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Hi-C reads, which represent ligation events between different regions of the genome, must be processed into matrices of interaction frequencies for downstream analysis. Here, I describe a procedure for mapping Hi-C reads to the genome and conversion of mapped reads into the HOMER tag directory format and interaction matrix format for visualization with Juicebox. The method is demonstrated for the mouse composite X chromosome in which reads from the active and inactive X chromosomes are combined after mock DMSO treatment or targeted degradation of cohesin., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cohesin complex is essential for cell division and regulating cell type-specific gene expression programs. Mutations in genes encoding the cohesin subunits are associated with hematological malignancies, preleukemia, and clonal hematopoiesis of indeterminate potential. In this study, we examined how cohesin mutation impacts hematopoiesis using adult zebrafish that carry heterozygous germline nonsense mutation in the cohesin subunit, rad21 ( rad21 +/- ) that is orthologous to human RAD21 . Single-cell RNA sequencing analyses showed that adult zebrafish harboring rad21 +/- mutation exhibit significant transcriptional dysregulation within the whole kidney marrow and have altered erythroid and granulocyte output. Erythroid progenitors were expanded in rad21 +/- and erythroid differentiation was altered. The expression profile of several erythroid genes, including gata1a , was dysregulated in rad21 +/- erythroid cells. Mature granulocyte population declined in rad21 +/- , and the transcriptional program of granulocytes was impaired but granulocytic maturation was maintained. Granulocytes from rad21 +/- showed upregulation of stress hematopoiesis factor, cebpb . These findings show that normal rad21 is required to maintain steady erythropoiesis and granulopoiesis in the adult zebrafish marrow. NEW & NOTEWORTHY Mutations in cohesin subunit genes are early events in leukemogenesis. This study characterizes the hematopoietic compartment of adult zebrafish that carry germline heterozygous mutation in cohesin subunit, rad21 . Our results show that despite normal appearance, rad21 mutant adult zebrafish exhibit transcriptional dysregulation and altered erythroid and granulocyte output. No obvious morphological dysplasia was observed in the rad21 mutant hematopoietic cells. These results suggest that rad21 mutation can cause underlying hematopoietic disturbances.

The Structural Maintenance of Chromosomes (SMC) protein complexes are DNA-binding molecular machines required to shape chromosomes into functional units and to safeguard the genome through cell division. These ring-shaped multi-subunit protein complexes, which are present in all kingdoms of life, achieve this by organizing chromosomes in three-dimensional space. Mechanistically, the SMC complexes hydrolyze ATP to either stably entrap DNA molecules within their lumen, or rapidly reel DNA into large loops, which allow them to link two stretches of DNA in cis or trans. In this chapter, the canonical structure of the SMC complexes is first introduced, followed by a description of the composition and general functions of the main types of eukaryotic and prokaryotic SMC complexes. Thereafter, the current model for how SMC complexes perform in vitro DNA loop extrusion is presented. Lastly, chromosome loop formation by SMC complexes is introduced, and how the DNA loop extrusion mechanism contributes to chromosome looping by SMC complexes in cells is discussed., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Optineurin (OPTN) is an autophagy receptor that participates in the degradation of damaged mitochondria, protein aggregates, and invading pathogens. OPTN is closely related to various types of neurodegenerative diseases. However, the role of OPTN in the central nervous system is unclear. Here, we found that OPTN dysregulation in the compact part of substantia nigra (SNc) led to motor and learning deficits in animal models. Knockdown of OPTN increased total and phosphorylated α-synuclein levels which induced microglial activation and dopaminergic neuronal loss in the SNc. Overexpression of OPTN can't reverse the motor and learning phenotypes. Mechanistic analysis revealed that upregulation of OPTN increased α-synuclein phosphorylation independent of its autophagy receptor activity, which further resulted in microglial activation and dopaminergic neuronal loss similar to OPTN downregulation. Our study uncovers the crucial role of OPTN in maintaining dopaminergic neuron survival and motor and learning functions which are disrupted in PD patients., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

The epiblast is a pluripotent cell population formed in the late blastocyst stage of preimplantation embryos. During the process of epiblast formation from the inner cell mass (ICM) of the early blastocyst, activation of the Hippo pathway transcription factor TEAD by the nuclear translocation of the coactivator protein YAP is required for the robust expression of pluripotency factors. However, the mechanisms that alter YAP localization during epiblast formation remain unknown. Here, we reveal two such mechanisms. Expansion of the blastocoel promotes nuclear YAP localization by increasing cytoplasmic F-actin and reducing YAP phosphorylation. Additionally, cell differentiation regulates YAP. Expression of the junctional Hippo component, AMOT, gradually decreases during epiblast formation through a tankyrase-mediated degradation. SOX2 expression in the ICM is necessary for the reduction of AMOT and YAP phosphorylation. These two mechanisms function in parallel. Thus, the blastocoel-F-actin and SOX2-AMOT axes cooperatively suppress YAP phosphorylation and promote YAP nuclear localization during epiblast formation. The cooperation of these two distinct mechanisms likely contributes to the robustness of epiblast cell differentiation., Competing Interests: Declarations of interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

