Assay For Transposase-accessible Chromatin Research Articles

Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells

Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.

Establishing an optimized ATAC-seq protocol for the maize.

The advent of next-generation sequencing in crop improvement offers unprecedented insights into the chromatin landscape closely linked to gene activity governing key traits in plant development and adaptation. Particularly in maize, its dynamic chromatin structure is found to collaborate with massive transcriptional variations across tissues and developmental stages, implying intricate regulatory mechanisms, which highlights the importance of integrating chromatin information into breeding strategies for precise gene controls. The depiction of maize chromatin architecture using Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) provides great opportunities to investigate cis-regulatory elements, which is crucial for crop improvement. In this context, we developed an easy-to-implement ATAC-seq protocol for maize with fewer nuclei and simple equipment. We demonstrate a streamlined ATAC-seq protocol with four key steps for maize in which nuclei purification can be achieved without cell sorting and using only a standard bench-top centrifuge. Our protocol, coupled with the bioinformatic analysis, including validation by read length periodicity, key metrics, and correlation with transcript abundance, provides a precise and efficient assessment of the maize chromatin landscape. Beyond its application to maize, our testing design holds the potential to be applied to other crops or other tissues, especially for those with limited size and amount, establishing a robust foundation for chromatin structure studies in diverse crop species.

Inhibition of HDAC2 sensitises antitumour therapy by promoting NLRP3/GSDMD-mediated pyroptosis in colorectal cancer.

Although numerous studies have indicated that activated pyroptosis can enhance the efficacy of antitumour therapy in several tumours, the precise mechanism of pyroptosis in colorectal cancer (CRC) remains unclear. Pyroptosis in CRC cells treated with antitumour agents was assessed using various techniques, including Western blotting, lactate dehydrogenase release assay and microscopy analysis. To uncover the epigenetic mechanisms that regulate NLRP3, chromatin changes and NLRP3 promoter histone modifications were assessed using Assay for Transposase-Accessible Chromatin using sequencing and RNA sequencing. Chromatin immunoprecipitation‒quantitative polymerase chain reaction was used to investigate the NLRP3 transcriptional regulatory mechanism. Additionally, xenograft and patient-derived xenograft models were constructed to validate the effects of the drug combinations. As the core molecule of the inflammasome, NLRP3 expression was silenced in CRC, thereby limiting gasdermin D (GSDMD)-mediated pyroptosis. Supplementation with NLRP3 can rescue pyroptosis induced by antitumour therapy. Overexpression of HDAC2 in CRC silences NLRP3 via epigenetic regulation. Mechanistically, HDAC2 suppressed chromatin accessibility by eliminating H3K27 acetylation. HDAC2 knockout promotes H3K27ac-mediated recruitment of the BRD4-p-P65 complex to enhance NLRP3 transcription. Inhibiting HDAC2 by Santacruzamate A in combination with classic antitumour agents (5-fluorouracil or regorafenib) in CRC xenograft-bearing animals markedly activated pyroptosis and achieved a significant therapeutic effect. Clinically, HDAC2 is inversely correlated with H3K27ac/p-P65/NLRP3 and is a prognostic factor for CRC patients. Collectively, our data revealed a crucial role for HDAC2 in inhibiting NLRP3/GSDMD-mediated pyroptosis in CRC cells and highlighted HDAC2 as a potential therapeutic target for antitumour therapy. Silencing of NLRP3 limits the GSDMD-dependent pyroptosis in colorectal cancer. HDAC2-mediated histone deacetylation leads to epigenetic silencing of NLRP3. HDAC2 suppresses the NLRP3 transcription by inhibiting the formation of H3K27ac/BRD4/p-P65 complex. Targeting HDAC2 activates pyroptosis and enhances therapeutic effect.

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Dependency-aware deep generative models for multitasking analysis of spatial omics data.

Spatially resolved transcriptomics (SRT) technologies have significantly advanced biomedical research, but their data analysis remains challenging due to the discrete nature of the data and the high levels of noise, compounded by complex spatial dependencies. Here, we propose spaVAE, a dependency-aware, deep generative spatial variational autoencoder model that probabilistically characterizes count data while capturing spatial correlations. spaVAE introduces a hybrid embedding combining a Gaussian process prior with a Gaussian prior to explicitly capture spatial correlations among spots. It then optimizes the parameters of deep neural networks to approximate the distributions underlying the SRT data. With the approximated distributions, spaVAE can contribute to several analytical tasks that are essential for SRT data analysis, including dimensionality reduction, visualization, clustering, batch integration, denoising, differential expression, spatial interpolation, resolution enhancement and identification of spatially variable genes. Moreover, we have extended spaVAE to spaPeakVAE and spaMultiVAE to characterize spatial ATAC-seq (assay for transposase-accessible chromatin using sequencing) data and spatial multi-omics data, respectively.

