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Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors worldwide. Brahma-related gene 1 (BRG1), as a catalytic ATPase, is a major regulator of gene expression and is known to mutate and overexpress in HCC. The purpose of this study was to investigate the mechanism of action of BRG1 in HCC cells. In our study, BRG1 was silenced or overexpressed in human HCC cell lines. Transwell and wound healing assays were used to analyze cell invasiveness and migration. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) detection were used to evaluate mitochondrial function in HCC cells. Colony formation and cell apoptosis assays were used to evaluate the effect of BRG1/TOMM40/ATP5A1 on HCC cell proliferation and apoptosis/death. Immunocytochemistry (ICC), immunofluorescence (IF) staining and western blot analysis were used to determine the effect of BRG1 on TOMM40, ATP5A1 pathway in HCC cells. As a result, knockdown of BRG1 significantly inhibited cell proliferation and invasion, promoted apoptosis in HCC cells, whereas BRG1 overexpression reversed the above effects. Overexpression of BRG1 can up-regulate MMP level, inhibit mPTP opening and activate TOMM40, ATP5A1 expression. Our results suggest that BRG1, as an oncogene, promotes HCC progression by regulating TOMM40 affecting mitochondrial function and ATP5A1 synthesis. Targeting BRG1 may represent a new and effective way to prevent HCC development.

The toxic effects of C9orf72-derived arginine-rich dipeptide repeats (R-DPRs) on cellular stress granules in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia remain unclear at the molecular level. Stress granules are formed through the switch of Ras GTPase-activating protein-binding protein 1 (G3BP1) by RNA from a closed inactive state to an open activated state, driving the formation of the organelle by liquid-liquid phase separation (LLPS). We show that R-DPRs bind G3BP1 a thousand times stronger than RNA and initiate LLPS much more effectively. Their pathogenic effect is underscored by the slow transition of R-DPR-G3BP1 droplets to aggregated, ThS-positive states that can recruit ALS-linked proteins hnRNPA1, hnRNPA2, and TDP-43. Deletion constructs and molecular simulations show that R-DPR binding and LLPS are mediated via the negatively charged intrinsically disordered region 1 (IDR1) of the protein, allosterically regulated by its positively charged IDR3. Bioinformatic analyses point to the strong mechanistic parallels of these effects with the interaction of R-DPRs with nucleolar nucleophosmin 1 (NPM1) and underscore that R-DPRs interact with many other similar nucleolar and stress-granule proteins, extending the underlying mechanism of R-DPR toxicity in cells. Our results also highlight characteristic differences between the two R-DPRs, poly-GR and poly-PR, and suggest that the primary pathological target of poly-GR is not NPM1 in nucleoli, but G3BP1 in stress granules in affected cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

BRG1 has been found to promote the generation of induced pluripotent stem cells (iPSCs) by regulating epigenetic modifications or binding to transcription factors, however, the role of BRG1 on the cellular metabolism during reprogramming has not been reported. In this study, we found that BRG1 improved the efficiency of porcine iPSC generation, and upregulated the expression of pluripotency-related factors. Further analysis revealed that BRG1 promoted cellular glycolysis, and increased levels of glycolysis-related metabolites. It enhanced the transcriptional activity of glycolysis-related gene HK2, PKM2, and PFK-1 promoters, and decreased the enrichment of H3K9me3 in glycolysis- and pluripotency-related gene promoters. BRG1 also increased the phosphorylation level at the Ser473 site of AKT protein. The specific PI3K/AKT signaling pathway inhibitor, LY294002, impaired the generation of porcine iPSCs, downregulated the expression of pluripotency-related factors, and inhibited cellular glycolysis, overexpressing BRG1 rescued those changes caused by LY294002 treatment. In addition, the glycolysis inhibitor 2-DG and BRG1 inhibitor PFI-3 had similar effects to LY294002. The above results suggest that overexpression of BRG1 promotes the generation of porcine iPSCs by facilitating glycolytic reprogramming through the PI3K/AKT signaling pathway., Competing Interests: Declaration. Conflict of interest: The authors declare that they have no competing interests. Ethical approval and consent to participate: Not applicable. Consent for publication: Not applicable., (© 2024. The Author(s).)

Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. Damaging mutations in the SWI/SNF chromatin remodeling complex, such as SMARCA4 (BRG1), are associated with improved response to immune checkpoint blockade; however, the mechanism by which this occurs is unclear. We found that SMARCA4 loss in OC models resulted in increased cancer cell-intrinsic immunogenicity, characterized by up-regulation of long-terminal RNA repeats, increased expression of interferon-stimulated genes, and up-regulation of antigen presentation machinery. Notably, this response was dependent on STING, MAVS, and IRF3 signaling but was independent of the type I interferon receptor. Mouse ovarian and melanoma tumors with SMARCA4 loss demonstrated increased infiltration and activation of cytotoxic T cells, NK cells, and myeloid cells in the tumor microenvironment. These results were recapitulated in BRG1 inhibitor-treated SMARCA4- proficient tumor models, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to overcome cancer immune evasion.

A G4C2 hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Bidirectional transcription and subsequent repeat-associated non-AUG (RAN) translation of sense and antisense transcripts leads to the formation of five dipeptide repeat (DPR) proteins. These DPRs are toxic in a wide range of cell and animal models. Therefore, decreasing RAN-DPRs may be of therapeutic benefit in the context of C9ALS/FTD. In this study, we found that C9ALS/FTD patients have reduced expression of the AAA+ family members RuvBL1 and RuvBL2, which have both been implicated in aggregate clearance. We report that overexpression of RuvBL1, but to a greater extent RuvBL2, reduced C9orf72-associated DPRs in a range of in vitro systems including cell lines, primary neurons from the C9-500 transgenic mouse model, and patient-derived iPSC motor neurons. In vivo, we further demonstrated that RuvBL2 overexpression and consequent DPR reduction in our Drosophila model was sufficient to rescue a number of DPR-related motor phenotypes. Thus, modulating RuvBL levels to reduce DPRs may be of therapeutic potential in C9ALS/FTD., (© 2024 Webster et al.)

