Moxibustion Regulates the BRG1/Nrf2/HO-1 Pathway by Inhibiting MicroRNA-222-3p to Prevent Oxidative Stress in Intestinal Epithelial Cells in Ulcerative Colitis and Colitis-Associated Colorectal Cancer.

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)-1ß 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.

NEAT1 promotes genome stability via m6A methylation-dependent regulation of CHD4.

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 (m6A) 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.

53BP1 deficiency leads to hyperrecombination using break-induced replication (BIR).

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.

FBW7-Mediated Degradation of CHD3 Suppresses Hepatocellular Carcinoma Metastasis and Stemness to Enhance Oxaliplatin Sensitivity.

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.

Concurrent EGFR mutation and SMARCA4 deficiency in non-small cell lung cancer: A case report and literature review.

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.

Coinactivation of the Switch/Sucrose Nonfermenting Complex SMARCA4/BRG1 and SMARCB1/INI1 in a Cervical Mixed Carcinoma: A Case Report.

SMARCB1/SMARCA4-deficient malignancies of the female genital tract are rare entities, characterized by similar histologic features, such as sheet-like growth patterns and rhabdoid cells. Previous studies have shown mutually exclusive loss of SMARCA4/BRG1 and SMARCB1/INI1. Herein, we describe a unique cervical mixed carcinoma in a 77-year-old patient. The tumor consisted of 3 components, gastric-type adenocarcinoma, squamous carcinoma, and undifferentiated carcinoma. While the undifferentiated carcinoma was negtive for CK7, CK5/6 and p63, it was positive for pan-CK. DNA-based next-generation sequencing revealed a nonsense mutation in SMARCA4, copy number loss in SMARCB1, and a nonsense mutation in ARID1A. Different molecular alterations of the switch/sucrose nonfermenting complex subunits in the present case may provide further insights into the functions of the switch/sucrose nonfermenting complex in the progression of tumors.

Promotion of DNA end resection by BRCA1-BARD1 in homologous recombination.

The licensing step of DNA double-strand break repair by homologous recombination entails resection of DNA ends to generate a single-stranded DNA template for assembly of the repair machinery consisting of the RAD51 recombinase and ancillary factors1. DNA end resection is mechanistically intricate and reliant on the tumour suppressor complex BRCA1-BARD1 (ref. 2). Specifically, three distinct nuclease entities-the 5'-3' exonuclease EXO1 and heterodimeric complexes of the DNA endonuclease DNA2, with either the BLM or WRN helicase-act in synergy to execute the end resection process3. A major question concerns whether BRCA1-BARD1 directly regulates end resection. Here, using highly purified protein factors, we provide evidence that BRCA1-BARD1 physically interacts with EXO1, BLM and WRN. Importantly, with reconstituted biochemical systems and a single-molecule analytical tool, we show that BRCA1-BARD1 upregulates the activity of all three resection pathways. We also demonstrate that BRCA1 and BARD1 harbour stand-alone modules that contribute to the overall functionality of BRCA1-BARD1. Moreover, analysis of a BARD1 mutant impaired in DNA binding shows the importance of this BARD1 attribute in end resection, both in vitro and in cells. Thus, BRCA1-BARD1 enhances the efficiency of all three long-range DNA end resection pathways during homologous recombination in human cells.

A model for polyomavirus helicase activity derived in part from the AlphaFold2 structure of SV40 T-antigen.

The mechanism used by polyomavirus and other viral SF3 helicases to unwind DNA at replication forks remains unknown. Using AlphaFold2, we have determined the structure of a representative SF3 helicase, the SV40 T-antigen (T-ag). This model has been analyzed in terms of the features of T-ag required for helicase activity, particularly the proximity of the T-ag origin binding domain (OBD) to the replication fork and the distribution of basic residues on the surface of the OBD that are known to play roles in DNA unwinding. These and related studies provide additional evidence that the T-ag OBDs have a role in the unwinding of DNA at the replication fork. Nuclear magnetic resonance and modeling experiments also indicate that protonated histidines on the surface of the T-ag OBD play an important role in the unwinding process, and additional modeling studies indicate that protonated histidines are essential in other SF3 and SF6 helicases. Finally, a model for T-ag's helicase activity is presented, which is a variant of the "rope climber." According to this model, the hands are the N-terminal OBD domains that interact with the replication fork, while the C-terminal helicase domains contain the feet that bind to single-stranded DNA. IMPORTANCE: Enzymes termed helicases are essential for the replication of DNA tumor viruses. Unfortunately, much remains to be determined about this class of enzymes, including their structures and the mechanism(s) they employ to unwind DNA. Herein, we present the full-length structure of a model helicase encoded by a DNA tumor virus. Moreover, this AI-based structure has been analyzed in terms of its basic functional properties, such as the orientation of the helicase at replication forks and the relative locations of the amino acid residues that are critical for helicase activity. Obtaining this information is important because it permits proposals regarding how DNA is routed through these model helicases. Also presented is structural evidence that the conclusions drawn from our detailed analyses of one model helicase, encoded by one class of tumor viruses, are likely to apply to other viral and eukaryotic helicases.

Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5'-terminated strands at DSB sites1,2. The breast cancer suppressor BRCA1-BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress1,3-9. BRCA1-BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear10-16. Using purified recombinant proteins, we show here that BRCA1-BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11-RAD50-NBS1 and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex17,18, which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1-C complex19-21, inhibits resection, showing that BRCA1-C is a functionally integrated ensemble. Whereas BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4-6,8). We show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1-BARD1 in various physiological contexts.

Expanding the clinicopathologic spectrum and genomic landscape of tumors with SMARCA2/4::CREM fusions.

CREB gene family (ATF1, CREB1, CREM) fusions with either EWSR1 or FUS gene partners drive the pathogenesis of a wide range of neoplasms, including various soft tissue tumors, intracranial myxoid mesenchymal tumors (IMMTs), hyalinizing clear cell carcinoma (HCCC), and rare mesotheliomas. Recently, a SMARCA2::CREM fusion was reported in one case each of IMMT and HCCC. In this study, we expand the clinicopathologic and molecular spectrum of these neoplasms by describing three additional cases with SMARCA2::CREM and one with a novel SMARCA4::CREM fusion, highlighting the recurrent potential of additional CREB gene fusion partners beyond FET family members. To evaluate if these fusions define a new pathologic entity, we performed a comprehensive genomic and methylation analysis and compared the results to other related tumors. Tumors occurred in children and young adults (median age 20 years) and spanned a broad anatomic distribution, including soft tissue, intracranial, head and neck, and prostatic urethra. Microscopically, the tumors shared an undifferentiated round to epithelioid cell phenotype and a hyalinized fibrous stroma. Immunohistochemically, a polyphenotypic profile was observed, with variable expression of SOX10, desmin, and/or epithelial markers. No targetable genomic alterations were found using panel-based DNA sequencing. By DNA methylation and transcriptomic analyses, tumors grouped closely to FET::CREB entities, but not with SMARCA4/SMARCB1-deficient tumors. High expression of CREM by immunohistochemistry was also documented in these tumors. Patients experienced local recurrence (n = 2), locoregional lymph node metastases (n = 2), and an isolated visceral metastasis (n = 1). Overall, our study suggests that SMARCA2/4::CREM fusions define a distinct group of neoplasms with round cell to epithelioid histology, a variable immunoprofile, and a definite risk of malignancy. Larger studies are needed to further explore the pathogenetic relationship with the FET::CREB family of tumors. © 2024 The Pathological Society of Great Britain and Ireland.

Bivalent chromatin accommodates survivin and BRG1/SWI complex to activate DNA damage response in CD4+ cells.

BACKGROUND: Bivalent regions of chromatin (BvCR) are characterized by trimethylated lysine 4 (H3K4me3) and lysine 27 on histone H3 (H3K27me3) deposition which aid gene expression control during cell differentiation. The role of BvCR in post-transcriptional DNA damage response remains unidentified. Oncoprotein survivin binds chromatin and mediates IFNγ effects in CD4+ cells. In this study, we explored the role of BvCR in DNA damage response of autoimmune CD4+ cells in rheumatoid arthritis (RA). METHODS: We performed deep sequencing of the chromatin bound to survivin, H3K4me3, H3K27me3, and H3K27ac, in human CD4+ cells and identified BvCR, which possessed all three histone H3 modifications. Protein partners of survivin on chromatin were predicted by integration of motif enrichment analysis, computational machine-learning, and structural modeling, and validated experimentally by mass spectrometry and peptide binding array. Survivin-dependent change in BvCR and transcription of genes controlled by the BvCR was studied in CD4+ cells treated with survivin inhibitor, which revealed survivin-dependent biological processes. Finally, the survivin-dependent processes were mapped to the transcriptome of CD4+ cells in blood and in synovial tissue of RA patients and the effect of modern immunomodulating drugs on these processes was explored. RESULTS: We identified that BvCR dominated by H3K4me3 (H3K4me3-BvCR) accommodated survivin within cis-regulatory elements of the genes controlling DNA damage. Inhibition of survivin or JAK-STAT signaling enhanced H3K4me3-BvCR dominance, which improved DNA damage recognition and arrested cell cycle progression in cultured CD4+ cells. Specifically, BvCR accommodating survivin aided sequence-specific anchoring of the BRG1/SWI chromatin-remodeling complex coordinating DNA damage response. Mapping survivin interactome to BRG1/SWI complex demonstrated interaction of survivin with the subunits anchoring the complex to chromatin. Co-expression of BRG1, survivin and IFNγ in CD4+ cells rendered complete deregulation of DNA damage response in RA. Such cells possessed strong ability of homing to RA joints. Immunomodulating drugs inhibited the anchoring subunits of BRG1/SWI complex, which affected arthritogenic profile of CD4+ cells. CONCLUSIONS: BvCR execute DNA damage control to maintain genome fidelity in IFN-activated CD4+ cells. Survivin anchors the BRG1/SWI complex to BvCR to repress DNA damage response. These results offer a platform for therapeutic interventions targeting survivin and BRG1/SWI complex in autoimmunity.

