Assay For Transposase-accessible Chromatin Research Articles

Multi-omics insights into the pathogenesis of diabetic cardiomyopathy: epigenetic and metabolic profiles.

Diabetic cardiomyopathy (DbCM), a complex metabolic disease, greatly threatens human health due to therapeutic limitations. Multi-omics approaches facilitate the elucidation of its intrinsic pathological changes. Metabolomics, RNA-seq, proteomics, and assay of transposase-accessible chromatin (ATAC-seq) were utilized to elucidate multidimensional molecular alterations in DbCM. In the heart and plasma of mice with DbCM, metabolomic analysis demonstrated significant differences in branched-chain amino acids (BCAAs) and lipids. Subsequent RNA-seq and proteomics showed that the key genes, including BCKDHB, PPM1K, Cpt1b, Fabp4, Acadm, Acadl, Acadvl, HADH, HADHA, HADHB, Eci1, Eci2, PDK4, and HMGCS2, were aberrantly regulated, contributing to the disorder of BCAAs and fatty acids. ATAC-seq analysis underscored the pivotal role of epigenetic regulation by revealing dynamic shifts in chromatin accessibility and a robust positive correlation with gene expression patterns in diabetic cardiomyopathy mice. Furthermore, motif analysis identified that KLF15 as a critical transcription factor in DbCM, regulating the core genes implicated with BCAAs metabolism. Our research delved into the metabolic alterations and epigenetic landscape and revealed that KLF15 may be a promising candidate for therapeutic intervention in DbCM.

M6A-Methylated NUTM2B-AS1 Promotes Hepatocellular Carcinoma Stemness Feature via Epigenetically Activating BMPR1A Transcription.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies in the world. Oncofetal proteins are the optimal diagnostic biomarkers and therapeutic targets for HCC. As the most abundant modification in RNA, N6-methyladenosine (m6A) has been reported to be involved in HCC initiation and progression. However, whether m6A has oncofetal characteristics remains unknown. Gene expression in HCC tissues and cells was detected using qPCR. The level of m6A methylation was determined using methylated RNA immunoprecipitation assay. The biological roles of NUTM2B-AS1 in HCC were detected using Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine incorporation, and spheroid formation assays. The mechanisms underlying the roles of NUTM2B-AS1 were explored using RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), chromatin immunoprecipitation (ChIP), and assay for transposase-accessible chromatin (ATAC). NUTM2B-AS1 was identified as a novel oncofetal long noncoding RNA that was upregulated in the fetal liver and HCC and silenced in adult liver tissues. METTL3 and METTL16 induce m6A hypermethylation of NUTM2B-AS1. The m6A methylation levels of NUTM2B-AS1 exhibit oncofetal characteristics. m6A methylation upregulates NUTM2B-AS1 expression by increasing NUTM2B-AS1 transcript stability. m6A-methylated NUTM2B-AS1 promotes HCC cell proliferation and stemness via epigenetically activating BMPR1A expression. NUTM2B-AS1 specifically binds to BMPR1A promoter. m6A-methylated NUTM2B-AS1 is recognized by the m6A reader YTHDC2, which further binds to the H3K4 methyltransferase MLL1. m6A-methylated NUTM2B-AS1 recruits YTHDC2 and MLL1 to BMPR1A promoter, leading to increased H3K4me3 and chromatin accessibility at BMPR1A promoter. Functional rescue assays suggest that BMPR1A is a critical mediator of the oncogenic role of m6A-methylated NUTM2B-AS1 in HCC. METTL3- and METTL16-mediated m6A methylation of NUTM2B-AS1 is a novel oncofetal molecular event in HCC that promotes HCC stemness via epigenetically activating BMPR1A transcription.

KAS-ATAC reveals the genome-wide single-stranded accessible chromatin landscape of the human genome.