V(D)J recombination secures the production of functional immunoglobulin (Ig) genes and antibody diversity during the early stages of B-cell development through long-distance interactions mediated by cis-regulatory elements and trans-acting factors. O-GlcNAcylation is a dynamic and reversible posttranslational modification of nuclear and cytoplasmic proteins that regulates various protein functions, including DNA-binding affinity and protein-protein interactions. However, the effects of O-GlcNAcylation on proteins involved in V(D)J recombination remain largely unknown. To elucidate this relationship, we downregulated O-GlcNAcylation in a mouse model by administering an O-GlcNAc inhibitor or restricting the consumption of a regular diet. Interestingly, the inhibition of O-GlcNAcylation in mice severely impaired Ig heavy-chain (IgH) gene rearrangement. We identified several factors crucial for V(D)J recombination, including YY1, CTCF, SMC1, and SMC3, as direct targets of O-GlcNAc modification. Importantly, O-GlcNAcylation regulates the physical interaction between SMC1 and SMC3 and the DNA-binding patterns of YY1 at the IgH gene locus. Moreover, O-GlcNAc inhibition downregulated DDX5 protein expression, affecting the functional association of CTCF with its DNA-binding sites at the IgH locus. Our results showed that locus contraction and long-range interactions throughout the IgH locus are disrupted in a manner dependent on the cellular O-GlcNAc level. In this study, we established that V(D)J recombination relies on the O-GlcNAc status of stage-specific proteins during early B-cell development and identified O-GlcNAc-dependent mechanisms as new regulatory components for the development of a diverse antibody repertoire., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Pancreatic cancer is a highly malignant tumor characterized by high mortality and low survival rates. The mitotic interactor and substrate of Plk1 (MISP) is a cancer-associated protein that regulates mitotic spindle localization and is highly expressed in several malignant tumors, contributing to tumor development. However, the function and regulatory mechanisms of MISP in pancreatic cancer remain unclear. In this study, we analyzed RNA sequencing data related to pancreatic cancer from the TCGA and GEO databases, identifying MISP as a potential prognostic marker for the disease. MISP was significantly upregulated in pancreatic cancer cells and tissues compared to normal pancreatic cells and tissues. Notably, in pancreatic cancer cells, high MISP protein expression promoted cell proliferation and growth. Mechanistically, the upregulation of MISP facilitated the nuclear accumulation of β-catenin, thereby activating the Wnt/β-catenin signaling pathway and promoting pancreatic cancer growth. In search of effective inhibitors of MISP expression, we screened an FDA-approved drug library and identified Fisetin as a potential suppressor of MISP expression. Fisetin was found to downregulate the transcription factor MYB, thereby reducing MISP expression. Further experiments demonstrated that Fisetin effectively inhibited the in vitro and in vivo growth of pancreatic cancer by suppressing the MISP/Wnt/β-catenin signaling axis. In summary, our research has identified MISP as a novel therapeutic target in pancreatic cancer and uncovered its associated regulatory mechanisms., Competing Interests: Declaration of competing interest All authors declare they have no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Background: Promoters serve as key elements in the regulation of gene transcription. In mammals, loop interactions between promoters and enhancers increase the complexity of the promoter-based regulatory networks. However, the identification of enhancer-promoter or promoter-related loops in Arabidopsis remains incomplete., Results: Here, we use promoter capture Hi-C to identify promoter-related loops in Arabidopsis, which shows that gene body, proximal promoter, and intergenic regions can interact with promoters, potentially functioning as distal regulatory elements or enhancers. We find that promoter-related loops mainly repress gene transcription and are associated with ordered chromatin structures, such as topologically associating domains and fountains-chromatin structures not previously identified in Arabidopsis. Cohesin binds to the center of fountains and is involved in their formation. Moreover, fountain strength is positively correlated with the number of promoter-related loops, and the maintenance of these loops is linked to H3K4me3. In atxr3 mutants, which lack the major H3K4me3 methyltransferases in Arabidopsis, the number of promoter-related loops at fountains is reduced, leading to upregulation of fountain-regulated genes., Conclusions: We identify promoter-related loops associated with ordered chromatin structures and reveal the molecular mechanisms involved in fountain formation and maintenance., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The CCCTC-binding factor (CTCF) is pivotal in orchestrating diverse biological functions across the human genome, yet the mechanisms driving its cell type-active DNA binding affinity remain underexplored. Here, we collected ChIP-seq data from 67 cell lines in ENCODE, constructed a unique dataset of cell type-active CTCF binding sites (CBS), and trained convolutional neural networks (CNN) to dissect the patterns of CTCF binding activity. Our analysis reveals that transcription factors RAD21/SMC3 and chromatin accessibility are more predictive compared to sequence motifs and histone modifications. Integrating them together achieved AUPRC values consistently above 0.868, highlighting their utility in deciphering CTCF transcription factor binding dynamics. This study provides a deeper understanding of the regulatory functions of CTCF via machine learning framework., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Polo-like kinase 1 (PLK1) protects against genome instability by ensuring timely and accurate mitotic cell division, and its activity is tightly regulated throughout the cell cycle. Although the pathways that initially activate PLK1 in G2 are well-characterized, the factors that directly regulate mitotic PLK1 remain poorly understood. Here, we identify that human PLK1 activity is sustained by the DNA damage response kinase Checkpoint kinase 2 (Chk2) in mitosis. Chk2 directly phosphorylates PLK1 T210, a residue on its T-loop whose phosphorylation is essential for full PLK1 kinase activity. Loss of Chk2-dependent PLK1 activity causes increased mitotic errors, including chromosome misalignment, chromosome missegregation, and cytokinetic defects. Moreover, Chk2 deficiency increases sensitivity to PLK1 inhibitors, suggesting that Chk2 status may be an informative biomarker for PLK1 inhibitor efficacy. This work demonstrates that Chk2 sustains mitotic PLK1 activity and protects genome stability through discrete functions in interphase DNA damage repair and mitotic chromosome segregation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

