in vitro, indicating that they may have acquired intrinsic defects, contributing to the disease and providing a mechanism for sustained patient muscle weakness despite efficient anti-inflammatory treatments. Here, we investigated the transcriptomic regulation of DM-derived MuSCs, with a focus on the H3.3 histone variant that regulates myogenesis progression. DM-derived MuSCs were unable to effectively execute the myogenic transcriptional program during in vitro differentiation. They exhibited an activated canonical tumor necrosis factor (TNF)-⍺ signaling. They also showed reduced expression of H3.3 and its chaperone genes, coupled with a decrease in H3.3 deposition across the entire genome, and particularly at myogenic regulatory factor loci. The loss of H3.3 combined with elevated TNF-⍺ signaling was associated with a failure of DM-derived MuSCs to achieve myogenesis, suggesting a mechanistic link between epigenetic dysregulation and defective muscle regeneration in humans.

The cGAS-cGAMP-STING pathway is crucial for antiviral immunity. While cytosolic cGAS detects viral DNA, most DNA viruses shield their genome and invade the nucleus, where chromatin restricts cGAS activation. How viruses may activate nuclear cGAS is not well understood. Here, we show that several herpesvirus proteins trigger nuclear cGAS activation by perturbing centromeres, where cGAS is enriched. The herpes simplex virus type 1 (HSV-1) ubiquitin ligase infected cell protein 0 (ICP0), which degrades centromeric proteins, promotes centromeric DNA amplification through the translesion DNA synthesis (TLS) pathway in quiescent monocyte-derived cells, thereby activating nuclear cGAS. During infection, HSV-1 evades this detection by also expressing UL36USP, a suppressor of TLS. Similarly to ICP0, the cytomegalovirus IE1 protein causes centromeric DNA amplification and cGAS activation. We define this mechanism as viral-induced centromeric DNA amplification and recognition (VICAR), uncovering a non-mitotic, immune-activating role of centromeres.

Loss of ATRX function, a diagnostic criterion of IDH-mutant astrocytoma, is closely associated with alternative lengthening of telomeres (ALT), a telomere maintenance mechanism (TMM). As immunohistochemical (IHC) assessment of ATRX is error-prone, sequencing has been integrated into clinical workflows. While frameshift and nonsense variants can be classified as loss-of-function (LOF) mutations, missense variants remain difficult to interpret. To address this, we analyzed ATRX-altered gliomas from TCGA (N = 539 tumors, 587 alterations) and a local cohort of 100 diffuse gliomas. Aside from IDH-mutant astrocytoma and H3.3-mutant glioma, glioblastoma (13–19

Cyclic GMP-AMP synthase (cGAS), initially identified as a cytosolic sensor for double-stranded DNA, is now widely recognized as a nuclear protein with distinct STING-independent functions. Its presence in the same compartment as genomic DNA highlights the critical need to regulate its nuclear levels to balance the risk of cell-intrinsic immune activation with the recognition of pathogenic DNA. The recent discovery of a proteasome-dependent degradation mechanism for chromatin-bound cGAS offers new insights into the regulation of nuclear cGAS. In this study, we examine the dynamics and stability of nuclear cGAS following DNA damage. We demonstrate that cGAS is released from chromatin in a process dependent on the p97 segregase, followed by its degradation. When protein degradation is blocked, cGAS accumulates in foci juxtaposed to PML nuclear bodies (PML NBs). We show that this juxtaposition is SUMO-dependent, with both SUMO and PML required for cGAS degradation, a process that also involves the Cullin3-RING E3 ubiquitin Ligase complex. Increasing cGAS levels on chromatin after damage by preventing its degradation expands its localization on chromatin, correlating with a dampened DNA damage response and impaired senescence entry. Overall, our findings show that a p97-PML NBs axis modulates cGAS abundance, ensuring proper regulation of the DNA damage response and balancing the possible cell-intrinsic activation of innate immune responses with senescence entry. ### Competing Interest Statement The authors have declared no competing interest. Centre National de la Recherche Scientifique, https://ror.org/02feahw73, CNRS Prematuration program 2021-2023 Inserm, https://ror.org/02vjkv261 Université Claude Bernard Lyon 1, https://ror.org/029brtt94 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, EPIPRO ANR-18-CE15-0014-01, CHROMACoV ANR-20-COV9-0004, IFN-Epi-IM ANR-21-CE17-0018 Labex DEV2CAN Association Francaise contre les Myopathies, https://ror.org/0162y2387, MyoNeurALP, MyoNeurALP2 La Ligue Contre le Cancer, https://ror.org/00rkrv905, Comité départemental du Rhône Fondation ARC pour la Recherche sur le Cancer, https://ror.org/0489qz649, 4th year PhD Institut Universitaire de France, https://ror.org/055khg266