Ras-GTPase-activating protein SH3-domain-binding proteins (G3BP) are critical for the formation of stress granules (SGs) through their RNA- and ribosome-binding properties. SARS-CoV-2 nucleocapsid (N) protein exhibits strong binding affinity for G3BP and inhibits infection-induced SG formation soon after infection. To study the impact of the G3BP-N interaction on viral replication and pathogenesis in detail, we generated a mutant SARS-CoV-2 (RATA) that specifically lacks the G3BP-binding motif in the N protein. RATA triggers a stronger and more persistent SG response in infected cells, showing reduced replication across various cell lines, and greatly reduced pathogenesis in K18-hACE2 transgenic mice. At early times of infection, G3BP and WT N protein strongly colocalise with dsRNA and with non-structural protein 3 (nsp3), a component of the pore complex in double membrane vesicles (DMVs) from which nascent viral RNA emerges. Furthermore, G3BP-N complexes promote highly localized translation of viral mRNAs in the immediate vicinity of the DMVs and thus contribute to efficient viral gene expression and replication. In contrast, G3BP is absent from the DMVs in cells infected with RATA and translation of viral mRNAs is less efficient. This work provides a fuller understanding of the multifunctional roles of G3BP in SARS-CoV-2 infection., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

SMARCA4-deficient undifferentiated tumor (SMARCA4-UT) in the chest is a high-grade malignant tumor that grows rapidly and often carries a poor prognosis. Unfortunately, there are currently no effective treatment available until now. Here, we report a case of SMARCA4-UT in a patient who showed a swift response to a combination treatment of penpulimab, anlotinib, and chemotherapy. A 55-year-old man was diagnosed with thoracic SMARCA4-UT along with metastases to multiple lymph nodes, the pleura, and bones. Immunohistochemical (IHC) testing indicated the absence of PD-L1 expression in tumor cells. He was given sintilimab and anlotinib as first line treatment. However, a follow-up chest CT revealed progressive disease (PD) after the first cycle treatment. Subsequently, the second line regimen was modified to etoposide and cisplatin (EP) combined with anlotinib and penpulimab. The effectiveness evaluation revealed partial remission (PR) following two cycles of the second-line regimen treatment. Notably, the patient's progress-free survival (PFS) exceeds 7 months and the overall survival up to 12 months. Our case implies that a combination of chemotherapy, anlotinib, and penpulimab might offer a promising therapeutic approach for PD-L1-negative thoracic SMARCA4-UT., (© 2024 The Author(s). The Clinical Respiratory Journal published by John Wiley & Sons Ltd.)

Some DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

The switch/sucrose non-fermenting (SWI/SNF) complex family includes important chromatin-remodeling factors that are frequently mutated in lung adenocarcinoma (LUAD). However, the role of one family member, SMARCA4, in LUAD prognosis and immunotherapy sensitivity remains unclear. In the present study, 6745 LUAD samples from the cBioPortal database were used to analyze the relationships between SMARCA4 mutations and patient prognoses and clinical characteristics. Additionally, we examined the correlation between SMARCA4 mutations and prognosis in patients treated with immunotherapy using two immune-related datasets. SMARCA4 mutations and low expression were associated with shorter survival, and mutations were associated with a high tumor mutational burden and high microsatellite instability. SMARCA4 mutations were accompanied by KRAS, KEAP1, TP53 and STK11 mutations. No significant difference was observed in the immunotherapy response between patients with and without SMARCA4 mutations. When KRAS or STK11 mutations were present, immunotherapy effectiveness was poorer; however, when both SMARCA4 and TP53 mutations were present, immunotherapy was more effective. Furthermore, low SMARCA4 expression predicted a higher immunophenoscore, and SMARCA4 expression was correlated with certain immune microenvironment features. Taken together, our results suggest that SMARCA4 mutations and low expression might be associated with poor LUAD prognosis. Additionally, immunotherapy efficacy in patients with SMARCA4 mutations depended on the co-mutant genes. Thus, SMARCA4 could be an important factor to be considered for LUAD diagnosis and treatment., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nucleosome remodelers at replication forks function in the assembly and maturation of chromatin post DNA synthesis. The ISWI chromatin remodeler SNF2L (or SMARCA1) travels with replication forks but its contribution to DNA replication remains largely unknown. We find that fork elongation is curtailed when SNF2L is absent. SNF2L deficiency elevates replication stress and causes fork collapse due to remodeling activities by fork reversal enzymes. Mechanistically, SNF2L regulates nucleosome assembly to suppress post-replicative ssDNA gap accumulation. Gap induction is not dependent on fork remodeling and PRIMPOL. Instead, gap synthesis is driven by MRE11 and EXO1 indicating susceptibility of nascent DNA to nucleolytic cleavage and resection when SNF2L is removed. Additionally, nucleosome remodeling by SNF2L protects nascent chromatin from MNase digestion and gap induction highlighting a critical role of SNF2L in chromatin assembly post DNA synthesis to maintain unperturbed replication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transcription-coupled repair (TCR) is the major pathway to remove transcription-blocking lesions. Although discovered for nearly 40 years, the mechanism and critical players of mammalian TCR remain unclear. STK19 is a factor affecting cell survival and recovery of RNA synthesis in response to DNA damage, however, whether it is a necessary component for TCR is unknown. Here, we demonstrated that STK19 is essential for human TCR. Mechanistically, STK19 is recruited to damage sites through direct interaction with CSA. It can also interact with RNA polymerase II in vitro. Once recruited, STK19 plays an important role in UVSSA ubiquitination which is needed for TCR. STK19 also promotes TCR independent of UVSSA ubiquitination by stimulating TFIIH recruitment through its direct interaction with TFIIH. In summary, our results suggest that STK19 is a key factor of human TCR that links CSA, UVSSA ubiquitination and TFIIH loading, shedding light on the molecular mechanisms of TCR., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mammalian RAD52 is a DNA repair factor with strand annealing and recombination mediator activities that appear important in both interphase and mitotic cells. Nonetheless, RAD52 is dispensable for cell viability. To query RAD52 synthetic lethal relationships, we performed genome-wide CRISPR knock-out screens and identified hundreds of candidate synthetic lethal interactions. We then performed secondary screening and identified genes for which depletion causes reduced viability and elevated genome instability (increased 53BP1 nuclear foci) in RAD52-deficient cells. One such factor was ERCC6L, which marks DNA bridges during anaphase, and hence is important for genome stability in mitosis. Thus, we investigated the functional interrelationship between RAD52 and ERCC6L. We found that RAD52 deficiency increases ERCC6L-coated anaphase ultrafine bridges, and that ERCC6L depletion causes elevated RAD52 foci in prometaphase and interphase cells. These effects were enhanced with replication stress (i.e. hydroxyurea) and topoisomerase IIα inhibition (ICRF-193), where post-treatment effect timings were consistent with defects in addressing stress in mitosis. Altogether, we suggest that RAD52 and ERCC6L co-compensate to protect genome stability in mitosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Osia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A female infant, aged 11 days, was admitted due to dyspnea for 11 days after birth, with the main clinical manifestations of inspiratory dyspnea, feeding difficulties, and unusual facies (micrognathia, high palatal arch, cleft palate, glossoptosis, and oblique mouth to the right), and the preliminary diagnosis was Pierre-Robin syndrome. There was no marked improvement after treatment such as ventilator-assisted ventilation, nutrition, and surgical ligation of patent ductus arteriosus. Whole-exome sequencing of the family showed a heterozygous mutation of c.3082A>G (p.Ile1028 Val) in the CHD7 gene, which was a pathogenic mutation of CHARGE syndrome. The neonate was ultimately diagnosed with CHARGE syndrome, and the family decided to withdraw treatment due to concerns about poor prognosis. This article reports a case of CHARGE syndrome caused by a mutation in the CHD7 gene and the multidisciplinary diagnosis and treatment of this disease, in order to provide help for early disease identification and guide clinical decision-making.