High-fat diet (HFD) induces low-grade chronic inflammation, contributing to obesity and insulin resistance. However, the precise mechanisms triggering obesity-associated metabolic inflammation remain elusive. In this study, we identified epigenetic factor Brd4 as a key player in this process by regulating the expression of Ccr2/Ccr5 in colonic macrophage. Upon 4-week HFD, myeloid-lineage-specific Brd4 deletion (Brd4-CKO) mice showed reduced colonic inflammation and macrophage infiltration with decreased expression of Ccr2 and Ccr5. Mechanistically, Brd4 was recruited by NF-κB to the enhancer regions of Ccr2 and Ccr5, promoting enhancer RNA expression, which facilitated Ccr2/Ccr5 expression and macrophage migration. Furthermore, decreased infiltration of Ccr2/Ccr5-positive colonic macrophages in Brd4-CKO mice altered gut microbiota composition and reduced intestinal permeability, thereby lowering metabolic endotoxemia. Finally, Brd4-CKO mice subjected to a 4-week LPS infusion exhibited restored susceptibility to HFD-induced obesity and insulin resistance. This study identifies Brd4 as a critical initiator of colonic macrophage-mediated inflammation and metabolic endotoxemia upon HFD, suggesting Brd4 as a potential target for mitigating HFD-induced inflammation, obesity, and its metabolic complications., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The overexpression of Polo-like kinase 1 (PLK1) is associated with poor clinical outcomes in various malignancies, making it an attractive target for anticancer therapies. Although recent studies suggest PLK1's involvement in homologous recombination (HR), the impact of its overexpression on HR remains unclear. In this study, we investigated the effect of PLK1 overexpression on HR using bioinformatics and experimental approaches. Analyzing The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) datasets with the Homologous Recombination Deficiency (HRD) score, we found a positive correlation between PLK1 expression and HRD score, indicating that increased PLK1 expression suppresses HR. To validate these findings, we performed cell line-based experiments, demonstrating that PLK1 overexpression attenuates RAD51 focus formation and HR, as measured by ASHRA in T47D cells. Since HR-deficient cells are hypersensitive to PARP inhibitors, we further confirmed that PLK1 overexpression increases sensitivity to PARP inhibitors, both in CCLE dataset analysis and experiments using T47D cells. Additionally, we found that the effects of PLK1 overexpression on HR suppression and increased PARP inhibitor sensitivity were mitigated by either a PLK1 kinase inhibitor or the kinase-dead mutant [T210A]. This suggests that PLK1's impact on HR and PARP inhibitor sensitivity is mediated through its kinase activity. Moreover, analysis of clinical ovarian cancer samples revealed that higher PLK1 expression correlates with increased sensitivity to PARP inhibitors. Our results suggest that PLK1 overexpression suppresses homologous recombination, leading to enhanced sensitivity to PARP inhibition, presenting a potential therapeutic strategy for targeting cancers with overexpression of PLK1., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Accurate and timely genetic material replication is essential for preserving genomic integrity. The replication process begins with chromatin licensing and DNA replication factor 1 (CDT1). It has been demonstrated that dysregulated CDT1 expression causes genomic instability, damages DNA, and may even cause cancer. Although this protein is overexpressed in several malignancies, its significance in gastric cancer is still unknown. This work aims to investigate the biological function and molecular mechanism of CDT1 in gastric cancer. Bioinformatics studies were carried out using TCGA database, Kaplan-Meier and GEPIA online data analysis software. The expression of CDT1 in stomach cancer tissues was determined by immunohistochemistry, and its relationship to clinicopathological features was examined. Using siRNA and cell function experiments, the impact of CDT1 expression on the capacity of gastric cancer cell lines to proliferate, migrate, and invade was investigated. In gastric cancer tissues, CDT1 is overexpressed and is linked to a dismal prognosis. Through the up-regulation of M-cyc, MCM5 (microchromosome maintenance protein 5), CyclinD1, N-cadherin, and the down-regulation of E-cadherin, CDT1 facilitated the proliferation and invasion of gastric cancer cells. The study's findings shed insight on the possible molecular processes behind CDT1's function in promoting the start and progression of gastric cancer and demonstrate the potential predictive and diagnostic use of CDT1 expression in gastric cancer., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Gastric cancer (GC) is one of the most common cancers worldwide, with increasing incidence and mortality rates. It is typically diagnosed at advanced stages, leading to a poor prognosis. GC is a highly heterogeneous disease and its progression is associated with complex interplay between genetic and environmental factors. Identifying novel genes and pathways involved in GC development is crucial for improving the therapeutic outcome. Regulator of Telomerase Length 1 (RTEL1) has been found to maintain telomere stability through its helicase activity, facilitating telomere reconstruction and repair. However, the precise role of RTEL1 in human cancers, particularly in GC, is not yet fully understood. In this study, we observed significantly increased RTEL1 expression in GC tissues, which was associated with a poor prognosis. Functionally, RTEL1 promotes GC cell proliferation both in vitro and in vivo. Additionally, RTEL1 appears to regulate multiple signaling pathways, with a particular promoting effect on the cell cycle progression. Notably, CDC23 and TRIP13 are potential downstream target genes of RTEL1, which may mediate its tumor-promoting effects in GC. These findings suggest that RTEL1 plays a critical role in GC tumorigenesis and could be a promising target for the therapy and prognosis of GC., Competing Interests: Declarations. Author statement: Zehua Bian and Zhaohui Huang designed the research study. Chunyu Yang, Suzeng Wang, Ge Gao, Peiwen Xu, Mengyuan Qian, Yuan Yin and Surui Yao carried out the experimental work. Chunyu Yang, Zehua Bian and Zhaohui Huang analyzed the data and wrote the paper. All the authors have read and approved the final manuscript. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Acute liver injury is characterized by the rapid onset of inflammation in the liver, which in turn plays a role in the development of hypertension. Additionally, hypertension increases susceptibility to liver diseases associated with inflammatory states. Recently, the antihypertensive drug diltiazem has demonstrated anti-inflammatory properties and has been shown to inhibit the expression of the thioredoxin-interacting protein (TXNIP), an upstream regulator of the NOD-like receptor pyrin-3 (NLRP3) inflammasome pathway. In our quest for an optimal therapeutic intervention for liver inflammatory diseases, we investigated the effects of diltiazem. Herein, we employed a multi-step approach integrating computational target prediction, network analysis, and molecular docking with experimental validation to explore potential interactions between diltiazem and TXNIP. Our investigations in rats with thioacetamide-induced liver injury revealed the anti-inflammatory potential of diltiazem, likely due to the suppression of the NLRP3 signaling pathway via targeting TXNIP. Furthermore, diltiazem suppressed the priming signal induced by nuclear factor kappa-B (NFκB) activation, as well as subsequent inflammasome components, including cleaved caspase-1, gasdermin D, IL-1β, and IL-18. Consequently, diltiazem exhibited anti-pyroptotic effects in the injured liver. Additionally, diltiazem was observed to reduce BCL-2-associated X-protein (Bax) levels, increase B-Cell Lymphoma-2 (BCL2) levels, and decrease the tissue expression of cleaved caspase-3, indicating potential anti-apoptotic effects. These effects were reflected in improved liver weight-to-body weight ratio, liver histology, oxidative stress parameters, and liver function. In conclusion, diltiazem shows promise as a protective agent for liver inflammatory diseases. Further research is warranted to translate these preclinical results into effective strategies for improving liver health., 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.)

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are key downstream effectors of the Hippo pathway that regulate organ size, tissue homeostasis, and cancer development. YAP/TAZ play crucial regulatory roles in organ growth, cell proliferation, cell renewal, and regeneration. Mechanistically, YAP/TAZ influence the occurrence and progression of liver regeneration (LR) through various signaling pathways, including Notch, Wnt/β-catenin, TGF-β/Smad. While the activation of YAP/TAZ can promote the regeneration of damaged liver tissue, their mechanisms of action may differ under various LR conditions. Furthermore, excessive activation of YAP/TAZ may also lead to severe liver damage, manifesting as alcoholic hepatitis, liver fibrosis, and even liver cancer. Here, we review the role and mechanisms of YAP/TAZ in LR and liver disease, highlighting the potential for advancements in clinical diagnosis and treatment targeting YAP/TAZ in these contexts., Competing Interests: Declarations. Conflict of interest: The author declares that no competing interests. Ethics approval: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Ovarian cancer (OC) is prone to adipose tissue metastasis. However, the underlying molecular mechanisms remain elusive. Here, we observed that omental adipocytes were induced into cancer-associated adipocytes (CAAs) by OC-derived TGF-β1 to establish a pre-metastatic niche (PMN) through collagen and fibronectin secretion. Mechanistically, OC-derived TGF-β1 binds to adipocyte membrane receptors and thus activates intracellular signaling by SMAD3 phosphorylation. The activation of TGF-β1/SMAD3 signaling pathway dedifferentiates adipocytes into CAAs by upregulating Tribbles homolog 3 (TRIB3), which suppresses the phosphorylation of CEBPβ. Additionally, CAAs secrete collagen I, collagen VI, and fibronectin to remodel the extracellular matrix and promote the adhesion of OC cells. Pharmacological inhibition of the TGF-β1/SMAD3 pathway significantly inhibits CAAs and PMN formation, thereby reducing the OC metastatic burden. Our findings indicate that the formation of CAAs and PMN in adipose tissues facilitates OC cell implantation and blocking the TGF-β1/SMAD3 signaling pathway could prevent OC omental metastasis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Proteins become asymmetrically distributed in the one-cell Caenorhabditis elegans embryo thanks to reaction-diffusion mechanisms that are often entangled in complex feedback loops. Cortical polarity drives the enrichment of the RNA-binding proteins MEX-5 and MEX-6 in the anterior cytoplasm through concentration gradients. MEX-5 and MEX-6 promote the patterning of other cytoplasmic factors, including that of the anteriorly enriched mitotic polo-like kinase PLK-1, but also contribute to proper cortical polarity. The gradient of MEX-5 forms through a differential-diffusion mechanism. How MEX-6 establishes a gradient and how MEX-5 and MEX-6 regulate cortical polarity is not known. Here, we reveal that the two MEX proteins develop concentration asymmetries via similar mechanisms, but despite their strong sequence homology, they differ in terms of how their concentration gradients are regulated. We find that PLK-1 promotes the enrichment of MEX-5 and MEX-6 at the anterior through different circuits: PLK-1 influences the MEX-5 gradient indirectly by regulating cortical polarity while it modulates the formation of the gradient of MEX-6 through its physical interaction with the protein. We thus propose a model in which PLK-1 mediates protein circuitries between MEX-5, MEX-6, and cortical proteins to faithfully establish and maintain polarity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Aneuploidy in eggs is a leading cause of miscarriages or viable developmental syndromes. Aneuploidy rates differ between individual chromosomes. For instance, chromosome 21 frequently missegregates, resulting in Down Syndrome. What causes chromosome-specific aneuploidy in meiosis is unclear. Chromosome 21 belongs to the class of acrocentric chromosomes, whose centromeres are located close to the chromosome end, resulting in one long and one short chromosome arm. We demonstrate that acrocentric chromosomes are generally more often aneuploid than metacentric chromosomes in porcine eggs. Kinetochores of acrocentric chromosomes are often partially covered by the short chromosome arm during meiosis I in human and porcine oocytes and orient less efficiently toward the spindle poles. These partially covered kinetochores are more likely to be incorrectly attached to the spindle. Additionally, sister chromatids of acrocentric chromosomes are held together by lower levels of cohesin, making them more vulnerable to age-dependent cohesin loss. Chromosome architecture and low cohesion therefore bias acrocentric chromosomes toward aneuploidy during mammalian meiosis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