Gene regulation in most eukaryotes involves two fundamental physical processes -- alterations in the packaging of the genome by nucleosomes, with active cis-regulatory elements (CREs) generally characterized by an open-chromatin configuration, and the activation of transcription. Mapping these physical properties and biochemical activities genome-wide -- through profiling chromatin accessibility and active transcription -- are key tools used to understand the logic and mechanisms of transcription and its regulation. However, the relationship between these two states has until now not been accessible to simultaneous measurement. To address this, we developed KAS-ATAC, a combination of the KAS-seq (Kethoxal-Assisted SsDNA sequencing and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) methods for mapping single-stranded DNA (and thus active transcription) and chromatin accessibility, respectively, enabling the genome-wide identification of DNA fragments that are simultaneously accessible and contain ssDNA. We use KAS-ATAC to evaluate levels of active transcription over different classes of regulatory elements in the human genome, to estimate the absolute levels of transcribed accessible DNA over CREs, to map the nucleosomal configurations associated with RNA polymerase activities, and to assess transcription factor association with transcribed DNA through transcription factor binding site (TFBS) footprinting. We observe lower levels of transcription over distal enhancers compared to promoters and distinct nucleosomal configurations around transcription initiation sites associated with active transcription. Most TFs associate equally with transcribed and nontranscribed DNA but a few factors specifically do not exhibit footprints over ssDNA-containing fragments. We anticipate KAS-ATAC to continue to derive useful insights into chromatin organization and transcriptional regulation in other contexts in the future.

Spatial multiomic landscape of the human placenta at molecular resolution.

Successful pregnancy relies directly on the placenta's complex, dynamic, gene-regulatory networks. Disruption of this vast collection of intercellular and intracellular programs leads to pregnancy complications and developmental defects. In the present study, we generated a comprehensive, spatially resolved, multimodal cell census elucidating the molecular architecture of the first trimester human placenta. We utilized paired single-nucleus (sn)ATAC (assay for transposase accessible chromatin) sequencing and RNA sequencing (RNA-seq), spatial snATAC-seq and RNA-seq, and in situ sequencing and hybridization mapping of transcriptomes at molecular resolution to spatially reconstruct the joint epigenomic and transcriptomic regulatory landscape. Paired analyses unraveled intricate tumor-like gene expression and transcription factor motif programs potentially sustaining the placenta in a hostile uterine environment; further investigation of gene-linked cis-regulatory elements revealed heightened regulatory complexity that may govern trophoblast differentiation and placental disease risk. Complementary spatial mapping techniques decoded these programs within the placental villous core and extravillous trophoblast cell column architecture while simultaneously revealing niche-establishing transcriptional elements and cell-cell communication. Finally, we computationally imputed genome-wide, multiomic single-cell profiles and spatially characterized the placental chromatin accessibility landscape. This spatially resolved, single-cell multiomic framework of the first trimester human placenta serves as a blueprint for future studies on early placental development and pregnancy.

Brca1 haploinsufficiency promotes early tumor onset and epigenetic alterations in a mouse model of hereditary breast cancer.

Germline BRCA1 mutation carriers face a high breast cancer risk; however, the underlying mechanisms for this risk are not completely understood. Using a new genetically engineered mouse model of germline Brca1 heterozygosity, we demonstrate that early tumor onset in a Brca1 heterozygous background cannot be fully explained by the conventional 'two-hit' hypothesis, suggesting the existence of inherent tumor-promoting alterations in the Brca1 heterozygous state. Single-cell RNA sequencing and assay for transposase-accessible chromatin with sequencing analyses uncover a unique set of differentially accessible chromatin regions in ostensibly normal Brca1 heterozygous mammary epithelial cells, distinct from wild-type cells and partially mimicking the chromatin and RNA-level changes in tumor cells. Transcription factor analyses identify loss of ELF5 and gain of AP-1 sites in these epigenetically primed regions; in vivo experiments further implicate AP-1 and Wnt10a as strong promoters of Brca1-related breast cancer. These findings reveal a previously unappreciated epigenetic effect of Brca1 haploinsufficiency in accelerating tumorigenesis, advancing our mechanistic understanding and informing potential therapeutic strategies.

Application of computational algorithms for single-cell RNA-seq and ATAC-seq in neurodegenerative diseases.