N 6 -methyladenosine (m 6 A)-modified mRNAs and their cytoplasmic reader YTHDFs are colocalized with stress granules (SGs) under stress conditions, but the interplay between m 6 A modification and SG stability remains unclear. Here, we presented a spatiotemporal m 6 A imaging system (SMIS) that can monitor the m 6 A modification and the translation of mRNAs with high specificity and sensitivity in a single live cell. SMIS showed that m 6 A-modified reporter mRNAs dynamically enriched into SGs under arsenite stress and gradually partitioned into the cytosol as SG disassembled. SMIS revealed that knockdown of YTHDF2 contributed to SG disassembly, resulting in the fast redistribution of mRNAs from SGs and rapid recovery of stalled translation. The mechanism is that YTHDF2 can regulate SG stability through the interaction with G3BP1 in m 6 A-modified RNA-dependent manner. Our results suggest a mechanism for the interplay between m 6 A modification and SG through YTHDF2 regulation.

RAD54L is a DNA motor protein with multiple roles in homologous recombination DNA repair. In vitro, RAD54L was shown to also catalyze the reversal and restoration of model replication forks. In cells, however, little is known about how RAD54L may regulate the dynamics of DNA replication. Here, we show that RAD54L restrains the progression of replication forks and functions as a fork remodeler in human cancer cell lines and non-transformed cells. Analogous to HLTF, SMARCAL1 and FBH1, and consistent with a role in fork reversal, RAD54L decelerates fork progression in response to replication stress and suppresses the formation of replication-associated ssDNA gaps. Interestingly, loss of RAD54L prevents nascent strand DNA degradation in both BRCA1/2- and 53BP1-deficient cells, suggesting that RAD54L functions in both pathways of RAD51-mediated replication fork reversal. In the HLTF/SMARCAL1 pathway, RAD54L is critical, but its ability to catalyze branch migration is dispensable, indicative of its function downstream of HLTF/SMARCAL1. Conversely, in the FBH1 pathway, branch migration activity of RAD54L is essential, and FBH1 engagement is dependent on its concerted action with RAD54L. Collectively, our results reveal disparate requirements for RAD54L in two distinct RAD51-mediated fork reversal pathways, positing its potential as a future therapeutic target., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Replication is initiated bidirectionally in the three domains of life by the assembly of two replication forks at an origin of replication. This is made possible by the recruitment of two replicative helicases to a nucleoprotein platform built at the origin of replication with the initiator protein. The reason why replication is initiated bidirectionally has never been experimentally addressed due to the lack of a suitable biological system. Using genetic and genomic approaches, we show that upon depletion of DciA, replication is no longer initiated bidirectionally at the origin of replication of Vibrio cholerae chromosome 1. We show that following unidirectional replication on the left replichore, nascent DNA strands at ori1 anneal to each other to form a double-stranded DNA end. While this DNA end can be efficiently resected in recB+ cells, only a few cells use it to trigger replication on the right replichore. In most DciA-depleted cells, chromosome 1 is degraded leading to cell death. Our results suggest that DciA is essential to ensuring bidirectional initiation of replication in bacteria, preventing a cascade of deleterious events following unidirectional replication initiation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Kallmann syndrome is a rare disorder characterized by hypogonadotropic hypogonadism and an impaired sense of smell (anosmia or hyposmia) caused by congenital defects in the development of the gonadotropin-releasing hormone (GnRH) and olfactory neurons. Mutations in several genes have been associated with Kallmann syndrome. However, genetic testing of this disorder often reveals variants of uncertain significance (VUS) that remain uninterpreted without experimental validation. The aim of this study was to analyze the functional consequences of a heterozygous missense VUS in the CHD7 gene (c.4354G>T, p.Val1452Leu), in a patient with Kallmann syndrome with reversal of hypogonadism. The variant, located in the first nucleotide of exon 19, was analyzed using minigene assays to determine its effect on ribonucleic acid (RNA) splicing. These showed that the variant generates two different transcripts: a full-length transcript with the missense change (p.Val1452Leu), and an abnormally spliced transcript lacking exon 19. The latter results in an in-frame deletion (p.Val1452_Lys1511del) that disrupts the helicase C-terminal domain of the CHD7 protein. The variant was reclassified as likely pathogenic. These findings demonstrate that missense variants can exert more extensive effects beyond simple amino acid substitutions and underscore the critical role of functional analyses in VUS reclassification and genetic diagnosis.