CELL DIVISION CYCLE 5 (CDC5) is a R2R3-type MYB transcription factor, serving as a key component of modifier of snc1, 4-associated complex/NineTeen complex, which is associated with plant immunity, RNA splicing, and miRNA biogenesis. In this study, we demonstrate that mutation of CDC5 accelerates flowering in Arabidopsis (Arabidopsis thaliana). CDC5 activates the expression of FLOWERING LOCUS C (FLC) by binding to and affecting the enrichment of RNA polymerase II on FLC chromatin. Moreover, genetic analysis confirmed that CDC5 regulates flowering in an FLC-dependent manner. Furthermore, we characterized the interaction of CDC5 with the RNA polymerase-associated factor 1 (Paf1) complex and confirmed that CDC5, as part of the spliceosome, mediates genome-wide alternative splicing, as revealed by RNA-seq. CDC5 affected the splicing of flowering-associated genes such as FLC, SEF, and MAFs. Additionally, we also demonstrated that CDC5 contributes to the regulation of histone modification of FLC chromatin, which further promotes FLC expression. In summary, our results establish CDC5 as a key factor regulating flowering. This provides valuable insight for future research into plant flowering., Competing Interests: Conflict of interest statement: The authors declare that they have no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Introduction: The aberrant up-regulation of meiotic nuclear division 1 (MND1) in somatic cells is considered as one of the driving factors of oncogenesis, whereas its expression and role in breast invasive cancer (BRCA) remain unclear. Hence, this study embarked on a comprehensive evaluation of MND1 across various cancers and identified its roles in BRCA., Methods: Based on publicly available databases, including but not limited to UCSC Xena, TCGA, GTEx, GEO, STRING, GeneMANIA, and CancerSEA, we evaluated the expression patterns, genomic features, and biological functions of MND1 from a pan-cancer viewpoint and delved into the implications of MND1 in the prognosis and treatment of BRCA. Further molecular biology experiments were undertaken to identify the role of MND1 in proliferation, migration, and apoptosis in BRCA cells., Results: Elevated levels of MND1 were notably observed in a wide array of tumor types, especially in BRCA, COAD, HNSC, LIHC, LUAD, LUSC, STAD, and UCEC. Elevated MND1 expression was markedly associated with shortened OS in several tumors, including BRCA (HR = 1.52 [95%CI, 1.10-2.09], P = 0.011). The up-regulation of MND1 in BRCA was validated in external cohorts and clinical samples. Survival analyses demonstrated that elevated MND1 expression was associated with decreased survival for patients with BRCA. Co-expressed genes of MND1 were identified, and subsequent pathway analyses based on significantly associated genes indicated that MND1 plays key roles in DNA replication, cell cycle regulation, and DNA damage repair. The observed abnormal elevation and activation of MND1 led to increased proliferation and migration, along with decreased apoptosis in BRCA cells., Conclusions: MND1 emerges as a promising biomarker for diagnostic and therapeutic targeting in various cancers, including BRCA. The abnormal up-regulation and activation of MND1 are linked to carcinogenesis and poor prognosis among BRCA patients, which may be attributed to its involvement in HR-dependent ALT, warranting further scrutiny., 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 Inc. All rights reserved.)

Background/Objectives : AML is an aggressive malignant disease characterized by aberrant proliferation and accumulation of immature blast cells in the patient's bone marrow. Chemotherapeutic treatment can effectively induce remission and re-establish functional hematopoiesis. However, many patients experience chemoresistance-associated relapse and disease progression with a poor prognosis. The identification of molecular determinants of chemoresistance that could serve as potential targets for the therapeutic restoration of chemosensitivity has proven to be challenging. Methods : To address this, we have analyzed longitudinal changes in the expression of microRNAs during disease progression in a small set of four AML patients, combined with gene ontology (GO) pathway analysis and evaluation of gene expression data in patient databases. Results : MicroRNA profiling of bone marrow samples at diagnosis and after relapse revealed significant differential expression of a large number of microRNAs between the two time points. Subsequent GO pathway analysis identified 11 signal transduction pathways likely to be affected by the differential miRNA signatures. Exemplary validation of the FoxO signaling pathway by gene expression analysis confirmed significant upregulation of FOXO1 and the target genes GADD45 and SOD2 . Conclusions : Here, we show how a microRNA-based pathway prediction strategy can be used to identify differentially regulated signaling pathways that represent potential targets for therapeutic intervention.

Background: Sperm-associated antigen 5 (SPAG5) is a mitotic spindle protein crucial for coordinating the separation of sister chromatids into daughter cells. Increasing evidence suggests that SPAG5 is overexpressed in various malignancies, functioning as an oncogene. However, research specifically examining SPAG5 in esophageal cancer remains limited., Methods: In this research, we leveraged bioinformatics techniques to evaluate the expression and prognostic significance of SPAG5 in a variety of cancer types. We conducted Gene Set Enrichment Analysis (GSEA) to elucidate the relationship between SPAG5 and cancer characteristics. Additionally, we investigated the correlation between SPAG5 expression and immune cell infiltration utilizing the TIMER2.0 platform. The TIDE platform was used to assess the impact of SPAG5 on the effectiveness of immunotherapy and to screen for potential therapeutic drugs. We employed qRT-PCR and immunohistochemistry staining to ascertain the expression of SPAG5 in esophageal cancer tissue. Through cellular functional experiments, we examined the influence of SPAG5 expression on the proliferation, apoptosis, invasion, and migration of esophageal cancer cells. The Pathscan Stress Signaling Antibody Array was utilized to probe the potential molecular mechanisms of SPAG5., Results: SPAG5 exhibits high levels of expression in various cancers, encompassing esophageal cancer, and its presence indicates an unfavorable prognosis. SPAG5 is primarily enriched in pathways associated with cellular proliferation and demonstrates a correlation with immune gene expression as well as the infiltration of immune cells. Suppression of SPAG5 expression in esophageal cancer cells not only inhibits cell proliferation, but also attenuates cell invasion and migration while inducing cellular apoptosis. The depletion of SPAG5 results in a decline in the levels of critical signaling proteins., Conclusion: SPAG5 plays a pivotal role in esophageal cancer cell proliferation, apoptosis, and metastasis within the tumor microenvironment, making it a promising therapeutic target., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Structural maintenance of chromosomes (SMC) protein complexes, including cohesin, condensin, and the Smc5/6 complex, are integral to various processes in chromosome biology. Despite their distinct roles, these complexes share two key properties: the ability to extrude DNA into large loop structures and the capacity to alter the superhelicity of the DNA double helix. In this review, we explore the influence of eukaryotic SMC complexes on DNA topology, debate its potential physiological function, and discuss new structural insights that may explain how these complexes mediate changes in DNA topology., (© 2024 The Author(s).)