We evaluated the Lattice di-SIM 3D structural illumination method with a focus on Promyelocytic Leukemia Nuclear Bodies (PML NBs). Lattice SIM2's performance was compared to its predecessor using biological samples and calibration beads. Optimized SIM2 parameters revealed PML NBs with a distinct ring-like morphology. Furthermore, we compared Lattice-SIM2 with dSTORM, finding a strong correlation between blinking events and the SIM2 mask. For 2D nanoscopic correlation, we used the long-lived imaging buffer Eternity, while the modified variant, Eternity-SIM, with a higher refractive index, provided improved 3D correlation for SIM2 imaging.

Herpes simplex virus 1 (HSV-1) latently infected neurons display diverse patterns in the distribution of the viral genomes within the nucleus. A key pattern involves quiescent HSV-1 genomes sequestered in promyelocytic leukemia nuclear bodies (PML NBs) forming viral DNA- containing PML-NBs (vDCP NBs). Using a cellular model that replicates vDCP NB formation, we previously demonstrated that these viral genomes are chromatinized with the H3.3 histone variant modified on its lysine 9 by trimethylation (H3.3K9me3), a mark associated with transcriptional repression. Here, we identify the HUSH complex and its effectors, SETDB1 and MORC2, as crucial for the acquisition of H3K9me3 on PML NB- associated HSV-1 and the maintenance of HSV-1 transcriptional repression. ChIP-seq analyses show H3K9me3 association with the entire viral genome. Inactivating the HUSH-SETDB1-MORC2 complex before infection significantly reduces H3K9me3 on the viral genome, with minimal impact on the cellular genome, aside from expected changes in LINE- 1 retroelements. Depletion of HUSH, SETDB1, or MORC2 alleviates HSV-1 repression in infected primary human fibroblasts and human induced pluripotent stem cell-derived sensory neurons (hiPSDN). We found that the viral protein ICP0 induces MORC2 degradation via the proteasome machinery. This process is concurrent with ICP0 and MORC2 depletion capability to reactivate silenced HSV-1 in hiPSDN. Overall, our findings underscore the robust antiviral function of the HUSH-SETDB1-MORC2 repressor complex against a herpesvirus by modulating chromatin marks linked to repression, thus presenting promising avenues for antiherpesvirus therapeutic strategies.

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.

Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal bodies, which are associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. RNAP1, Fibrillarin, and nucleolar DNA are exported to the periphery of the nucleolus after genotoxic stress and, once DNA repair is fully completed, the organization of the nucleolus is restored. We find that SMN is required for the restoration of the nucleolar structure after genotoxic stress. During DNA repair, SMN shuttles from the Cajal bodies to the nucleolus. This shuttling is important for nucleolar homeostasis and relies on the presence of Coilin and the activity of PRMT1. DNA damage causes a major reorganization of the nucleolus. Here, the authors find that this structural restoration depends on the shuttling of the protein SMN from the Cajal bodies to the nucleolus, which requires coilin and PRMT1.

The survival of motor neuron (SMN) protein is depleted in spinal muscular atrophy (SMA) pathology and herein defined as the first reader of histone H3 mono-methylated on lysine 79 through its central TUDOR domain. Spinal muscular atrophy is the leading genetic cause of infant mortality and results from depleted levels of functional survival of motor neuron (SMN) protein by either deletion or mutation of the SMN1 gene. SMN is characterized by a central TUDOR domain, which mediates the association of SMN with arginine methylated (R me ) partners, such as coilin, fibrillarin, and RNA pol II (RNA polymerase II). Herein, we biochemically demonstrate that SMN also associates with histone H3 monomethylated on lysine 79 (H3K79 me1 ), defining SMN as not only the first protein known to associate with the H3K79 me1 histone modification but also the first histone mark reader to recognize both methylated arginine and lysine residues. Mutational analyzes provide evidence that SMN TUDOR associates with H3 via an aromatic cage. Importantly, most SMN TUDOR mutants found in spinal muscular atrophy patients fail to associate with H3K79 me1 .

