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BioRxiv : the Preprint Server For... Dec 2023Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory...
Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects and programmability of such DNA sequences remain difficult to map or predict because we have lacked scalable methods to precisely edit regulatory DNA and quantify the effects in an endogenous genomic context. Here we present an approach to measure the quantitative effects of hundreds of designed DNA sequence variants on gene expression, by combining pooled CRISPR prime editing with RNA fluorescence hybridization and cell sorting (Variant-FlowFISH). We apply this method to mutagenize and rewrite regulatory DNA sequences in an enhancer and the promoter of in two immune cell lines. Of 672 variant-cell type pairs, we identify 497 that affect expression. These variants appear to act through a variety of mechanisms including disruption or optimization of existing transcription factor binding sites, as well as creation of sites. Disrupting a single endogenous transcription factor binding site often led to large changes in expression (up to -40% in the enhancer, and -50% in the promoter). The same variant often had different effects across cell types and states, demonstrating a highly tunable regulatory landscape. We use these data to benchmark performance of sequence-based predictive models of gene regulation, and find that certain types of variants are not accurately predicted by existing models. Finally, we computationally design 185 small sequence variants (≤10 bp) and optimize them for specific effects on expression . 84% of these rationally designed edits showed the intended direction of effect, and some had dramatic effects on expression (-100% to +202%). Variant-FlowFISH thus provides a powerful tool to map the effects of variants and transcription factor binding sites on gene expression, test and improve computational models of gene regulation, and reprogram regulatory DNA.
PubMed: 38187584
DOI: 10.1101/2023.12.20.572268 -
Frontiers in Endocrinology 2023The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources....
INTRODUCTION
The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources. Even a single DNA double-strand break (DSB) can trigger oocyte apoptosis, which can lead to accelerated depletion of the ovarian reserve, early loss of fertility and menopause. Therefore, repair of DNA damage is important for preserving the quality of oocytes to sustain fertility across the reproductive lifespan. This study aimed to evaluate the role of KU80 (encoded by the XRCC5 gene) - an essential component of the non-homologous end joining (NHEJ) pathway - in the repair of oocyte DNA DSBs during reproductive ageing, and following insult caused by the DNA-damaging chemotherapies cyclophosphamide and cisplatin.
METHODS
To investigate the importance of KU80 following endogenous and exogenous DNA damage, ovaries from conditional oocyte-specific knockout ( cKO) and wildtype (WT) mice that were aged or exposed to DNA damage-inducing chemotherapy were compared. Ovarian follicles and oocytes were quantified, morphologically assessed and analysed via immunohistochemistry for markers of DNA damage and apoptosis. In addition, chemotherapy exposed mice were superovulated, and the numbers and quality of mature metaphase- II (MII) oocytes were assessed.
RESULTS
The number of healthy follicles, atretic (dying) follicles, and corpora lutea were similar in Xrcc5 cKO and WT mice at PN50, PN200 and PN300. Additionally, primordial follicle number and ovulation rates were similar in young adult Xrcc5 cKO and WT mice following treatment with cyclophosphamide (75mg/kg), cisplatin (4mg/kg), or vehicle control (saline). Furthermore, KU80 was not essential for the repair of exogenously induced DNA damage in primordial follicle oocytes.
DISCUSSION
These data indicate that KU80 is not required for maintenance of the ovarian reserve, follicle development, or ovulation during maternal ageing. Similarly, this study also indicates that KU80 is not required for the repair of exogenously induced DSBs in the prophase-arrested oocytes of primordial follicles.
Topics: Animals; Female; Mice; Cisplatin; Cyclophosphamide; DNA; Oocytes; Ovarian Follicle; Prophase; Ku Autoantigen
PubMed: 37900135
DOI: 10.3389/fendo.2023.1268009 -
Biomedicine & Pharmacotherapy =... Sep 2023Cordycepin (with a molecular formula of CHNO), a natural adenosine isolated from Cordyceps militaris, has an important regulatory effect on skeletal muscle remodelling...