It is well known that genome organizers, like mammalian CCCTC-binding factor (CTCF) or Drosophila architectural proteins CP190 and BEAF-32, contribute to the three-dimensional (3D) organization of the genome and ensure normal gene transcription. However, bona fide genome organizers have not been identified in plants. Here, we show that EMBRYONIC FLOWER1 (EMF1) functions as a genome modulator in Arabidopsis. EMF1 interacts with the cohesin component SISTER CHROMATIN COHESION3 (SCC3), and both proteins are enriched at compartment domain (CD) boundaries. Accordingly, emf1 and scc3 show a strength decrease at the CD boundary in which these proteins colocalize. EMF1 maintains CD boundary strength, either independently or in cooperation with histone modifications. Moreover, EMF1 is required to maintain gene-resolution interactions and to block long-range aberrant chromatin loops. These data unveil a key role of EMF1 in regulating 3D chromatin structure., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The primary cilium is a cellular organelle whose assembly and disassembly are closely linked to the cell cycle. The centriole distal appendage (DA) is essential for the early stages of ciliogenesis by anchoring the mother centriole to the cell surface. Despite the identification of over twelve proteins constituting the DA, including CEP83, CEP89, CEP164, FBF1, and SCLT1, their specific functions in ciliary dynamics are not fully understood. Here, we elucidate the precise role of DA proteins in ciliary assembly and disassembly. While Cep89 mutant cells exhibit normal ciliogenesis, the kinetics of ciliary disassembly is significantly delayed. Through siRNA-mediated knockdown of DA proteins, we identified two functional subgroups within DA proteins: CEP83, SCLT1, and CEP164, which are primarily essential for ciliary assembly and centriole docking, and CEP89 and FBF1, which specifically regulate ciliary disassembly. Notably, the depletion of CEP89 and FBF1 not only impedes ciliary disassembly but also disrupts the Aurora A kinase signaling pathway, leading to its downregulation and mislocalization at the basal body during serum-induced cell cycle re-entry. These findings suggest that DA components can be functionally categorized into two modules responsible for distinct aspects of ciliary dynamics, with broad implications for cellular signaling, homeostasis, and development., Competing Interests: Decleration. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

During infection the autonomous parvovirus minute virus of mice (MVM) generates extensive DNA damage which facilitates virus replication and induces a cellular DNA damage response (DDR) driven by the ataxia telangiectasia mutated (ATM) kinase. Atypically, the ataxia telangiectasia and Rad-3-related (ATR) DDR pathway remains inactive. Upon DNA damage ATR is normally recruited to single-stranded DNA sequences formed at genomic DNA damage sites, and while within a multiprotein complex activates, via phosphorylation, the key DDR regulator checkpoint kinase 1 (Chk1). Inactivation of ATR during MVM infection leads to the accumulation of damaged DNA and enhancement of virus replication. Although ATR is inactivated, we show that during infection, the Chk1 activation pathway downstream of the initial ATR activating events remained functional. Activation of ATR, and consequently of Chk1, requires interaction with TopBP1, which itself is maintained in proximity to ATR by interaction with the phosphorylated S387 residue of Rad9, part of the Rad9-Hus1-Rad1 (911) complex. Both MVM infection and MVM NS1 overexpression inhibited Rad9 S387 phosphorylation and subsequent ATR activation. ATR inactivation during infection was suppressed by expression of Rad9 bearing a phosphomimetic 387 residue, indicating that this site, and the function it served, was the target of NS1 inhibition. NS1 interaction with CK2α and CK2α enzymatic activity was both required to prevent ATR activation, indicating MVM retargeted this kinase's activity during infection. Inhibition of the protein phosphatase 2C (PP2C) prevented Rad9 S387 dephosphorylation and Chk1 inactivation during MVM infection and NS1 overexpression revealing its role in the pathway's suppression., Importance: Infection by the parvovirus minute virus of mice (MVM) causes significant DNA damage and induces a potent DNA damage response (DDR) which the virus exploits to further its replication. The cell responds to infection with an ATM-regulated DDR; however, atypically, the ATR-regulated DDR pathway is disabled during infection. This prevents Chk1 activation, thus allowing the accumulation of damaged DNA which facilitates virus replication. We describe here how MVM, and specifically the main viral replication protein NS1, inhibits ATR activation. Activation of ATR, and consequently Chk1, requires TopBP1 localization into the activating complex via its interaction with a phosphorylated residue of Rad9. We show that NS1 redirects casein kinase 2 to activate a phosphatase in the PP2C family which causes dephosphorylation of this critical residue, thus inhibiting ATR activation. This work provides mechanistic insight into one of the ways by which parvoviruses modify the host DDR response to facilitate their replication., Competing Interests: The authors declare no conflict of interest.

In spite of being dispensable for catalysis, Dpb2, the second largest subunit of leading strand DNA polymerase (Polymerase ε) is essential for cell survival in budding yeast. Dpb2 physically connects polymerase epsilon with the replicative helicase (CMG,Cdc45-Mcm-GINS) by interacting with its Psf1 subunit. Dpb2-Psf1 interaction has been shown to be critical for incorporating polymerase ε into the replisome. Site-directed mutagenesis studies on conserved amino acid residues within the N-terminal domain of Dpb2 led to identification of key amino acid residues involved in interaction with Psf1 subunit of GINS. These amino acid residues are found to be well conserved among Dpb2 orthologues in higher eukaryotes thereby indicating the protein-protein interaction to be evolutionarily conserved. Replicating cells are known to mount a strong replicative stress response and DNA damage response upon exposure to diverse range of stressors. Here, we show that the absence of the N-terminal domain of Dpb2 increases the vulnerability of the budding yeast cells towards the cytotoxic effects of hydroxyurea (HU) and methyl methane sulphonate (MMS). Our results illustrate the importance of N-terminal domain of Dpb2 not only during replisome assembly but also in coordinating stress response in budding yeast. Considering high degree of sequence conservation across eukaryotes, Dpb2 subunit of leading-strand DNA polymerase appears to have important implications in maintenance of genome integrity., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern, severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript, the small nuclear RNA U4atac, is involved in the excision of ~850 minor introns. Here, we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC, instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts, we analysed neural stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and cell death. In cortical organoids, we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes, by observing delayed apico-basal polarization of NSC. Altogether, these defects contributed to a marked delay of rosette formation in RTTN-mutated organoids, thus impeding their overall growth and shedding light on mechanisms leading to microcephaly., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Guguin 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 pathogenesis of neurodegenerative diseases results from the interplay between genetic and environmental factors. Aging and chronic oxidative stress are critical contributors to neurodegeneration. UBQLN2, a ubiquitin-related protein, aids in protein degradation and protects against oxidative stress. In ALS neurons harboring UBQLN2 mutations, oxidative stress accelerates pathological changes, yet the precise mechanisms remain unclear. Using induced motor neurons (iMNs) derived from UBQLN2 P497H iPSCs, we observed ALS-like phenotypes, including TDP-43 mislocalization, increased cell death, and reduced viability. Sodium arsenite (SA)-induced oxidative stress triggered stress granule formation, while autophagy dysfunction exacerbated neuronal degeneration. CHX and bosutinib treatments reduced ubiquitinated protein accumulation and alleviated degeneration, highlighting potential therapeutic pathways. These findings emphasize the role of chronic oxidative stress and stress granule formation in UBQLN2 ALS, offering insights into novel therapeutic targets.