Abstract The establishment of latent herpes simplex virus 1 (HSV-1) infection is controlled by promyelocytic leukemia nuclear bodies (PML NBs). Viral genomes are recruited to PML NBs structures and chromatinized by repressive H3.3K9me3 modified H3.3 histone variant to form viral DNA-containing PML-NBs (vDCP NBs). Exactly how this occurs is unclear. Here we identify an essential role for the HUSH complex and its SETDB1 and MORC2 effectors in the establishment and maintenance of latent herpes simplex virus 1 (HSV-1) infection. We show that the formation of repressive heterochromatin is dependent on HUSH, SETDB1 and MORC2 in vDCP NBs, and depletion of any of these components prior to viral infection decreases H3K9me3 levels over latent/quiescent HSV-1 genomes. Once latency is established, depletion of HUSH, SETDB1, or MORC2 by shRNAs or the HIV-2 Vpx protein, induces the reactivation of HSV-1 in infected primary human fibroblasts as well as human induced pluripotent stem cell-derived sensory neurons (hiPSDN). Our data demonstrate the potent antiviral restriction activity of the HUSH/SETDB1/ MORC2 complex to a non-integrated human herpesvirus, its close association with PML NBs, and introduces a new target for anti-herpesvirus therapy.

Spinal muscular atrophy is the leading genetic cause of infant mortality and results from depleted levels of functional survival of motor neuron (SMN) protein by either deletion or mutation of the SMN1 gene. SMN is characterized by a central TUDOR domain, which mediates the association of SMN with arginine methylated (Rme) partners, such as coilin, fibrillarin, and RNA pol II (RNA polymerase II). Herein, we biochemically demonstrate that SMN also associates with histone H3 monomethylated on lysine 79 (H3K79me1), defining SMN as not only the first protein known to associate with the H3K79me1 histone modification but also the first histone mark reader to recognize both methylated arginine and lysine residues. Mutational analyzes provide evidence that SMNTUDOR associates with H3 via an aromatic cage. Importantly, most SMNTUDOR mutants found in spinal muscular atrophy patients fail to associate with H3K79me1.

Primary infection with herpes simplex type 1 (HSV-1) occurring around the mouth and nose switches rapidly to lifelong latent infection in sensitive trigeminal ganglia (TG) neurons. Sporadic reactivation of these latent reservoirs later in life is the cause of acute infections of the corneal epithelium, which can cause potentially blinding herpes simplex keratitis (HSK). There is no effective vaccine to protect against HSK, and antiviral drugs provide only partial protection against recurrences. We previously engendered an acute disease-free, non-reactivating latent state in mice when challenged with virulent HSV-1 in orofacial mucosa, by priming with non-neurovirulent HSV-1 (TK del ) before the challenge. Herein, we define the local immune infiltration and inflammatory chemokine production changes after virulent HSV-1 challenge, which were elicited by TK del prime. Heightened immunosurveillance before virulent challenge, and early enhanced lymphocyte-enriched infiltration of the challenged lip were induced, which corresponded to attenuation of inflammation in the TG and enhanced viral control. Furthermore, classical latent-phase T cell persistence around latent HSV-1 reservoirs were severely reduced. These findings identify the immune processes that are likely to be responsible for establishing non-reactivating latent HSV-1 reservoirs. Stopping reactivation is essential for development of efficient vaccine strategies against HSV-1.