Recent advancements in single-cell technologies, including single-cell RNA sequencing (scRNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), have greatly improved our insight into the epigenomic landscapes across various biological contexts and diseases. This paper reviews key computational tools and machine learning approaches that integrate scRNA-seq and scATAC-seq data to facilitate the alignment of transcriptomic data with chromatin accessibility profiles. Applying these integrated single-cell technologies in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, reveals how changes in chromatin accessibility and gene expression can illuminate pathogenic mechanisms and identify potential therapeutic targets. Despite facing challenges like data sparsity and computational demands, ongoing enhancements in scATAC-seq and scRNA-seq technologies, along with better analytical methods, continue to expand their applications. These advancements promise to revolutionize our approach to medical research and clinical diagnostics, offering a comprehensive view of cellular function and disease pathology.

Understanding the complex chromatin dynamics in primary human neutrophils during PMA-induced NET formation.

Primary human neutrophils play a pivotal role in innate immunity, mainly through the formation of neutrophil extracellular traps (NETs) in a process known as NETosis. This cell-death pathway is crucial for combating infections but is also implicated in many inflammatory diseases, such as sepsis, systemic lupus erythematosus, and rheumatoid arthritis. The study presented here investigates chromatin dynamics during NET formation by stimulating primary human neutrophils with phorbol 12-myristate 13-acetate (PMA). We adapt the ATAC-Seq (assay for transposase-accessible chromatin using sequencing) method to isolated neutrophils and characterize a time-dependent chromatin response. We found that chromatin accessibility patterns are consistent across individual donors and most chromatin changes occur within 30 min, with many continuing across the 90 min assessed in this study. Regulatory regions gaining accessibility were associated with the activity of pathways that have been implicated in NOX-dependent NET formation. Our findings increase the understanding of the chromatin changes underlying NET formation and also identify potential early-acting targets for modulating this process in inflammatory diseases.

Postoperative Innate Immune Dysregulation, Proteomic, and Monocyte Epigenomic Changes After Colorectal Surgery: A Substudy of a Randomized Controlled Trial.

Colorectal surgery is associated with moderate-to-severe postoperative complications in over 25% of patients, predominantly infections. Monocyte epigenetic alterations leading to immune tolerance could explain postoperative increased susceptibility to infections. This research explores whether changes in monocyte DNA accessibility contribute to postoperative innate immune dysregulation. Damage-associated molecular patterns (DAMPs) and ex vivo cytokine production capacity were measured in a randomized controlled trial (n = 100) in colorectal surgery patients, with additional exploratory subgroup proteomic (proximity extension assay; Olink) and epigenomic analyses (Assay for Transposase-Accessible Chromatin [ATAC sequencing]). Monocytes of healthy volunteers were used to study the effect of high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70) on cytokine production capacity in vitro. Plasma DAMPs were increased after surgery. HMGB1 showed a mean 235% increase from before- (preop) to the end of surgery (95% confidence interval [CI] [166 - 305], P < .0001) and 90% increase (95% CI [63-118], P = .0004) preop to postoperative day 1 (POD1). HSP70 increased by a mean 12% from preop to the end of surgery (95% CI [3-21], not significant) and 30% to POD1 (95% CI [18-41], P < .0001). Nuclear deoxyribonucleic acid (nDNA) increases by 66% (95% CI [40-92], P < .0001) at the end of surgery and 94% on POD1 (95% CI [60-127], P < .0001). Mitochondrial DNA (mtDNA) increases by 370% at the end of surgery (95% CI [225-515], P < .0001) and by 503% on POD1 (95% CI [332-673], P < .0001). In vitro incubation of monocytes with HSP70 decreased cytokine production capacity of tumor necrosis factor (TNF) by 46% (95% CI [29-64], P < .0001), IL-6 by 22% (95% CI [12-32], P = .0004) and IL-10 by 19% (95% CI [12-26], P = .0015). In vitro incubation with HMGB1 decreased cytokine production capacity of TNF by 34% (95% CI [3-65], P = .0003), interleukin 1β (IL-1β) by 24% (95% CI [16-32], P < .0001), and IL-10 by 40% (95% CI [21-58], P = .0009). Analysis of the inflammatory proteome alongside epigenetic shifts in monocytes indicated significant changes in gene accessibility, particularly in inflammatory markers such as CXCL8 (IL-8), IL-6, and interferon-gamma (IFN-γ). A significant enrichment of interferon regulatory factors (IRFs) was found in loci exhibiting decreased accessibility, whereas enrichment of activating protein-1 (AP-1) family motifs was found in loci with increased accessibility. These findings illuminate the complex epigenetic modulation influencing monocytes' response to surgical stress, shedding light on potential biomarkers for immune dysregulation. Our results advocate for further research into the role of anesthesia in these molecular pathways and the development of personalized interventions to mitigate immune dysfunction after surgery.