Deep learning has gained increasing attention in recent years, yielding promising results in hit screening and molecular optimization. Herein, we employed an efficient strategy based on multiple deep learning techniques to optimize Wee1 inhibitors, which involves activity interpretation, scaffold-based molecular generation, and activity prediction. Starting from our in-house Wee1 inhibitor GLX0198 (IC 50  = 157.9 nM), we obtained three optimized compounds (IC 50  = 13.5 nM, 33.7 nM, and 47.1 nM) out of five picked molecules. Further minor modifications on these compounds led to the identification of potent Wee1 inhibitors with desirable inhibitory effects on multiple cancer cell lines. Notably, the best compound 13 exhibited superior cancer cell inhibition, with IC 50 values below 100 nM in all tested cancer cells. These results suggest that deep learning can greatly facilitate decision-making at the stage of molecular optimization., 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 Masson SAS. All rights reserved.)

Aims: During embryonic development, arteriovenous (AV) differentiation ensures proper blood vessel formation and maturation. Defects in arterial or venous identity cause inappropriate fusion of vessels, resulting in atypical shunts, so-called AV malformations (AVMs). Currently, the mechanism behind AVM formation remains unclear, and treatment options are fairly limited. Mammalian AV differentiation is initiated before the onset of blood flow in the embryo; however, this pre-flow mechanism is poorly understood. Here, we aimed to unravel the role of Smad1/5 signalling in pre-flow arterial identity and, in the process, uncovered an unexpected control mechanism of Smad1/5 signalling., Methods and Results: We establish that despite Notch1 being expressed in the pre-flow mouse embryo, it is not activated, nor is it necessary for the expression of the earliest arterial genes in the dorsal aortae (i.e. Hey1 and Gja4). Furthermore, interrupting blood flow by using the Ncx1 KO model completely prevents the activation of Notch1 signalling, suggesting a strong role of shear stress in maintaining arterial identity. We demonstrate that early expression of Hey1 and Gja4 requires SMAD1/5 signalling. Using embryo cultures, we show that Smad1/5 signalling is activated through the Alk1/Alk5/transforming growth factor (TGF)βR2 receptor complex, with TGFβ1 as a necessary ligand. Furthermore, our findings demonstrate that early arterial gene expression requires the acetylation of Smad1/5 proteins, rendering them more sensitive to TGFβ1 stimulation. Blocking acetyl-CoA production prevents pre-flow arterial expression of Hey1 and Gja4, while stabilizing acetylation rescues their expression., Conclusion: Our findings highlight the importance of the acetyl-CoA production in the cell and provide a novel control mechanism of Smad1/5 signalling involving protein acetylation. As disturbed canonical Smad1/5 signalling is involved in several vascular conditions, our results offer new insights in treatment options for circumventing canonical Smad1/5 signalling., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Eukaryotes carry three types of structural maintenance of chromosome (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion (LE). SMCs modulate DNA supercoiling but remains incompletely understood how this is achieved. Using a single-molecule magnetic tweezers assay that directly measures how much twist is induced by individual SMCs in each LE step, we demonstrate that all three SMC complexes induce the same large negative twist (i.e., linking number change [Formula: see text] of ~-0.6 at each LE step) into the extruded loop, independent of step size and DNA tension. Using ATP hydrolysis mutants and nonhydrolyzable ATP analogs, we find that ATP binding is the twist-inducing event during the ATPase cycle, coinciding with the force-generating LE step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-LE mechanism among these SMC complexes.

The spatiotemporal configuration of genes with distal regulatory elements is believed to be crucial for transcriptional control, but full mechanistic understanding is lacking. We combine simultaneous live tracking of pairs of genomic loci and nascent transcripts with molecular dynamics simulations to assess the Sox2 gene and its enhancer. We find that both loci exhibit more constrained mobility than control sequences due to stalled cohesin at CCCTC-binding factor sites. Strikingly, enhancer mobility becomes constrained on transcriptional firing, homogenizing its dynamics with the gene promoter, suggestive of their cotranscriptional sharing of a nuclear microenvironment. Furthermore, we find transcription and loop extrusion to be antagonistic processes constraining regulatory loci. These findings indicate that modulating chromatin mobility can be an additional, underestimated means for effective gene regulation.

Pan-BD2 inhibitors have been shown to retain an antileukemia effect and display less dose-limiting toxicities than pan-BET inhibitors. However, it is necessary to consider the potential off-target toxicity associated with the inhibition of four BET BD2 proteins. To date, no BRD4 BD2 domain selective inhibitor has been reported. Based on our previous pan-BD2 inhibitor 12 (XY153), we successfully identified 16o (XY221) as the first BRD4 BD2-selective inhibitor. 16o demonstrated potent binding affinity for BRD4 BD2 (IC 50 = 5.8 nM), along with high pan-BD2 selectivity (667-fold over BRD4 BD1) and BRD4 BD2 domain selectivity (9-32-fold over BRD2/3/T BD2). The BRD4 BD2 selectivity of 16o was further confirmed by the BLI assay, showing 66-144-fold selectivity over other BET BD2 domains. 16o exhibited good liver microsomal stability ( T 1/2 > 120 min) and pharmacokinetic properties ( F = 13.1%). These data indicate that 16o may serve as a valuable candidate for BRD4 BD2 advancing epigenetic research.

Bromodomain-containing protein 4 (BRD4) is the most promising target for the treatment of triple-negative breast cancer (TNBC). However, its inherent resistant and acquired drug resistance limits its potential clinical application. Recently it has been shown that cyclin-dependent kinases 4/6 (CDK4/6) inhibitors can reincrease the sensitivity of TNBC cells to BRD4 inhibitors by combination therapy, so we designed a series of dual target CDK6/BRD4 inhibitors. Among the newly synthesized compounds, BC13 exhibited potent inhibitory activity against CDK6 and BRD4. It also displayed potent antiproliferative activity against TNBC cells. In vivo experiments showed that BC13 has potent antitumor activity in the MDA-MB-231 xenograft mouse model, without observable side effects. BC13 demonstrates profound synergistic antitumor effects with ferroptosis inducer in TNBC cells. Therefore, BC13 is a novel dual inhibitor of CDK6/BRD4 for the treatment of TNBC either as a single agent or in combination with RSL3.