Microrchidia CW-type zinc finger 2 (MORC2) gene encodes a protein expressed in all tissues and enriched in the brain. MORC2 protein is composed of a catalytic ATPase domain, three coil-coiled domains allowing dimerization or protein complex interaction, a zinc-finger CW domain allowing DNA interaction, and a CHROMO-like (CHRromatin Organization Modifier) domain. Recently, de novo or dominantly inherited heterozygous mutations have been associated with a spectrum of disorders affecting the peripheral nervous system such as the Charcot-Marie-Tooth disease, spinal muscular atrophy-like phenotype disorder, or a neurodevelopmental syndrome associated with developmental delay, impaired growth, dysmorphic facies, and axonal neuropathy (DIGFAN). In this review, we detail the various mutations of MORC2 and their consequences on clinical manifestations. Possible genotype-phenotype correlations as well as intra and inter-family variability are discussed. MORC2 molecular functions such as transcriptional modulation, DNA damage repair, and lipid metabolism are then reviewed. We further discuss the impact of MORC2 mutations on the epigenetic landscape in the neuromuscular system and hypothesize probable pathophysiological mechanisms underlying the phenotypic variability observed.

Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology that originates from the loss of the SMN1 gene in most cases or mutations in rare cases. Interestingly, several SMN1 mutations occur within the TUDOR methylarginine reader domain of SMN. We hypothesized that in SMN1 mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a BioID proteomic approach, we have identified and validated a number of SMN-interacting proteins, including fragile X mental retardation protein (FMRP) family members (FMRFM). Importantly, SMA-linked SMNTUDOR mutant forms (SMNST) failed to interact with FMRFM In agreement with the recent work, we define biochemically that SMN forms droplets in vitro and these droplets are stabilized by RNA, suggesting that SMN could be involved in the formation of membraneless organelles, such as Cajal nuclear bodies. Finally, we found that SMN and FMRP co-fractionate with polysomes, in an RNA-dependent manner, suggesting a potential role in localized translation in motor neurons.

Background: In glioma, TERT promoter mutation and loss of ATRX (ATRX loss) are associated with reactivation of telomerase or alternative lengthening of telomeres (ALT), respectively, i.e. the two telomere maintenance mechanisms (TMM). Strangely, 25% of gliomas have been reported to display neither or both of these alterations. Materials and methods: The C-circle (CC) assay was adapted to tumor (formalin-fixed paraffin-embedded and frozen) and blood samples to investigate the TMM. Results: We constructed a CC-based algorithm able to identify the TMM and reported a sensitivity of 100% and a specificity of 97.3% (n = 284 gliomas). By combining the TMM, the mutational status of the isocitrate dehydrogenase 1/2 (IDH) gene (IDHmt), and the histological grading, we propose a new classification tool: TeloDIAG. This classification defined five subtypes: tOD, tLGA, tGBM_IDHmt, tGBM, and tAIV, corresponding to oligodendroglioma, IDHmt low-grade astrocytoma, IDHmt glioblastoma, and IDHwt glioblastoma (GBM), respectively; the last class gathers ALTthorn IDHwt gliomas that tend to be related to longer survival (21.2 months) than tGBM (16.5 months). The TeloDIAG was 99% concordant with the World Health Organization classification (n = 312), and further modified the classification of 55 of 144 (38%) gliomas with atypical molecular characteristics. As an example, 14 of 69 (20%) of TERTwt, ATRXwt, and IDHwt GBM were actually tAIV. Outstandingly, CC in blood sampled from IDHmt astrocytoma patients was detected with a sensitivity of 56% and a specificity of 97% (n = 206 gliomas and 30 healthy donors). Conclusion: The TeloDIAG is a new, simple, and effective tool helping in glioma diagnosis and a promising option for liquid biopsy.

Herpes simplex virus-1 (HSV-1) establishes latency preferentially in sensory neurons of peripheral ganglia. A variety of stresses can induce recurrent reactivations of the virus, which spreads and then actively replicates to the site of primary infection (usually the lips or eyes). Viral particles produced following reactivation can also reach the brain, causing a rare but severe form of diffuse acute infection, namely herpes simplex encephalitis. Most of the time, this infection is clinically asymptomatic. However, it was recently correlated with the production and accumulation of neuropathological biomarkers of Alzheimer's disease. In this review we discuss the different cellular and molecular mechanisms underlying the acute and long-term damage caused by HSV-1 infection in the brain.