Chromatin accessibility is associated with therapeutic response in prostate cancer.

Treatment of advanced prostate cancer is challenging due to a lack of effective therapies. Therefore, it is important to understand the molecular mechanisms underlying therapeutic resistance in prostate cancer and to identify promising drug targets offering significant clinical advantages. Given the pivotal role of dysregulated transcriptional programs in the therapeutic response, it is essential to prioritize translational efforts targeting cancer-associated transcription factors (TFs). The present study investigated whether chromatin accessibility was associated with therapeutic resistance in prostate cancer using Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) data. The bioinformatics analysis identified differences in chromatin accessibility between the drug response (Remission) and drug resistance (Disease) groups. Additionally, a significant association was observed between chromatin accessibility, transcriptional output and TF activity. Among TFs, forkhead box protein M1 (FOXM1) was identified as a TF with high activity and expression in the Disease group. Notably, the results of the computational analysis were validated by FOXM1 knockdown experiments, which resulted in suppressed cell proliferation and enhanced therapeutic sensitivity in prostate cancer cells. The present findings demonstrated that chromatin accessibility and TF activity may be associated with therapeutic resistance in prostate cancer. Additionally, these results provide the basis for future investigations aimed at understanding the molecular mechanisms of drug resistance and developing novel therapeutic approaches for prostate cancer.

CREB3L1 deficiency impairs odontoblastic differentiation and molar dentin deposition partially through the TMEM30B

Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins, which are crucial for dentinogenesis. Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1 (Creb3l1) was specifically enriched in the terminal differentiated odontoblasts. In this study, deletion of Creb3l1 in the Wnt1+ lineage led to insufficient root elongation and dentin deposition. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells (mDPCs), the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation, protein biosynthesis and transport, all of which were evidenced by a diminished ability of odontoblastic differentiation, a significant reduction in intracellular proteins, and an even greater decline in extracellular supernatant proteins. Dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp), and transmembrane protein 30B (Tmem30b) were identified as direct transcriptional regulatory targets. TMEM30B was intensively expressed in the differentiated odontoblasts, and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro. Deletion of Tmem30b impaired the ability of odontoblastic differentiation, protein synthesis, and protein secretion in mDPCs. Moreover, overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion. Collectively, our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1, Dspp, and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B.

Gene regulatory dynamics during the development of a paleopteran insect, the mayfly Cloeon dipterum.

The evolution of insects has been marked by the appearance of key body plan innovations that promoted the outstanding ability of this lineage to adapt to new habitats, boosting the most successful radiation in animals. To understand the evolution of these new structures, it is essential to investigate which genes and gene regulatory networks participate during the embryonic development of insects. Great efforts have been made to fully understand gene expression and gene regulation during the development of holometabolous insects, in particular Drosophila melanogaster. Conversely, functional genomics resources and databases in other insect lineages are scarce. To provide a new platform to study gene regulation in insects, we generated ATAC-seq for the first time during the development of the mayfly Cloeon dipterum, which belongs to Paleoptera, the sister group to all other winged insects. With these comprehensive datasets along six developmental stages, we characterized pronounced changes in accessible chromatin between early and late embryogenesis. The application of ATAC-seq in mayflies provides a fundamental resource to understand the evolution of gene regulation in insects.

Analysis of chromatin accessibility in peripheral blood mononuclear cells from patients with early-stage breast cancer.