Suppression of single-stranded DNA (ssDNA) gap accumulation at replication forks has emerged as a potential determinant of chemosensitivity in homologous recombination (HR)-deficient tumors, as ssDNA gaps are transformed into cytotoxic double-stranded DNA breaks. We have previously shown that the histone chaperone CAF-1's nucleosome deposition function is vital to preventing degradation of stalled replication forks correlating with HR-deficient cells' response to genotoxic drugs. Here we report that the CAF-1-ASF1 pathway promotes ssDNA gap accumulation at replication forks in both wild-type and breast cancer (BRCA)-deficient backgrounds. We show that this is independent of CAF-1's nucleosome deposition function but instead may rely on its proper localization to replication forks. Moreover, we show that the efficient localization to nascent DNA of PrimPol, the enzyme responsible for repriming upon replication stress, is dependent on CAF-1. As PrimPol has been shown to be responsible for generating ssDNA gaps as a byproduct of its repriming function, CAF-1's role in its recruitment could directly impact ssDNA gap formation. We also show that chemoresistance observed in HR-deficient cells when CAF-1 or ASF1A are lost correlates with suppression of ssDNA gaps rather than protection of stalled replication forks. Overall, this work identifies an unexpected role of CAF-1 in regulating PrimPol recruitment and ssDNA gap generation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The phosphorylation of histone H2A.X into γH2A.X is a crucial early event in the DNA damage response, marking DNA damage sites and initiating repair processes. While ATM kinase is traditionally recognized as the primary mediator of H2A.X phosphorylation, our study identifies serine/threonine kinase 39 (STK39) as a novel enhancer of this critical signaling pathway. We demonstrate that after DNA damage, STK39 undergoes phosphorylation by the ATM kinase, facilitating its interaction with the Mre11-Rad50-Nbs1 complex and subsequent recruitment to chromatin. This recruitment enables STK39 to further phosphorylate H2A.X, thus amplifying γH2A.X production and promoting homologous recombination repair. Notably, we observe a significant upregulation of STK39 in pancreatic adenocarcinoma (PAAD) tissues, correlating with heightened resistance to PARPi therapy. Furthermore, we demonstrate the synergistic efficacy of combining STK39 inhibition with PARP inhibitors in suppressing and reversing PAAD growth. This study not only provides new insights into the molecular dynamics of H2A.X phosphorylation but also highlights the therapeutic potential of targeting STK39 to enhance PARPi sensitivity in PAAD (created with BioRender)., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Conformational changes in a transcription factor can significantly affect its transcriptional activity. The activated form of the FOXM1 transcription factor regulates the transcriptional network of genes essential for cell cycle progression and carcinogenesis. However, the mechanism and impact of FOXM1 conformational change on its transcriptional activity in vivo throughout the cell cycle progression remain unexplored. Here, we demonstrate that FOXM1 proteins form novel intermolecular homodimerizations in vivo, and these conformational changes in FOXM1 homodimers impact activity during the cell cycle. Specifically, during the G1 phase, FOXM1 undergoes autorepressive homodimerization, wherein the αβα motif in the C-terminal transcriptional activation domain interacts with the ββαβ motif in the N-terminal repression domain, as evidenced by FRET imaging. Phosphorylation of the αβα motif by PLK1 at S715/S724 disrupts ββαβ-αβα hydrophobic interactions, thereby facilitating a conserved αβα motif switch binding partner to the novel intrinsically disordered regions, leading to FOXM1 autostimulatory homodimerization persisting from the S phase to the G2/M phase in vivo. Furthermore, we identified a minimal ββαβ motif peptide that effectively inhibits cancer cell proliferation both in cell culture and in a mouse tumor model, suggesting a promising autorepression approach for targeting FOXM1 in cancer therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12-15 hr, after which cells 'adapt' to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2 ATRIP , Rad9, Rad24, or Rad53 CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and maintain the arrest. Dun1 is required for the establishment, but not the maintenance, of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with two persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hr these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2's binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest require overlapping but different sets of factors., Competing Interests: FZ, DW, MA, SC, GM, VE, JH No competing interests declared, (© 2024, Zhou, Waterman et al.)

PLK1 inhibitors are emerging anticancer agents that are being tested as monotherapy and combination therapies for various cancers. Although PLK1 inhibition in experimental models has shown potent antitumor effects, translation to the clinic has been hampered by low antitumor activity and tumor relapse. Here, we report the identification of mitochondrial protein signatures that determine the sensitivity to approaches targeting PLK1 in human melanoma cell lines. In response to PLK1 inhibition or gene silencing, resistant cells adopt a pro-inflammatory and dedifferentiated phenotype, whereas sensitive cells undergo apoptosis. Mitochondrial DNA depletion and silencing of the ABCD1 transporter sensitize cells to PLK1 inhibition and attenuate the associated pro-inflammatory response. We also found that nonselective inhibitors of the p90 ribosomal S6 kinase (RSK) exert their antiproliferative and pro-inflammatory effects via PLK1 inhibition. Specific inhibition of RSK, on the other hand, is anti-inflammatory and promotes a program of antigen presentation. This study reveals the overlooked effects of PLK1 on phenotype switching and suggests that mitochondrial precision medicine can help improve the response to targeted therapies., (© 2024 Lavallée et al.)

During meiosis, programmed DNA double-strand breaks (DSBs) are formed by the topoisomerase-like enzyme, Spo11, activating the DNA damage response (DDR) kinase Mec1ATR via the checkpoint clamp loader, Rad24RAD17. At single loci, loss of Mec1 and Rad24 activity alters DSB formation and recombination outcome, but their genome-wide roles have not been examined in detail. Here, we utilise two strategies-deletion of the mismatch repair protein, Msh2, and control of meiotic prophase length via regulation of the Ndt80 transcription factor-to help characterise the roles Mec1 and Rad24 play in meiotic recombination by enabling genome-wide mapping of meiotic progeny. In line with previous studies, we observe severely impacted spore viability and a reduction in the frequency of recombination upon deletion of RAD24-driven by a shortened prophase. By contrast, loss of Mec1 function increases recombination frequency, consistent with its role in DSB trans-interference, and has less effect on spore viability. Despite these differences, complex multi-chromatid events initiated by closely spaced DSBs-rare in wild-type cells-occur more frequently in the absence of either Rad24 or Mec1, suggesting a loss of spatial regulation at the level of DSB formation in both. Mec1 and Rad24 also have important roles in the spatial regulation of crossovers (COs). Upon loss of either Mec1 or Rad24, CO distributions become more random-suggesting reductions in the global manifestation of interference. Such effects are similar to, but less extreme than, the phenotype of 'ZMM' mutants such as zip3Δ, and may be driven by reductions in the proportion of interfering COs. Collectively, in addition to shared roles in CO regulation, our results highlight separable roles for Rad24 as a pro-CO factor, and for Mec1 as a regulator of recombination frequency, the loss of which helps to suppress any broader defects in CO regulation caused by abrogation of the DDR., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Crawford 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.)

Cornelia de Lange syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder. Pathogenic variants in genes encoding the structural subunits and regulatory proteins of the cohesin complex ( NIPBL , SMC1A , SMC3 , HDAC8 , and RAD21 ) are the primary contributors to the pathogenesis of CdLS. Pathogenic variations in these genes disrupt normal cohesin function, leading to the syndrome's diverse and complex clinical presentation. In this study, we discovered that cells harboring variants in the NIPBL , SMC1A and HDAC8 genes exhibit spontaneous genome instability, elevated oxidative stress and premature cellular aging. These findings suggest that cohesin plays a critical role in maintaining proper cellular function and highlight its contribution to the pathophysiology seen in the related diagnoses.