Purpose: Upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) share histomorphological and therapeutic features but distinct epidemiologic and clinicopathologic characteristics. We examined alterations of chromatin regulatory genes in molecular subtypes, clonal relatedness, and T-cell receptor (TCR) diversity in UTUC and UCB., Materials and Methods: Targeted next-generation sequencing or whole-exome DNA sequencing and TCR sequencing were conducted with 34 UTUC and 49 UCB specimens from 63 patients. Tumors were subtyped based on the expression of CK5 and GATA3. Results of tissue microarray of 78 muscle-invasive bladder cancer (MIBC) samples were used as prognostic factors of different subtypes of MIBC., Results: Chromatin regulatory genes were frequently mutated in both UTUC and UCB. Rapid relapse and progression of non-MIBC are correlated with alterations of KMT2C and EP300. Frequency of alterations in chromatin regulatory genes is higher in UTUC patients with SBS22 and SBS2 signatures and lower in UCB patients with SBS2 and SBS6 signatures. GATA3 and CK5 double-positive patients with higher frequencies of SMARCA4, ARID1A, and EP300 mutations have better prognoses than patients with basal subtypes. Although UTUC and UCB in the same patient can be either clonally related or developed independently, mutated genes in chromatin pathway were enriched in the related clones. Compared to UTUC, UCB had more deleterious mutations in DNA damage repair (DDR) genes, higher levels of tumor mutation burden (TMB) and copy number variations (CNVs), as well as higher TCR clonality and lower TCR diversity., Conclusions: Since genetic alterations of the chromatin pathway genes are important in both UTUC and UCB, they could serve as potential biomarkers for predicting disease progression and therapeutic targets. Differences in mutation frequencies of DDR pathway, TMB, CNV, and TCR might be the contributing factors for the distinct responses to immune checkpoint inhibitor (ICI) between UTUC and UCB., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Helicases are ubiquitous motors involved in almost all aspects of nucleic acid metabolism; therefore, revealing their unwinding behaviors and mechanisms is fundamentally and medically essential. In recent decades, single-molecule applications have revolutionized our ability to study helicases by avoiding the averaging of bulk assays and bridging the knowledge gap between dynamics and structures. This advancement has updated our understanding of the biochemical properties of helicases, such as their rate, directionality, processivity, and step size, while also uncovering unprecedented mechanistic insights. Among these, repetitive motion, a new feature of helicases, is one of the most remarkable discoveries. However, comprehensive reviews and comparisons are still lacking. Consequently, the present review aims to summarize repetitive helicases, compare the repetitive phenomena, and discuss the underlying molecular mechanisms. This review may provide a systematic understanding of repetitive helicases and help understand their cellular functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these membraneless organelles is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs form SGs following stress-induced translational arrest. Three G3BP paralogues (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogues to SG formation and gene expression changes is incompletely understood. Here, we probed the functions of G3BPs by identifying important residues for SG assembly at their N-terminal domain such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that a G3BP V11A mutant leads to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially forms SGs and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Furthermore, our work is a resource for researchers to study gene expression changes under cellular stress. Together, this work suggests that perturbing protein-protein interactions mediated by G3BPs affect SG assembly and gene expression during the ISR, and such functions are differentially regulated by G3BP paralogues under ER stress., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Over a lifetime, hematopoietic stem and progenitor cells (HSPCs) are forced to repeatedly proliferate to maintain hematopoiesis, increasing their susceptibility to DNA damaging replication stress. However, the proteins that mitigate this stress, protect HSPC replication, and prevent aging-driven dysregulation are unknown. We report two evolutionarily conserved, ubiquitously expressed chromatin remodeling enzymes with similar DNA replication fork reversal biochemical functions, Zranb3 and Smarcal1, have surprisingly specialized roles in distinct HSPC populations. While both proteins actively mitigate replication stress and prevent DNA damage and breaks during lifelong hematopoiesis, the loss of either resulted in distinct biochemical and biological consequences. Notably, defective long-term HSC function, revealed with bone marrow transplantation, caused hematopoiesis abnormalities in young mice lacking Zranb3. Aging significantly worsened these hematopoiesis defects in Zranb3-deficient mice, including accelerating the onset of myeloid-biased hematopoietic dysregulation to early in life. Such Zranb3-deficient HSPC abnormalities with age were driven by accumulated DNA damage and replication stress. Conversely, Smarcal1 loss primarily negatively affected progenitor cell functions that were exacerbated with aging, resulting in a lymphoid bias. Simultaneous loss of both Zranb3 and Smarcal1 compounded HSPC defects. Additionally, HSPC DNA replication fork dynamics had unanticipated HSPC type and age plasticity that depended on the stress and Zranb3 and/or Smarcal1. Our data reveal both Zranb3 and Smarcal1 have essential HSPC cell intrinsic functions in lifelong hematopoiesis that protect HSPCs from replication stress and DNA damage in unexpected, unique ways., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

We report that the eukaryotic replicative helicase CMG (Cdc45-MCM-GINS) is required for differential gene expression in cells produced by asymmetric cell divisions in C. elegans. We found that the C. elegans CMG component, PSF-2 GINS2, is necessary for transcriptional upregulation of the pro-apoptotic gene egl-1 BH3-only that occurs in cells programmed to die after they are produced through asymmetric cell divisions. We propose that CMG's histone chaperone activity causes epigenetic changes at the egl-1 locus during replication in mother cells, and that these changes are required for egl-1 upregulation in cells programmed to die. We find that PSF-2 is also required for the divergence of other cell fates during C. elegans development, suggesting that this function is not unique to egl-1 expression. Our work uncovers an unexpected role of CMG in cell fate decisions and an intrinsic mechanism for gene expression plasticity in the context of asymmetric cell division., (© 2024. The Author(s).)

CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase. The structures reveal how the effector complexes recognize the protospacer adjacent motif and target-strand DNA to form an R-loop structure. Additionally, we reveal differences between types IV-A1 and IV-A3 in DNA interactions and structural motifs that allow for in trans recruitment of DinG. Our study provides a detailed view of type IV-A mediated DNA-interference and presents a structural foundation for engineering type IV-A-based genome editing tools., (© 2024. The Author(s).)

MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Williams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Ubiquitination plays a key role in various cancers, and F-box and WD repeat domain containing 7 (FBW7) is a tumor suppressor that targets several cancer-causing proteins for ubiquitination. This paper set out to pinpoint the role of FBW7 in hepatocellular carcinoma (HCC)., Methods: The target proteins of FBW7 and the expression of hromodomain helicase DNA binding protein 3 ( CHD3 ) were analyzed in liver HCC (LIHC) samples using the BioSignal Data website. The effects of CHD3 and FBW7 on HCC cell viability, migration, invasion and stemness were investigated through cell counting kit (CCK)-8, wound healing, transwell and sphere formation assays. Detection on CHD3 and FBW7 expressions as well as their relationship was performed employing quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunoprecipitation, ubiquitination and western blot analyses., Results: The prediction of Ubibrowser revealed CHD3 as a target protein of FBW7. The data of starBase exhibited a higher expression level of CHD3 in LIHC samples relative to normal samples. CHD3 was upregulated in HCC cells. CHD3 knockdown inhibited HCC cell proliferation, migration, invasion, stemness and oxaliplatin sensitivity. FBW7 targeted CHD3 for ubiquitination. FBW7 overexpression restrained HCC cell migration, invasion and stemness, and attenuated the effects of overexpressed CHD3 on promoting migration, invasion, stemness and oxaliplatin resistance in HCC cells., Conclusion: FBW7 overexpression suppresses HCC cell metastasis, stemness and oxaliplatin resistance via targeting CHD3 for ubiquitylation and degradation., (© 2024 The Author(s). Published by IMR Press.)

The paraventricular hypothalamus (PVH) is crucial for food intake control, yet the presynaptic mechanisms underlying PVH neurons remain unclear. Here, we show that RUVBL2 in the PVH is significantly reduced during energy deficit, and knockout (KO) of PVH RUVBL2 results in hyperphagic obesity in mice. RUVBL2-expressing neurons in the PVH (PVH RUVBL2 ) exert the anorexigenic effect by projecting to the arcuate hypothalamus, the dorsomedial hypothalamus, and the parabrachial complex. We further demonstrate that PVH RUVBL2 neurons form the synaptic connections with POMC and AgRP neurons in the ARC. PVH RUVBL2 KO impairs the excitatory synaptic transmission by reducing presynaptic boutons and synaptic vesicles near active zone. Finally, RUVBL2 overexpression in the PVH suppresses food intake and protects against diet induced obesity. Together, this study demonstrates an essential role for PVH RUVBL2 in food intake control, and suggests that modulation of synaptic plasticity could be an effective way to curb appetite and obesity., (© 2024. The Author(s).)

Long noncoding (lnc)RNAs emerge as regulators of genome stability. The nuclear-enriched abundant transcript 1 (NEAT1) is overexpressed in many tumors and is responsive to genotoxic stress. However, the mechanism that links NEAT1 to DNA damage response (DDR) is unclear. Here, we investigate the expression, modification, localization, and structure of NEAT1 in response to DNA double-strand breaks (DSBs). DNA damage increases the levels and N6-methyladenosine (m 6 A) marks on NEAT1, which promotes alterations in NEAT1 structure, accumulation of hypermethylated NEAT1 at promoter-associated DSBs, and DSB signaling. The depletion of NEAT1 impairs DSB focus formation and elevates DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves the release of the chromodomain helicase DNA binding protein 4 (CHD4) from NEAT1 to fine-tune histone acetylation at DSBs. Our data suggest a direct role for NEAT1 in DDR., (© 2024 Mamontova et al.; Published by Cold Spring Harbor Laboratory Press.)

Several protein ensembles facilitate crossover recombination and the associated assembly of synaptonemal complex (SC) during meiosis. In yeast, meiosis-specific factors including the DNA helicase Mer3, the "ZZS" complex consisting of Zip4, Zip2, and Spo16, the RING-domain protein Zip3, and the MutSγ heterodimer collaborate with crossover-promoting activity of the SC component, Zip1, to generate crossover-designated recombination intermediates. These ensembles also promote SC formation - the organized assembly of Zip1 with other structural proteins between aligned chromosome axes. We used proximity labeling to investigate spatial relationships between meiotic recombination and SC proteins in S. cerevisiae. We find that recombination initiation and SC factors are dispensable for proximity labeling of Zip3 by ZZS components, but proteins associated with early steps in recombination are required for Zip3 proximity labeling by MutSγ, suggesting that MutSγ joins Zip3 only after a recombination intermediate has been generated. We also find that zip1 separation-of-function mutants that are crossover deficient but still assemble SC fail to generate protein ensembles where Zip3 can engage ZZS and/or MutSγ. The SC structural protein Ecm11 is proximity labeled by ZZS proteins in a Zip4-dependent and Zip1-independent manner, but labeling of Ecm11 by Zip3 and MutSγ requires, at least in part, Zip1. Finally, mass spectrometry analysis of biotinylated proteins in eleven proximity labeling strains uncovered shared proximity targets of SC and crossover-associated proteins, some of which have not previously been implicated in meiotic recombination or SC formation, highlighting the potential of proximity labeling as a discovery tool., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Voelkel-Meiman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The INO80 protein is the main catalytic subunit of the INO80-chromatin remodeling complex, which is critical for DNA repair and transcription regulation in murine spermatocytes. In this study, we explored the role of INO80 in silencing genes on meiotic sex chromosomes in male mice. INO80 immunolocalization at the XY body in pachytene spermatocytes suggested a role for INO80 in the meiotic sex body. Subsequent deletion of Ino80 resulted in high expression of sex-linked genes. Furthermore, the active form of RNA polymerase II at the sex chromosomes of Ino80-null pachytene spermatocytes indicates incomplete inactivation of sex-linked genes. A reduction in the recruitment of initiators of meiotic sex chromosome inhibition (MSCI) argues for INO80-facilitated recruitment of DNA repair factors required for silencing sex-linked genes. This role of INO80 is independent of a common INO80 target, H2A.Z. Instead, in the absence of INO80, a reduction in chromatin accessibility at DNA repair sites occurs on the sex chromosomes. These data suggest a role for INO80 in DNA repair factor localization, thereby facilitating the silencing of sex-linked genes during the onset of pachynema., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chakraborty, Magnuson. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recently, a novel two-gene bacterial defense system against phages, encoding a SIR2 NADase and a HerA ATPase/helicase, has been identified. However, the molecular mechanism of the bacterial SIR2-HerA immune system remains unclear. Here, we determine the cryo-EM structures of SIR2, HerA and their complex from Paenibacillus sp. 453MF in different functional states. The SIR2 proteins oligomerize into a dodecameric ring-shaped structure consisting of two layers of interlocked hexamers, in which each subunit exhibits an auto-inhibited conformation. Distinct from the canonical AAA+ proteins, HerA hexamer alone in this antiphage system adopts a split spiral arrangement, which is stabilized by a unique C-terminal extension. SIR2 and HerA proteins assemble into a ∼1.1 MDa torch-shaped complex to fight against phage infection. Importantly, disruption of the interactions between SIR2 and HerA largely abolishes the antiphage activity. Interestingly, binding alters the oligomer state of SIR2, switching from a dodecamer to a tetradecamer state. The formation of the SIR2-HerA binary complex activates NADase and nuclease activities in SIR2 and ATPase and helicase activities in HerA. Together, our study not only provides a structural basis for the functional communications between SIR2 and HerA proteins, but also unravels a novel concerted antiviral mechanism through NAD+ degradation, ATP hydrolysis, and DNA cleavage., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