Objective: This study was aimed at exploring a specific open region of chromatin in the peripheral blood mononuclear cells (PBMCs) of patients with breast cancer and evaluating its feasibility as a biomarker for diagnosing and predicting breast cancer prognosis. Methods: We obtained PBMCs from breast cancer patients and healthy people for the assay for transposase-accessible chromatin (ATAC) sequencing (n = 3) and obtained the GSE27562 chip sequencing data for secondary analyses. Through bioinformatics analysis, we mined the pattern changes for chromatin accessibility in the PBMCs of breast cancer patients. Results: A total of 1,906 differentially accessible regions (DARs) and 1,632 differentially expressed genes (DEGs) were identified via ATAC sequencing. The upregulated DEGs in the disease group were mainly distributed in the cells, organelles, and cell-intima-related structures and were mainly responsible for biological functions such as cell nitrogen complex metabolism, macromolecular metabolism, and cell communication, in addition to functions such as nucleic acid binding, enzyme binding, hydrolase reaction, and transferase activity. Combined with microarray data analysis, the following set of nine DEGs showed intersection between the ATAC and microarray data: JUN, MSL2, CDC42, TRIB1, SERTAD3, RAB14, RHOB, RAB40B, and PRKDC. HOMER predicted and identified five transcription factors that could potentially bind to these peak sites, namely NFY, Sp 2, GFY, NRF, and ELK 1. Conclusion: Chromatin accessibility analysis of the PBMCs from patients with early-stage breast cancer underscores its potential as a significant avenue for biomarker discovery in breast cancer diagnostics and treatment. By screening the transcription factors and DEGs related to breast cancer, this study provides a comprehensive theoretical foundation that is expected to guide future clinical applications and therapeutic developments.

Review and Evaluate the Bioinformatics Analysis Strategies of ATAC-seq and CUT&Tag Data.

Efficient and reliable profiling methods are essential to study epigenetics. Tn5, one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency, is widely adopted in different genomic and epigenomic protocols for high-throughputly exploring the genome and epigenome. Based on Tn5, the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and the Cleavage Under Targets and Tagmentation (CUT&Tag) were developed to measure chromatin accessibility and detect DNA-protein interactions. These methodologies can be applied to large amounts of biological samples with low-input levels, such as rare tissues, embryos, and sorted single cells. However, fast and proper processing of these epigenomic data has become a bottleneck because massive data production continues to increase quickly. Furthermore, inappropriate data analysis can generate biased or misleading conclusions. Therefore, it is essential to evaluate the performance of Tn5-based ATAC-seq and CUT&Tag data processing bioinformatics tools, many of which were developed mostly for analyzing chromatin immunoprecipitation followed by sequencing (ChIP-seq) data. Here, we conducted a comprehensive benchmarking analysis to evaluate the performance of eight popular software for processing ATAC-seq and CUT&Tag data. We compared the sensitivity, specificity, and peak width distribution for both narrow-type and broad-type peak calling. We also tested the influence of the availability of control IgG input in CUT&Tag data analysis. Finally, we evaluated the differential analysis strategies commonly used for analyzing the CUT&Tag data. Our study provided comprehensive guidance for selecting bioinformatics tools and recommended analysis strategies, which were implemented into Docker/Singularity images for streamlined data analysis.

Targeting TREX1 Induces Innate Immune Response in Drug-Resistant Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation sequencing revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLCs. Analyses of human SCLC tumors and patient-derived xenografts (PDX) also showed an increase in TREX1 expression in postchemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase stimulator of interferon gene pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug-resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs. Significance: In this study, we show that targeting TREX1 induces an innate immune response and resensitizes SCLC cells to chemotherapy, representing a promising novel target for "immunologically" cold tumors, such as SCLC.

Integrated transcriptomic and epigenomic analyses to disclose the transcriptional regulatory mechanisms of lipid and energy metabolism under cold stress in grass carp

Cold-induced injuries significantly restrict the potential and efficacy of suivival and growth, necessitating a deeper understanding of cold response in ectothermic fish. Understanding the mechanisms by which fish respond to low-temperature stress has thus become a critical research focus in aquaculture. This study aims to investigate the effects of different temperature gradients (28 °C, 10 °C, and 2 °C) on global gene transcription in grass carp Ctenopharyngodon idellus. Additionally, the study examines cold-induced chromatin accessibility and transcriptional regulation through an integration of RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analyses. Transcriptome analysis revealed that mitochondrial damage and endoplasmic reticulum stress are major factors contributing to grass carp mortality under cold stress. The dataset also disclosed hepatocyte nuclear factor 1 beta (Hnf1b) and peroxisome proliferator-activated receptor alpha (PPARa) as key transcription factors for lipid and energy metabolism in grass carp under cold stress. Notably, Hnf1b and PPARa form a complex transcriptional regulatory network with other transcription factors, decreasd binding activity inactivates the expression of genes related to lipid and energy metabolism under cold stress. These results highlight the transcriptional regulation of lipid and energy metabolism in response to cold stress in grass carp. Overall, this study enhances our understanding of low-temperature response mechanisms in grass carp, providing valuable insights for further investigations.