Chemically induced proximity modalities such as targeted protein degradation (TPD) hold promise for expanding the number of proteins that can be manipulated pharmacologically. However, current TPD strategies are often limited to proteins with preexisting ligands. Molecular glues (e.g. glutarimide ligands for CUL4 CRBN ), offer the potential to target undruggable proteins. Yet, their rational design is largely unattainable due to the unpredictability of the 'gain-of-function' nature of the glue interaction upon chemical modification of ligands. We recently reported a covalent trans-labelling glue mechanism which we named 'Template-assisted covalent modification', where an electrophile decorated BRD4 inhibitor was effectively delivered to a cysteine residue on DCAF16 due to an electrophile-induced BRD4-DCAF16 interaction. Herein, we report our efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 recruitment and subsequent BRD4 degradation efficiency. We discovered a moderate correlation between the electrophile-induced BRD4-DCAF16 ternary complex formation and BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation optimally recruits DCAF16 and promotes BRD4 degradation. The diversity of covalent attachments in this class of BRD4 degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a new avenue for rational glue design by introducing covalent warheads to known binders., 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., (Published by Elsevier Masson SAS.)

Various DNA damage checkpoint control mechanisms in eukaryotic cells help maintain genomic integrity. Among these, NBS1, a key component of the MRE11-RAD50-NBS1 (MRN) complex, is an essential protein involved in the DNA damage response (DDR). In this study, we discovered that DNA damage-binding protein 1 (DDB1) interacts with NBS1. DDB1 is a DDR sensor protein found in UV-induced DNA replication blocks. Through pull-down and immunoprecipitation assays conducted in Xenopus egg extracts and human cell lines, we demonstrated a specific interaction between NBS1 and DDB1. DDB1 was also found to associate with several proteins that interact with NBS1, including DNA topoisomerase 2-binding protein 1 (TopBP1) and Mediator of DNA damage checkpoint protein 1 (MDC1). Notably, the interaction between DDB1 and NBS1 is disrupted in MDC1-depleted egg extracts, indicating that MDC1 is necessary for this interaction. Furthermore, the depletion of DDB1 leads to increased Chk1 activation upon DNA damage. These novel findings regarding the interaction between NBS1 and DDB1 provide new insights into how DDB1 regulates DNA damage pathways.

In recent years, methamphetamine (METH) misuse in the US has been rapidly increasing, and there is no FDA-approved pharmacotherapy for METH use disorder (MUD). In addition to being dependent on the drug, people with MUD develop a variety of neurological problems related to the toxicity of this drug. A variety of molecular mechanisms underlying METH neurotoxicity has been identified, including the dysfunction of the neuroprotective protein parkin. However, it is not known whether parkin loss of function within striatal dopaminergic (DAergic) terminals translates into decreased DA storage capacity. This study examined the relationship between parkin, its substrate cell division cycle related-1 (CDCrel-1) associated with synaptic vesicles, and vesicular monoamine transporter-2 (VMAT2) responsible for packaging DA in an in vivo model of METH neurotoxicity. To assess the individual differences in response to METH's neurotoxic effects, a large group of male Sprague Dawley rats were treated with binge METH or saline and sacrificed 1 h or 24 h later. This study is the first to show that CDCrel-1 interacts with VMAT2 in the rat striatum and that binge METH can alter this interaction as well as the levels and subcellular localization of CDCrel-1. The proteomic analysis of VMAT-2-associated proteins revealed the upregulation of several proteins involved in the exocytosis/endocytosis cycle and responses to stress. The results suggest that DAergic neurons are engaged in counteracting METH-induced toxic effects, including attempts to increase endocytosis and autophagy at 1 h after the METH binge, with the responses varying widely between individual rats. Studying CDCrel-1, VMAT2, and other proteins in large groups of outbred rats can help define individual genetic and molecular differences in responses to METH neurotoxicity, which, in turn, may aid treating humans suffering from MUD and its neurological consequences.

Extracellular vesicles (EVs) are known to mediate cell communications and shape tumor microenvironment. Compared to the well-studied small EVs, the function of large microvesicles (MVs) during tumorigenesis is poorly understood. Here we show the proteome of MVs in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC), and identify olfactomedin 4 (OLFM4) is induced by EBV infection and secreted via MVs to promote tumor progression through Hippo signaling. Specifically, OLFM4 is a target gene of the cGAS-STING pathway, and EBV infection activates cGAS-STING pathway and increases OLFM4 expression. Moreover, MV-carried OLFM4 binds with the extracellular cadherin domain of FAT1, thereby impairing its intracellular interaction with MST1 and leading to YAP activation in recipient cells. Together, our study not only reveals a regulatory mechanism though which viral infection is coupled via MVs with intercellular control of the Hippo signaling, but also highlights the OLFM4-Hippo axis as a therapeutic target for EBV-associated cancers., Competing Interests: Competing interests: The authors have filed patents (CN2024116656372) regarding the potential diagnostic application of OLFM4-containing MVs., (© 2024. The Author(s).)

The fork protection complex (FPC), composed of Mrc1, Tof1, and Csm3, supports rapid and stable DNA replication. Here, we show that FPC activity also introduces DNA damage by increasing DNA topological stress during replication. Mrc1 action increases DNA topological stress during plasmid replication, while Mrc1 or Tof1 activity causes replication stress and DNA damage within topologically constrained regions. We show that the recruitment of Top1 to the fork by Tof1 suppresses the DNA damage generated in these loci. While FPC activity introduces some DNA damage due to increased topological stress, the FPC is also necessary to prevent DNA damage in long replicons across the genome, indicating that the FPC is required for complete and faithful genome duplication. We conclude that FPC regulation must balance ensuring full genome duplication through rapid replication with minimizing the consequential DNA topological stress-induced DNA damage caused by rapid replication through constrained regions., Competing Interests: Competing interests statement:The authors declare no competing interest.

Advances in bladder cancer (BCa) treatment have been hampered by the lack of predictive biomarkers and targeted therapies. Here, we demonstrate that loss of the tumor suppressor NUMB promotes aggressive bladder tumorigenesis and worsens disease outcomes. Retrospective cohort studies show that NUMB-loss correlates with poor prognosis in post-cystectomy muscle-invasive BCa patients and increased risk of muscle invasion progression in non-muscle invasive BCa patients. In mouse models, targeted Numb ablation induces spontaneous tumorigenesis and sensitizes the urothelium to carcinogenic insults, accelerating tumor onset and progression. Integrative transcriptomic and functional analyses in mouse and human BCa models reveal that upregulation of YAP transcriptional activity via a RHOA/ROCK-dependent pathway is a hallmark of NUMB-deficient BCa. Pharmacological or genetic inhibition of this molecular pathway selectively inhibits proliferation and invasion of NUMB-deficient BCa cells in 3D-Matrigel organoids. Thus, NUMB-loss could serve as a biomarker for identifying high-risk patients who may benefit from targeted anti-RHOA/ROCK/YAP therapies., Competing Interests: Competing interests: G. Renne, G. Vago, D. Tosoni and S. Pece are co-inventors of a patent (EP24191940.6) related to this work, pending to the European Institute of Oncology and University of Milan. All the other authors declare no competing interests., (© 2024. The Author(s).)