FANCM is a DNA repair protein that recognizes stalled replication forks, and recruits downstream repair factors. FANCM activity is also essential for the survival of cancer cells that utilize the Alternative Lengthening of Telomeres (ALT) mechanism. FANCM efficiently recognizes stalled replication forks in the genome or at telomeres through its strong affinity for branched DNA structures. In this study, we demonstrate that the N-terminal translocase domain drives this specific branched DNA recognition. The Hel2i subdomain within the translocase is crucial for effective substrate engagement and couples DNA binding to catalytic ATP-dependent branch migration. Removal of Hel2i or mutation of key DNA-binding residues within this domain diminished FANCM's affinity for junction DNA and abolished branch migration activity. Importantly, these mutant FANCM variants failed to rescue the cell cycle arrest, telomere-associated replication stress, or lethality of ALT-positive cancer cells depleted of endogenous FANCM. Our results reveal the Hel2i domain is key for FANCM to properly engage DNA substrates, and therefore plays an essential role in its tumour-suppressive functions by restraining the hyperactivation of the ALT pathway., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Rationale: SMARCA4-deficient non-small cell lung cancer (NSCLC) represents a highly aggressive subtype with poor prognosis. While clinical studies have identified common co-mutations in TP53, LRP1B, STK11, KEAP1, and KRAS, actionable driver mutations such as EGFR or ALK are rarely reported in conjunction with SMARCA4 deficiency. This case presents a rare instance of NSCLC featuring both an EGFR exon 21 L858R mutation and SMARCA4 deficiency, highlighting the challenges in treatment and the need for novel therapeutic strategies., Patient Concerns: A 79-year-old female patient presented with concerns of a lung mass, suspected to be peripheral lung cancer based on diagnostic imaging., Diagnoses: Histopathological evaluation confirmed SMARCA4-deficient NSCLC. Molecular genetic analysis further revealed an EGFR exon 21 L858R mutation., Interventions: The patient was initially treated with osimertinib, an EGFR tyrosine kinase inhibitor. Upon disease progression, treatment was adjusted to include anlotinib in combination with ongoing osimertinib., Outcomes: The initial treatment with osimertinib led to partial remission. However, disease progression necessitated a change in therapy. The combination treatment stabilized the disease temporarily, achieving a stable disease status., Lessons: This case underscores the transient efficacy of targeted therapy in SMARCA4-deficient NSCLC with concurrent EGFR mutations. It highlights the need for continuous therapeutic adjustments and emphasizes the importance of further research into effective strategies for treating this complex and challenging subset of NSCLC, as current modalities have limitations in sustained efficacy., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process., (© 2024. The Author(s).)

Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA-protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG-PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L's role., (© 2024 Riggs et al.)

The introduction of new sequencing approaches into clinical practice has radically changed the diagnostic approach to mitochondrial diseases, significantly improving the molecular definition rate in this group of neurogenetic disorders. At the same time, there have been no equal successes in the area of in-depth understanding of disease mechanisms and few innovative therapeutic approaches have been proposed recently. In this regard, the identification of the molecular basis of phenotypic variability in primary mitochondrial disorders represents a key aspect for deciphering disease mechanisms with important therapeutic implications. In this study, we present data from proteomic investigations in two subjects affected by mitochondrial disease characterized by a different clinical severity and associated with the same variant in the TWNK gene, encoding the mitochondrial DNA and RNA helicase with a specific role in the mtDNA replisome. Heterozygous pathogenic variants in this gene are associated with progressive external ophthalmoplegia and ptosis, usually with adult onset. The overall results suggest an imbalance in glucose metabolism and ROS production/regulation, with possible consequences on the phenotypic manifestations of the enrolled subjects. Although the data will need to be validated in a large cohort, proteomic investigations have proven to be a valid approach for a deep understanding of these neurometabolic disorders.

Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment., (© 2024. The Author(s).)

Cockayne syndrome (CS) group B (CSB), which results from mutations in the excision repair cross-complementation group 6 ( ERCC6 ) genes, which produce CSB protein, is an autosomal recessive disease characterized by multiple progressive disorders including growth failure, microcephaly, skin photosensitivity, and premature aging. Clinical data show that brain atrophy, demyelination, and calcification are the main neurological manifestations of CS, which progress with time. Neuronal loss and calcification occur in various brain areas, particularly the cerebellum and basal ganglia, resulting in dyskinesia, ataxia, and limb tremors in CSB patients. However, the understanding of neurodevelopmental defects in CS has been constrained by the lack of significant neurodevelopmental and functional abnormalities observed in CSB-deficient mice. In this review, we focus on elucidating the protein structure and distribution of CSB and delve into the impact of CSB mutations on the development and function of the nervous system. In addition, we provide an overview of research models that have been instrumental in exploring CS disorders, with a forward-looking perspective on the substantial contributions that brain organoids are poised to further advance this field.