A comprehensive in silico investigation into the pathogenic SNPs in the RTEL1 gene and their biological consequences.

The Regulator of Telomere Helicase 1 (RTEL1) gene encodes a critical DNA helicase intricately involved in the maintenance of telomeric structures and the preservation of genomic stability. Germline mutations in the RTEL1 gene have been clinically associated with Hoyeraal-Hreidarsson syndrome, a more severe version of Dyskeratosis Congenita. Although various research has sought to link RTEL1 mutations to specific disorders, no comprehensive investigation has yet been conducted on missense mutations. In this study, we attempted to investigate the functionally and structurally deleterious coding and non-coding SNPs of the RTEL1 gene using an in silico approach. Initially, out of 1392 nsSNPs, 43 nsSNPs were filtered out through ten web-based bioinformatics tools. With subsequent analysis using nine in silico tools, these 43 nsSNPs were further shortened to 11 most deleterious nsSNPs. Furthermore, analyses of mutated protein structures, evolutionary conservancy, surface accessibility, domains & PTM sites, cancer susceptibility, and interatomic interaction revealed the detrimental effect of these 11 nsSNPs on RTEL1 protein. An in-depth investigation through molecular docking with the DNA binding sequence demonstrated a striking change in the interaction pattern for F15L, M25V, and G706R mutant proteins, suggesting the more severe consequences of these mutations on protein structure and functionality. Among the non-coding variants, two had the highest likelihood of being regulatory variants, whereas one variant was predicted to affect the target region of a miRNA. Thus, this study lays the groundwork for extensive analysis of RTEL1 gene variants in the future, along with the advancement of precision medicine and other treatment modalities.

Single-molecule imaging of SWI/SNF chromatin remodelers reveals bromodomain-mediated and cancer-mutants-specific landscape of multi-modal DNA-binding dynamics.

Despite their prevalent cancer implications, the in vivo dynamics of SWI/SNF chromatin remodelers and how misregulation of such dynamics underpins cancer remain poorly understood. Using live-cell single-molecule tracking, we quantify the intranuclear diffusion and chromatin-binding of three key subunits common to all major human SWI/SNF remodeler complexes (BAF57, BAF155 and BRG1), and resolve two temporally distinct stable binding modes for the fully assembled complex. Super-resolved density mapping reveals heterogeneous, nanoscale remodeler binding "hotspots" across the nucleoplasm where multiple binding events (especially longer-lived ones) preferentially cluster. Importantly, we uncover distinct roles of the bromodomain in modulating chromatin binding/targeting in a DNA-accessibility-dependent manner, pointing to a model where successive longer-lived binding within "hotspots" leads to sustained productive remodeling. Finally, systematic comparison of six common BRG1 mutants implicated in various cancers unveils alterations in chromatin-binding dynamics unique to each mutant, shedding insight into a multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers.

Synergistic Roles of Non-Homologous End Joining and Homologous Recombination in Repair of Ionizing Radiation-Induced DNA Double Strand Breaks in Mouse Embryonic Stem Cells.

DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PKcs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PKcs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PKcs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PKcs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PKcs or both DNA-PKcs and Rad54 exhibit an increased nuclear size approximately 18-24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PKcs, we generated DNA-PKcs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PKcs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PKcs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PKcs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PKcs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes.

Cell-free analysis reveals the role of RG/RGG motifs in DDX3X phase separation and their potential link to cancer pathogenesis.

The DEAD-box RNA helicase DDX3X is a multifunctional protein involved in RNA metabolism and stress responses. In this study, we investigated the role of RG/RGG motifs in the dynamic process of liquid-liquid phase separation (LLPS) of DDX3X using cell-free assays and explored their potential link to cancer development through bioinformatic analysis. Our results demonstrate that the number, location, and composition of RG/RGG motifs significantly influence the ability of DDX3X to undergo phase separation and form self-aggregates. Mutational analysis revealed that the spacing between RG/RGG motifs and the number of glycine residues within each motif are critical factors in determining the extent of phase separation. Furthermore, we found that DDX3X is co-expressed with the stress granule protein G3BP1 in several cancer types and can undergo co-phase separation with G3BP1 in a cell-free system, suggesting a potential functional interaction between these proteins in phase-separated structures. DDX3X and G3BP1 may interact through their RG/RGG domains and subsequently exert important cellular functions under stress situation. Collectively, our findings provide novel insights into the role of RG/RGG motifs in modulating DDX3X phase separation and their potential contribution to cancer pathogenesis.