P12 Transcriptional and epigenetic changes drive increased cellular plasticity in keloid fibroblasts

Abstract Introduction and aims Keloids are fibroproliferative scars that progressively grow beyond the original wound site. They occur as a result of genetic, epigenetic and environmental abnormalities. Fibroblasts are best characterized for their role in depositing and remodelling the extracellular matrix (ECM) and are believed to have a crucial role in keloid formation. However, the detailed molecular signatures that promote this pathogenic phenotype are still unknown. Methods To investigate the epigenetic and genetic changes in keloid fibroblasts, we performed assay for transposase-accessible chromatin (ATAC)-Seq and RNA-Seq on a patient-matched set of keloid and normal fibroblasts. ATAC-Seq is a high-throughput sequencing method that defines chromatin accessible regions in which the binding of sites of transcription factors that regulate gene expression can be mapped. Identified candidates are validated in keloid fibroblasts via quantitative polymerase chain reaction and their functional role is analysed in various in vitro assays. Results ATAC-Seq analysis revealed more accessibility in coding regions of keloid fibroblasts compared with normal fibroblasts. Pathways enriched in these regions include transcriptional regulation, embryonic development, cell differentiation, calcium-binding and collagen remodelling. Intriguingly, several developmental epigenetic modulators were identified that may alter the transcription and chromatin landscape of keloid fibroblasts to a more plastic state and have the capacity to influence cell differentiation choices, potentially promoting chondrogenic and adipogenic cell features. Currently, we are investigating the ability of keloid fibroblasts to be more readily differentiated into other cell types including induced pluripotent stem cells, and the contribution of those candidates on fibrotic features to the cell are also being explored. Conclusions Our analysis reveals that the chromatin of keloid fibroblasts is more accessible, especially in genomic regions associated with embryonic development and keloid fibroblasts show an increased plasticity in vitro. These findings may support the development of fibroblast-targeted therapies for keloid scars and possibly other connective tissue disorders.

O16 Understanding the pleiotropic effects of the Rho-kinase inhibitor Y-27632 on human keratinocytes

Abstract Introduction and aims Successful epidermal transplant generation depends on sufficiently high numbers of self-renewing stem cells that give rise to clonal colonies termed ‘holoclones’. Treatment with Y-27632, a nonspecific Rho-kinase 1/2 inhibitor, conditionally immortalizes epidermal keratinocyte cultures. However, the pleiotropic signalling mechanisms downstream of Y-27632 remain largely unexplored. We have therefore characterized the effects of continuous long-term Y-27632 treatment on chromatin regulation, and investigated whether Y-27632 reprogrammes a holoclone-like state. Methods Neonatal foreskin-derived human epidermal keratinocytes were cultured from passage p5 to p30, with or without Y treatment. Samples were collected for RNA sequencing analysis from progenitor-enriched middle passage cells (p5, MPK), progenitor-depleted late-passage cells (p8, LPK) and Y-treated cells at p8 (LPK-Y). Samples were also collected for assay for transposase-accessible chromatin (ATAC)-Seq analysis to assess open and closed chromatin regions. Results Untreated cultures became depleted of proliferating cells by p11, whereas Y maintained a highly proliferative culture up to p30 and beyond. Y withdrawal at p11 resulted in rapid loss of proliferative capacity within a single passage. ATAC-Seq identified 196 differentially regulated chromatin sites in MPK vs. LPK. Y treatment maintained accessibility in approximately 40% of these MPK-enriched chromatin regions. Approximately 3000 differentially expressed genes (DEGs) were identified in MPK vs. LPK, while Y treatment induced approximately 11 000 DEGs. Activated genes in LPK-Y were enriched for a holoclone signature, with no evidence for activation of a precancerous transcriptome. Y treatment altered the expression of various genes associated with amino acid metabolism, enabling cells to overcome serine/glycine autotrophy. Conclusions Y acts by preserving accessibility of progenitor-enriched chromatin sites and further reprogrammes the keratinocyte transcriptome towards a more holoclone-like state. However, keratinocytes retain a ‘memory’ of their doubling number, which is conditionally suppressed by Y. The observed global transcriptomic changes are indicative of the highly pleiotropic nature of the Y-induced signalling changes, warranting further deconvolution.