Tumor cell heterogeneity defines therapy responsiveness in neuroblastoma (NB), a cancer derived from neural crest cells. NB consists of two primary subtypes: adrenergic and mesenchymal. Adrenergic traits predominate in NB tumors, while mesenchymal features becomes enriched post-chemotherapy or after relapse. The interconversion between these subtypes contributes to NB lineage plasticity, but the underlying mechanisms driving this phenotypic switching remain unclear. Here, we demonstrate that SWI/SNF chromatin remodeling complex ATPases are essential in establishing an mesenchymal gene-permissive chromatin state in adrenergic-type NB, facilitating lineage plasticity. Targeting SWI/SNF ATPases with SMARCA2/4 dual degraders effectively inhibits NB cell proliferation, invasion, and notably, cellular plasticity, thereby preventing chemotherapy resistance. Mechanistically, depletion of SWI/SNF ATPases compacts cis-regulatory elements, diminishes enhancer activity, and displaces core transcription factors (MYCN, HAND2, PHOX2B, and GATA3) from DNA, thereby suppressing transcriptional programs associated with plasticity. These findings underscore the pivotal role of SWI/SNF ATPases in driving intrinsic plasticity and therapy resistance in neuroblastoma, highlighting an epigenetic target for combinational treatments in this cancer., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The Helicase-like Transcription Factor (HLTF) is a member of the SNF2-family of fork remodelers, primarily studied for its capacity to provide DNA Damage Tolerance (DDT) and to induce replication fork reversal (RFR). HLTF is recruited at stalled forks where both its ATPase motor and HIP116 Rad5p N-terminal (HIRAN) domains are necessary for regulating its interaction with DNA. HIRAN bestows specificity to ssDNA 3'-end and imparts branch migration as well as DNA remodeling capabilities facilitating damage repair. Both expression regulation and mutation rate affect HLTF activity. Gene hypermethylation induces loss of HLTF function, in particular in colorectal cancer (CRC), implying a tumour suppressor role. Surprisingly, a correlation between hypermethylation and HLTF mRNA upregulation has also been observed, even within the same cancer type. In many cancers, both complex mutation patterns and the presence of gene Copy Number Variations (CNVs) have been reported. These conditions affect the amount of functional HLTF and question the physiological role of this fork remodeler. This review offers a systematic collection of the presently strewed information regarding HLTF, its structural and functional characteristics, the multiple roles in DDT and the regulation in cancer progression highlighting new research perspectives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study describes genomic findings among individuals with both orofacial clefts (OC) and microphthalmia/anophthalmia/coloboma (MAC) recorded in the Brazilian Database on Craniofacial Anomalies (BDCA). Chromosomal microarray analysis (CMA) and Whole Exome Sequencing (WES) were performed in 17 individuals with OC-MAC. Clinical interpretation of molecular findings was based on data available at the BDCA and on re-examination. No copy number variants (CNVs) classified as likely pathogenic or pathogenic were detected by CMA. WES allowed a conclusive diagnosis in six individuals (35.29%), two of them with variants in the CHD7 gene, and the others with variants in the TFAP2A, POMT1, PTPN11, and TP63 genes with the following syndromes: CHARGE, CHD7-spectrum, Branchiooculofacial, POMT1-spectrum, LEOPARD, and ADULT. Variants of uncertain significance (VUS) possibly associated to the phenotypes were found in six other individuals. Among the individuals with VUSes, three individuals presented variants in genes associated to defects of cilia structure and/or function, including DYNC2H1, KIAA0586, WDR34, INTU, RPGRIP1L, KIF7, and LMNA. These results show that WES was the most effective molecular approach for OC-MAC in this cohort. This study also reinforces the genetic heterogeneity of OC-MAC, and the importance of genes related to ciliopathies in this phenotype., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

Premature ovarian insufficiency (POI) is the main cause of infertility in women. Some cases of POI are thought to be caused by genetic defects and the clinical outcomes of these patients are unknown. Here, we performed whole-exome sequencing of the peripheral blood of a cohort of 55 subjects with POI and identified one heterozygous missense variant in FOXL2 (c.1045G>C; p.Arg349Gly) and two heterozygous missense variants in ERCC6 (c.379G>A; p.Val127Ile and c.4223A>C; p.Glu1408 Ala) in four POI patients. All of these heterozygous mutations were predicted to be deleterious and were parentally inherited from their heterozygous fathers. The mRNA and protein expression of FOXL2 and ERCC6 were absent or decreased in the patients. The patients carrying the variants of FOXL2 (c.1045G>C; p.Arg349Gly) and ERCC6 (c.379G>A; p.Val127Ile) failed to conceive in two and four assisted reproductive cycles, respectively. Another patient and her sister carrying the ERCC6 (c.4223A>C; p.Glu1408 Ala) variant achieved good clinical outcomes after assisted reproductive therapy. Our findings support the possible roles of FOXL2 and ERCC6 in POI and might contribute to the genetic counseling of POI patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The molecular mechanism by which inborn errors of the human RNA lariat-debranching enzyme 1 (DBR1) underlie brainstem viral encephalitis is unknown. We show here that the accumulation of RNA lariats in human DBR1-deficient cells interferes with stress granule (SG) assembly, promoting the proteasome degradation of at least G3BP1 and G3BP2, two key components of SGs. In turn, impaired assembly of SGs, which normally recruit PKR, impairs PKR activation and activity against viruses, including HSV-1. Remarkably, the genetic ablation of PKR abolishes the corresponding antiviral effect of DBR1 in vitro. We also show that Dbr1Y17H/Y17H mice are susceptible to similar viral infections in vivo. Moreover, cells and brain samples from Dbr1Y17H/Y17H mice exhibit decreased G3BP1/2 expression and PKR phosphorylation. Thus, the debranching of RNA lariats by DBR1 permits G3BP1/2- and SG assembly-mediated PKR activation and cell-intrinsic antiviral immunity in mice and humans. DBR1-deficient patients are prone to viral disease because of intracellular lariat accumulation, which impairs G3BP1/2- and SG assembly-dependent PKR activation., (© 2024 Ru et al.)