Getah virus Nsp3 binds G3BP to block formation of bona fide stress granules.

Stress granules (SGs) are cytoplasmic aggregates of proteins and mRNA that form in response to diverse environmental stressors, including viral infections. Several viruses possess the ability to block the formation of stress granules by targeting the SGs marker protein G3BP. However, the molecular functions and mechanisms underlying the regulation of SGs formation by Getah virus (GETV) remain unclear. In this study, we found that GETV infection triggered the formation of Nsp3-G3BP aggregates, which differed in composition from SGs. Further studies revealed that the presence of these aggregates was dependent on the activation of the PKR/eIF2α signaling pathway. Interestingly, we found that Nsp3 HVD domain blocked the formation of SGs by binding to G3BP NTF2 domain. Moreover, knockout of G3BP in NCI-H1299 cells had no effect on GETV replication, while overexpression of G3BP to form the genuine SGs significantly inhibited GETV replication. Overall, our study elucidates a novel role GETV Nsp3 to change the composition of SG as well as cellular stress response.

Structural basis for the concerted antiphage activity in the SIR2-HerA system.

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.

Profound T Lymphocyte and DNA Repair Defect Characterizes Schimke Immuno-Osseous Dysplasia.

Schimke immuno-osseous dysplasia is a rare multisystemic disorder caused by biallelic loss of function of the SMARCAL1 gene that plays a pivotal role in replication fork stabilization and thus DNA repair. Individuals affected from this disease suffer from disproportionate growth failure, steroid resistant nephrotic syndrome leading to renal failure and primary immunodeficiency mediated by T cell lymphopenia. With infectious complications being the leading cause of death in this disease, researching the nature of the immunodeficiency is crucial, particularly as the state is exacerbated by loss of antibodies due to nephrotic syndrome or immunosuppressive treatment. Building on previous findings that identified the loss of IL-7 receptor expression as a possible cause of the immunodeficiency and increased sensitivity to radiation-induced damage, we have employed spectral cytometry and multiplex RNA-sequencing to assess the phenotype and function of T cells ex-vivo and to study changes induced by in-vitro UV irradiation and reaction of cells to the presence of IL-7. Our findings highlight the mature phenotype of T cells with proinflammatory Th1 skew and signs of exhaustion and lack of response to IL-7. UV light irradiation caused a severe increase in the apoptosis of T cells, however the expression of the genes related to immune response and regulation remained surprisingly similar to healthy cells. Due to the disease's rarity, more studies will be necessary for complete understanding of this unique immunodeficiency.

CHD1L Regulates Cell Survival in Breast Cancer and Its Inhibition by OTI-611 Impedes the DNA Damage Response and Induces PARthanatos.

The Chromodomain helicase DNA-binding protein 1-like (CHD1L) is a nucleosome remodeling enzyme, which plays a key role in chromatin relaxation during the DNA damage response. Genome editing has shown that deletion of CHD1L sensitizes cells to PARPi, but the effect of its pharmacological inhibition has not been defined. Triple-negative breast cancer SUM149PT, HCC1937, and MDA-MB-231 cells were used to assess the mechanism of action of the CHD1Li OTI-611. Cytotoxicity as a single agent or in combination with standard-of-care treatments was assessed in tumor organoids. Immunofluorescence was used to assess the translocation of PAR and AIF to the cytoplasm or the nucleus and to study markers of DNA damage or apoptosis. Trapping of PARP1/2 or CHD1L onto chromatin was also assessed by in situ subcellular fractionation and immunofluorescence and validated by Western blot. We show that the inhibition of CHD1L's ATPase activity by OTI-611 is cytotoxic to triple-negative breast cancer tumor organoids and synergizes with PARPi and chemotherapy independently of the BRCA mutation status. The inhibition of the remodeling function blocks the phosphorylation of H2AX, traps CHD1L on chromatin, and leaves PAR chains on PARP1/2 open for hydrolysis. PAR hydrolysis traps PARP1/2 at DNA damage sites and mediates PAR translocation to the cytoplasm, release of AIF from the mitochondria, and induction of PARthanatos. The targeted inhibition of CHD1L's oncogenic function by OTI-611 signifies an innovative therapeutic strategy for breast cancer and other cancers. This approach capitalizes on CHD1L-mediated DNA repair and cell survival vulnerabilities, thereby creating synergy with standard-of-care therapies.

Targeting SWI/SNF ATPases reduces neuroblastoma cell plasticity.

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.