Cordycepin (with a molecular formula of CHNO), a natural adenosine isolated from Cordyceps militaris, has an important regulatory effect on skeletal muscle remodelling and quality maintenance. The aim of this study was to investigate the effect of cordycepin on myoblast differentiation and explore the underlying molecular mechanisms of this effect. Our results showed that cordycepin inhibited myogenesis by downregulating myogenic differentiation (MyoD) and myogenin (MyoG), preserved undifferentiated reserve cell pools by upregulating myogenic factor 5 (Myf5) and retinoblastoma-like protein p130 (p130), and enhanced energy reserves by decreasing intracellular reactive oxygen species (ROS) and enhancing mitochondrial membrane potential, mitochondrial mass, and ATP content. The effect of cordycepin on myogenesis was associated with increased phosphorylation of extracellular signal-regulated kinase 1/2 (p-ERK1/2). PD98059 (a specific inhibitor of p-ERK1/2) attenuated the inhibitory effect of cordycepin on C2C12 differentiation. The present study reveals that cordycepin inhibits myogenesis through ERK1/2 MAPK signalling activation accompanied by an increase in skeletal muscle energy reserves and improving skeletal muscle oxidative stress, which may have implications for its further application for the prevention and treatment of degenerative muscle diseases caused by the depletion of depleted muscle stem cells.
Topics: MAP Kinase Signaling System; Cell Differentiation; Deoxyadenosines; Muscle Development
PubMed: 37453196
DOI: 10.1016/j.biopha.2023.115163 -
Methodist DeBakey Cardiovascular Journal 2023A 75-year-old patient was incidentally found to have an intracardiac mass by echocardiography. Subsequent cardiac magnetic resonance imaging and cardiac positron...
A 75-year-old patient was incidentally found to have an intracardiac mass by echocardiography. Subsequent cardiac magnetic resonance imaging and cardiac positron emission tomography confirmed a large and possibly malignant mass extending from the right atrium into the coronary sinus. The patient underwent an intracardiac echocardiography guided biopsy, which revealed diffuse B-cell lymphoma, and is currently undergoing rituximab, etoposide, vincristine, cyclophosphamide, and doxorubicin (R-EPOCH)-based chemotherapy.
Topics: Humans; Aged; Lymphoma, Large B-Cell, Diffuse; Rituximab; Cyclophosphamide; Vincristine; Prednisone; Antineoplastic Combined Chemotherapy Protocols; Doxorubicin; Positron-Emission Tomography; Echocardiography
PubMed: 38161506
DOI: 10.14797/mdcvj.1316 -
Proceedings of the National Academy of... Jul 2023Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA...
Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2β) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2β to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.
Topics: Humans; Mutagens; Topoisomerase II Inhibitors; Etoposide; DNA Topoisomerases, Type II; DNA Damage; DNA; Neoplasms
PubMed: 37406101
DOI: 10.1073/pnas.2302064120 -
Biomedicine & Pharmacotherapy =... Apr 2024Diffuse large B-cell lymphoma (DLBCL), a heterogeneous lymphoid malignancy, poses a significant threat to human health. The standard therapeutic regimen for patients... (Review)
Review
Diffuse large B-cell lymphoma (DLBCL), a heterogeneous lymphoid malignancy, poses a significant threat to human health. The standard therapeutic regimen for patients with DLBCL is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), with a typical cure rate of 50-70%. However, some patients either relapse after complete remission (CR) or exhibit resistance to R-CHOP treatment. Therefore, novel therapeutic approaches are imperative for managing high-risk or refractory DLBCL. Ferroptosis is driven by iron-dependent phospholipid peroxidation, a process that relies on the transition metal iron, reactive oxygen species (ROS), and phospholipids containing polyunsaturated fatty acids-containing phospholipids (PUFA-PLs). Research indicates that ferroptosis is implicated in various carcinogenic and anticancer pathways. Several hematological disorders exhibit heightened sensitivity to cell death induced by ferroptosis. DLBCL cells, in particular, demonstrate an increased demand for iron and an upregulation in the expression of fatty acid synthase. Additionally, there exists a correlation between ferroptosis-associated genes and the prognosis of DLBCL. Therefore, ferroptosis may be a promising novel target for DLBCL therapy. In this review, we elucidate ferroptosis mechanisms, its role in DLBCL, and the potential therapeutic targets in DLBCL. This review offers novel insights into the application of ferroptosis in treatment strategies for DLBCL.
Topics: Humans; Ferroptosis; Neoplasm Recurrence, Local; Rituximab; Vincristine; Cyclophosphamide; Prednisone; Doxorubicin; Lymphoma, Large B-Cell, Diffuse; Iron; Antineoplastic Combined Chemotherapy Protocols; Treatment Outcome
PubMed: 38492438
DOI: 10.1016/j.biopha.2024.116386 -
EMBO Reports Aug 202353BP1 is a chromatin-binding protein that promotes DNA double-strand break repair through the recruitment of downstream effectors including RIF1, shieldin, and CST. The...