The single-cell chromatin landscape in gonadal cell lineage specification

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

The enriched expression of aquaporin-2 in the renal inner medulla is mediated by increased chromatin accessibility

Introduction: The osmotic gradient between renal cortex (CTX) and renal inner medulla (IM) is important for the generation of a concentrated urine. In addition, the unique hypertonic environment in the IM induces a specific gene expression pattern. For example the expression of aquaporin-2 (Aqp2) is enriched in the IM compared to CTX. We postulated that these changes must be associated with epigenetic mechanisms that could regulate the spatial expression pattern in CTX and IM. We performed assay for transposase-accessible chromatin (ATAC) sequencing to identify genomic regions with differences in accessibility in CTX and IM. Methods: We used kidneys from control mice and collecting duct principal cell specific nuclear factor of activated t cells (Nfat5)-KO mice and dissected CTX and IM. These samples were subjected for ATAC-seq analysis. Accessible genomic regions were identified with MACS2. We next compared differentially accessible regions between CTX and IM in control mice and between control and Nfat5-KO mice. The results were correlated with corresponding gene expression profiles that have been analyzed in a former study. Results: There are massive differences in the accessibility of genomic regions between CTX and IM. In CTX, more than 100,000 regions are differentially accessible compared to IM, with 48,698 regions having higher accessibility and 55,453 regions having lower accessibility. Genes with higher accessibility in CTX show also higher or even specific gene expression in CTX and vice versa. Since loss of NFAT5 is associated with a massive change in gene expression in kidneys from Nfat5-KO mice, we analyzed if this is affected by differences in chromatin accessibility. Our data shows that not only spatial localization in the kidney affects chromatin accessibility, but also the function of NFAT5. Loss of NFAT5 results in 10,456 regions with higher accessibility and 3,858 regions with closed chromatin in the CTX and 16,452 regions losing accessibility and 9,494 regions with increased accessibility in the IM. The accessibility of the Aqp2 gene is higher in IM compared with CTX. The open chromatin of the Aqp2 promoter region correlates with higher expression of Aqp2 in the IM. This suggests that hypertonicity leads to an improvement in the accessibility of the promoter region of Aqp2. In Nfat5-KO IM the accessibility of this region was decreased, which could explain the reduced Aqp2 expression in Nfat5-KO mice. Conclusions: To date, studying hypertonicity-dependent regulation of chromatin accessibility and associated gene expression in CTX and IM in the kidney were missing. Here we show that the differences in gene expression between CTX and IM might be due to changes in accessibility of genomic regions. We also show that the renal expression of Aqp2 might be a result of an epigenetic mechanism. In this context, NFAT5 was found to be an important regulator of chromatin accessibility and gene expression in the kidney. The authors have nothing to disclose. This project was funded by the Deutsche Forschungsgemeinschaft(ED181/9-3). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Histone β-hydroxybutyrylation-mediated Chromatin Remodeling Upregulates β-oxidation of Fatty Acids and Attenuates Metabolic Syndrome