The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma.

Despite recent advances in therapeutic treatments, multiple myeloma (MM) remains an incurable malignancy. Epigenetic factors contribute to the initiation, progression, relapse, and clonal heterogeneity in MM, but our knowledge on epigenetic mechanisms underlying MM development is far from complete. The SAGA complex serves as a coactivator in transcription and catalyzes acetylation and deubiquitylation. Analyses of data sets in the Cancer Dependency Map Project revealed that many SAGA components are selective dependencies in MM. To define SAGA-specific functions, we focused on ADA2B, the only subunit in the lysine acetyltransferase (KAT) module that specifically functions in SAGA. Integration of RNA sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), and cleavage under targets and release using nuclease assay (CUT&RUN) results identified pathways directly regulated by ADA2B including MTORC1 signaling and oncogenic programs driven by MYC, E2F, and MM-specific MAF. We discovered that ADA2B is recruited to MAF and MYC gene targets, and that MAF shares a majority of its targets with MYC in MM cells. Furthermore, we found that the SANT domain of ADA2B is required for interaction with both GCN5 and PCAF acetyltransferases, incorporation into SAGA, and ADA2B protein stability. Our findings uncover previously unknown SAGA KAT module-dependent mechanisms controlling MM cell growth, revealing a vulnerability that might be exploited for future development of MM therapy.

CloudATAC: a cloud-based framework for ATAC-Seq data analysis.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) generates genome-wide chromatin accessibility profiles, providing valuable insights into epigenetic gene regulation at both pooled-cell and single-cell population levels. Comprehensive analysis of ATAC-seq data involves the use of various interdependent programs. Learning the correct sequence of steps needed to process the data can represent a major hurdle. Selecting appropriate parameters at each stage, including pre-analysis, core analysis, and advanced downstream analysis, is important to ensure accurate analysis and interpretation of ATAC-seq data. Additionally, obtaining and working within a limited computational environment presents a significant challenge to non-bioinformatic researchers. Therefore, we present Cloud ATAC, an open-source, cloud-based interactive framework with a scalable, flexible, and streamlined analysis framework based on the best practices approach for pooled-cell and single-cell ATAC-seq data. These frameworks use on-demand computational power and memory, scalability, and a secure and compliant environment provided by the Google Cloud. Additionally, we leverage Jupyter Notebook's interactive computing platform that combines live code, tutorials, narrative text, flashcards, quizzes, and custom visualizations to enhance learning and analysis. Further, leveraging GPU instances has significantly improved the run-time of the single-cell framework. The source codes and data are publicly available through NIH Cloud lab https://github.com/NIGMS/ATAC-Seq-and-Single-Cell-ATAC-Seq-Analysis. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

TGF-β1 mediates hypoxia-preconditioned olfactory mucosa mesenchymal stem cells improved neural functional recovery in Parkinson’s disease models and patients