Background: Müllerian duct anomalies (MDAs) are congenital developmental disorders that present as a series of abnormalities within the reproductive tracts of females. Genetic factors are linked to MDAs and recent advancements in whole-exome sequencing (WES) provide innovative perspectives in this field. However, relevant mechanism has only been investigated in a restricted manner without clear elucidation of respective observations., Methods: Our previous study reported that 2 of 12 patients with MDAs harbored the CHD1L variant c.348-1G>C. Subsequently, an additional 85 MDAs patients were recruited. Variants in CHD1L were screened through the in-house database of WES performed in the cohort and two cases were identified. One presented with partial septate uterus with left renal agenesis and the other with complete septate uterus, duplicated cervices and longitudinal vaginal septum. The pathogenicity of the discovered variants was further assessed by molecular dynamics simulation and various functional assays., Results: Ultimately, two novel heterozygous CHD1L variants, including a missense variant c.956G>A (p.R319Q) and a nonsense variant c.1831C>T (p.R611*) were observed. The variants were absent in 100 controls. Altogether, the contribution yield of CHD1L to MDAs was calculated as 4.12% (4/97). All three variants were assessed as pathogenic through various functional analysis. The splice-site variant c.348-1G>C resulted in a 11 bp sequence skipping in exon 4 of CHD1L and led to nonsense mediated decay of its transcripts. Unlike WT CHD1L, the truncated R611* protein mislocalized to the cytoplasm, abolish the ability of CHD1L to promote cell migration and failed to interact with PARP1 owing to the loss of macro domain. The R319Q variant exhibited conformational disparities and showed abnormal protein recruitment behavior through laser microirradiation comparing with the WT CHD1L. All these variants impaired the CHD1L function in DNA damage repair, thus participating in MDAs., Conclusions: The current study not only expands the mutational spectrum of CHD1L in MDAs but determines three variants as pathogenic according to ACMG guidelines with reliable functional evidence. Additionally, the impairment in DNA damage repair is an underlying mechanism involved in MDAs., (© 2024. The Author(s).)

Oxidative stress is crucial in ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC). Intestinal epithelial cells (IECs) are an important component of the intestinal barrier. In previous studies, we have demonstrated that suppressing microRNA-222-3p (miR-222-3p) can protect against oxidative stress in IECs, which ameliorates colonic injuries in UC mice and prevents the conversion of UC to CAC. In this case, we hope to explore whether moxibustion can alleviate UC and CAC by inhibiting miR-222-3p based on mouse models of UC and CAC. After herb-partitioned moxibustion (HPM) intervention, the disease activity index (DAI) and colon macroscopic damage index (CMDI) were significantly reduced in UC mice, and the number and volume of intestinal tumors were decreased considerably in CAC mice. Meanwhile, we found that HPM suppressed miR-222-3p expression and upregulated the mRNA and protein expression of Brahma-related gene 1 (BRG1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), while inhibiting Kelch-like ECH-associated protein 1 (Keap1) expression in IECs of UC and CAC mice. With changes in reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and inflammatory cytokines interleukin (IL)-1 β and tumor necrosis factor (TNF)- α ), we verified that HPM protects against oxidative stress and inflammation in IECs of UC and CAC mice. The effect of HPM was inhibited in miR-222-3p overexpression mice, further demonstrating that the protective effect of HPM on UC and CAC mice was through inhibiting miR-222-3p. In summary, HPM regulates the BRG1/Nrf2/HO-1 pathway by inhibiting miR-222-3p to attenuate oxidative stress in IECs in UC and CAC., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2024 Xuejun Wang et al.)

Pachynema progression contributes to the completion of prophase I. Nevertheless, the regulation of this significant meiotic process remains poorly understood. In this study, we identified a novel testis-specific protein HSF5, which regulates pachynema progression during male meiosis in a manner dependent on chromatin-binding. Deficiency of HSF5 results in meiotic arrest and male infertility, characterized as unconventional pachynema arrested at the mid-to-late stage, with extensive spermatocyte apoptosis. Our scRNA-seq data confirmed consistent expressional alterations of certain driver genes (Sycp1, Msh4, Meiob, etc.) crucial for pachynema progression in Hsf5-/- individuals. HSF5 was revealed to primarily bind to promoter regions of such key divers by CUT&Tag analysis. Also, our results demonstrated that HSF5 biologically interacted with SMARCA5, SMARCA4 and SMARCE1, and it could function as a transcription factor for pachynema progression during meiosis. Therefore, our study underscores the importance of the chromatin-associated HSF5 for the differentiation of spermatocytes, improving the protein regulatory network of the pachynema progression., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A major function of the DNA damage responses (DDRs) that act during the replicative phase of the cell cycle is to inhibit initiation and elongation of DNA replication. It has been shown that DNA replication of the polyomavirus, SV40, is inhibited and its replication fork is slowed by cellular DDR responses. The inhibition of SV40 DNA replication is associated with enhanced DDR kinase phosphorylation of SV40 Large T-antigen (LT), the viral DNA helicase. Mass spectroscopy was used to identify a novel highly conserved DDR kinase site, T518, on LT. In cell-based assays expression of a phosphomimetic form of LT at T518 (T518D) resulted in dramatically decreased levels of SV40 DNA replication, but LT-dependent transcriptional activation was unaffected. Purified WT and LT T518D were analyzed in vitro. In concordance with the cell-based data, reactions using SV40 LT-T518D, but not T518A, showed dramatic inhibition of SV40 DNA replication. A myriad of LT protein-protein interactions and LT's biochemical functions were unaffected by the LT T518D mutation; however, LT's DNA helicase activity was dramatically decreased on long, but not very short, DNA templates. These results suggest that DDR phosphorylation at T518 inhibits SV40 DNA replication by suppressing LT helicase activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)