53BP1 is a chromatin-binding protein that promotes DNA double-strand break repair through the recruitment of downstream effectors including RIF1, shieldin, and CST. The structural basis of the protein-protein interactions within the 53BP1-RIF1-shieldin-CST pathway that are essential for its DNA repair activity is largely unknown. Here, we used AlphaFold2-Multimer (AF2) to predict all possible pairwise combinations of proteins within this pathway and provide structural models of seven previously characterized interactions. This analysis also predicted an entirely novel binding interface between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3. Extensive interrogation of this interface through both in vitro pulldown analysis and cellular assays supports the AF2-predicted model and demonstrates that RIF1-SHLD3 binding is essential for shieldin recruitment to sites of DNA damage, and for its role in antibody class switch recombination and PARP inhibitor sensitivity. Direct physical interaction between RIF1 and SHLD3 is therefore essential for 53BP1-RIF1-shieldin-CST pathway activity.
Topics: Furylfuramide; Tumor Suppressor p53-Binding Protein 1; DNA-Binding Proteins; DNA Repair; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Telomere-Binding Proteins
PubMed: 37306046
DOI: 10.15252/embr.202356834 -
Ecotoxicology and Environmental Safety Jun 2024This study explores the eco-geno-toxic impact of Acyclovir (ACV), a widely used antiviral drug, on various freshwater organisms, given its increasing detection in...
This study explores the eco-geno-toxic impact of Acyclovir (ACV), a widely used antiviral drug, on various freshwater organisms, given its increasing detection in surface waters. The research focused on non-target organisms, including the green alga Raphidocelis subcapitata, the rotifer Brachionus calyciflorus, the cladoceran crustacean Ceriodaphnia dubia, and the benthic ostracod Heterocypris incongruens, exposed to ACV to assess both acute and chronic toxicity. The results indicate that while acute toxicity occurs at environmentally not-relevant concentrations, a significant chronic toxicity for C. dubia (EC = 0.03 µg/L, NOEC = 0.02·10 µg/L), highlighted substantial environmental concern. Furthermore, DNA strand breaks and reactive oxygen species detected in C. dubia indicate significant increase at concentrations exceeding 200 µg/L. Regarding environmental risk, the authors identified chronic exposures to acyclovir causing inhibitory effects on reproduction in B. calyciflorus at hundreds of µg/L and hundredths of µg/L for C. dubia as environmentally relevant environmental concentrations. The study concludes by quantifying the toxic and genotoxic risks of ACV showing a chronic risk quotient higher than the critical value of 1and a genotoxic risk quotient reaching this threshold, highlighting the urgent need for a broader risk assessment of ACV for its significant implications for aquatic ecosystems.
Topics: Animals; Water Pollutants, Chemical; Fresh Water; Antiviral Agents; Acyclovir; Rotifera; Reactive Oxygen Species; Cladocera; Aquatic Organisms; Toxicity Tests, Acute; DNA Damage; Reproduction; Toxicity Tests, Chronic; Mutagens; Chlorophyta
PubMed: 38718728
DOI: 10.1016/j.ecoenv.2024.116437 -
Acta Neuropathologica Communications Feb 2024
Topics: Humans; Mutagens; Guam; Methylazoxymethanol Acetate; Mutagenesis; Amyotrophic Lateral Sclerosis
PubMed: 38383591
DOI: 10.1186/s40478-024-01725-y -
Cancer Cell Mar 2024Co-culture of intestinal organoids with a colibactin-producing pksE. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli...
Co-culture of intestinal organoids with a colibactin-producing pksE. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli Nissle 1917 (EcN) remains a commonly used probiotic, despite harboring the pks operon and inducing double strand DNA breaks. We determine the mutagenicity of EcN and three CRC-derived pksE. coli strains with an analytical framework based on sequence characteristic of colibactin-induced mutations. All strains, including EcN, display varying levels of mutagenic activity. Furthermore, a machine learning approach attributing individual mutations to colibactin reveals that patients with colibactin-induced mutations are diagnosed at a younger age and that colibactin can induce a specific APC mutation. These approaches allow the sensitive detection of colibactin-induced mutations in ∼12% of CRC genomes and even in whole exome sequencing data, representing a crucial step toward pinpointing the mutagenic activity of distinct pksE. coli strains.
Topics: Humans; Escherichia coli; Mutation; DNA Damage; Mutagens; Colorectal Neoplasms; Organoids; Peptides; Polyketides
PubMed: 38471458
DOI: 10.1016/j.ccell.2024.02.009