BackgroundParkinson’s disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra (SN). Activation of the neuroinflammatory response has a pivotal role in PD. Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic approach for various nerve injuries, but there are limited reports on their use in PD and the underlying mechanisms remain unclear.MethodsWe investigated the effects of clinical-grade hypoxia-preconditioned olfactory mucosa (hOM)-MSCs on neural functional recovery in both PD models and patients, as well as the preventive effects on mouse models of PD. To assess improvement in neuroinflammatory response and neural functional recovery induced by hOM-MSCs exposure, we employed single-cell RNA sequencing (scRNA-seq), assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) combined with full-length transcriptome isoform-sequencing (ISO-seq), and functional assay. Furthermore, we present the findings from an initial cohort of patients enrolled in a phase I first-in-human clinical trial evaluating the safety and efficacy of intraspinal transplantation of hOM-MSC transplantation into severe PD patients.ResultsA functional assay identified that transforming growth factor-β1 (TGF-β1), secreted from hOM-MSCs, played a critical role in modulating mitochondrial function recovery in dopaminergic neurons. This effect was achieved through improving microglia immune regulation and autophagy homeostasis in the SN, which are closely associated with neuroinflammatory responses. Mechanistically, exposure to hOM-MSCs led to an improvement in neuroinflammation and neural function recovery partially mediated by TGF-β1 via activation of the anaplastic lymphoma kinase/phosphatidylinositol-3-kinase/protein kinase B (ALK/PI3K/Akt) signaling pathway in microglia located in the SN of PD patients. Furthermore, intraspinal transplantation of hOM-MSCs improved the recovery of neurologic function and regulated the neuroinflammatory response without any adverse reactions observed in patients with PD.ConclusionsThese findings provide compelling evidence for the involvement of TGF-β1 in mediating the beneficial effects of hOM-MSCs on neural functional recovery in PD. Treatment and prevention of hOM-MSCs could be a promising and effective neuroprotective strategy for PD. Additionally, TGF-β1 may be used alone or combined with hOM-MSCs therapy for treating PD.

Chromatin accessibility and transcriptional landscape in PK-15 cells during early exposure to Aflatoxin B1

Aflatoxin B1 (AFB1) not only causes significant losses in livestock production but also poses a serious threat to human health. It is the most carcinogenic among known chemicals. Pigs are more susceptible to AFB1 and experience a higher incidence. However, the molecular mechanism of the toxic effect of AFB1 remains unclear. In this study, we used assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA-seq to uncover chromatin accessibility and gene expression dynamics in PK-15 cells during early exposure to AFB1. We observed that the toxic effects of AFB1 involve signaling pathways such as p53, PI3K-AKT, Hippo, MAPK, TLRs, apoptosis, autophagy, and cancer pathways. Basic leucine zipper (bZIP) transcription factors (TFs), including AP-1, Fos, JunB, and Fra2, play a crucial role in regulating the biological processes involved in AFB1 challenge. Several new TFs, such as BORIS, HNF1b, Atf1, and KNRNPH2, represent potential targets for the toxic mechanism of AFB1. In addition, it is crucial to focus on the concentration of intracellular zinc ions. These findings will contribute to a better understanding of the mechanisms underlying AFB1-induced nephrotoxicity and offer new molecular targets.

Characterization and integrated analysis of extrachromosomal DNA amplification in hematological malignancies

The study of extrachromosomal DNA (ecDNA), an element existing beyond classical chromosomes, contributes to creating a more comprehensive map of the cancer genome. In hematological malignancies, research on ecDNA has lacked comprehensive investigation into its frequency, structure, function, and mechanisms of formation. We re-analyzed WGS data from 208 hematological cancer samples across 11 types, focusing on ecDNA characteristics. Amplification of ecDNA was observed in 7 of these cancer types, with no instances found in normal blood cells. Patients with leukemia carrying ecDNA showed a low induction therapy remission rate (<30 %), a high relapse rate (75 %) among those who achieved complete remission, and a significantly lower survival rate compared to the general leukemia population, even those with complex chromosomal karyotypes. Among the 55 identified ecDNA amplicons, 268 genes were detected, of which 38 are known cancer-related genes exhibiting significantly increased copy numbers. By integrating RNA-Seq data, we discovered that the increased copy number, resulting in a higher amount of available DNA templates, indeed leads to the elevated expression of genes encoded on ecDNA. Additionally, through the integration of H3K4me3/H3K27ac chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin with sequencing, and high-throughput chromosome conformation capture data, we identified that ecDNA amplifications can also facilitate efficient, copy number-independent amplification of oncogenes. This process is linked to active histone modifications, improved chromatin accessibility, and enhancer hijacking, all of which are effects of ecDNA amplification. Mechanistically, chromothripsis and dysfunction of the DNA repair pathway can, to some extent, explain the origin of ecDNA.