Center for Hypertension and Precision Medicine, Department of Physiology and Pharmacology, University of Toledo, Toledo, OH Introduction: Metabolic syndrome (MetS) is a cluster of metabolic dysregulations including high blood pressure (BP), dyslipidemia, central obesity, hyperglycemia, and insulin resistance. Regular exercise is one of the lifestyle modifications known to prevent MetS by the generation of ketone bodies. Precisely how ketone bodies operate to lower MetS is largely unknown. Previously, we demonstrated that the ketone body, β-hydroxybutyrate, is a potent anti-hypertensive metabolite. β-hydroxybutyrate has been recently recognized to epigenetically modify histones by β-hydroxybutyrylation. Using assay for transposase-accessible chromatin with sequencing and chromatin immunoprecipitation assays, we found that histone β-hydroxybutyrylation causes chromatin remodeling and exposes regions for transcription of genes for β-oxidation of fatty acids. Therefore, we hypothesized that β-hydroxybutyrate alleviates MetS via histone β-hydroxybutyrylation-mediated transcriptional upregulation of β-oxidation of fatty acids. To test this hypothesis, we examined the epigenetic effect of increasing systemic β-hydroxybutyrate in a genetic rat model of MetS inbred in our laboratory, the low-capacity runner (LCR/Tol) rats. Methods: Randomly selected two groups of six months old female LCR/Tol rats (n=5/group) were surgically implanted with radiotelemetry transmitters for BP measurement. The control group was given water, whereas the experimental group was given water containing 20% v/v of 1,3-butanediol (1,3-BD), which is a precursor of β-hydroxybutyrate for 10 weeks. Following euthanasia, sera and tissues were harvested. Histones were isolated and subjected to western blotting for quantitation of β-hydroxybutyrylation. Results: Compared to the control group, rats given 1,3-BD had a significantly increased circulating β-hydroxybutyrate (mean 1.98 mM vs 0.71 mM, p<0.05), lower body weight (mean 231.6 gm vs 248.4 gm, p<0.05), lower fasting glucose (mean 79.60 mg/dl vs 100.8 mg/dl, p<0.05) and lower 24-hour average systolic BP (116±2 mmHg vs. 120±2 mmHg, p<0.05). Interestingly, histones isolated from the experimental group with higher circulating β-hydroxybutyrate also displayed a marked increase in β-hydroxybutyrylation (mean 0.82 vs 0.22, p<0.01). Further, the target transcriptional genes of histone β-hydroxybutyrylation, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) and cytochrome P450 family 2 subfamily d polypeptide 4 (Cyp2d4) were both significantly upregulated in the experimental group (p<0.01). However, markers of liver injury, alanine transaminase, alkaline phosphatase, aspartate transaminase, and total bile acid levels, were comparable between the two groups, indicating no liver damage. Conclusion: This is the first study to demonstrate that enhanced systemic β-hydroxybutyrate attenuates MetS by histone β-hydroxybutyrylation-mediated chromatin remodeling to upregulate the transcription of Hmgcs2 and Cyp2d4 to promote β-oxidation of fatty acids. Further, our results demonstrate that for individuals with MetS who may be unable to exercise, utilizing 1,3-BD could serve as an alternate source to combat the MetS ameliorating benefits of the ketone body β-hydroxybutyrate. Funding Sources: Authors acknowledge the National Institutes of Health for funding support to Bina Joe (R01HL1430820). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MMGAT: a graph attention network framework for ATAC-seq motifs finding.

Motif finding in Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) data is essential to reveal the intricacies of transcription factor binding sites (TFBSs) and their pivotal roles in gene regulation. Deep learning technologies including convolutional neural networks (CNNs) and graph neural networks (GNNs), have achieved success in finding ATAC-seq motifs. However, CNN-based methods are limited by the fixed width of the convolutional kernel, which makes it difficult to find multiple transcription factor binding sites with different lengths. GNN-based methods has the limitation of using the edge weight information directly, makes it difficult to aggregate the neighboring nodes' information more efficiently when representing node embedding. To address this challenge, we developed a novel graph attention network framework named MMGAT, which employs an attention mechanism to adjust the attention coefficients among different nodes. And then MMGAT finds multiple ATAC-seq motifs based on the attention coefficients of sequence nodes and k-mer nodes as well as the coexisting probability of k-mers. Our approach achieved better performance on the human ATAC-seq datasets compared to existing tools, as evidenced the highest scores on the precision, recall, F1_score, ACC, AUC, and PRC metrics, as well as finding 389 higher quality motifs. To validate the performance of MMGAT in predicting TFBSs and finding motifs on more datasets, we enlarged the number of the human ATAC-seq datasets to 180 and newly integrated 80 mouse ATAC-seq datasets for multi-species experimental validation. Specifically on the mouse ATAC-seq dataset, MMGAT also achieved the highest scores on six metrics and found 356 higher-quality motifs. To facilitate researchers in utilizing MMGAT, we have also developed a user-friendly web server named MMGAT-S that hosts the MMGAT method and ATAC-seq motif finding results. The advanced methodology MMGAT provides a robust tool for finding ATAC-seq motifs, and the comprehensive server MMGAT-S makes a significant contribution to genomics research. The open-source code of MMGAT can be found at https://github.com/xiaotianr/MMGAT , and MMGAT-S is freely available at https://www.mmgraphws.com/MMGAT-S/ .

Integrated analysis of ATAC-seq and transcriptomic reveals the ScDof3-ScproC molecular module regulating the cold acclimation capacity of potato

Low temperature severely affects the geographical distribution and production of potato, which may incur cold damage in early spring or winter. Cultivated potatoes, mainly derived from Solanum tuberosum, are sensitive to freezing stress, but wild species of potato such as S. commersonii exhibit both constitutive freezing tolerance and/or cold acclimation tolerance. Hence, such wild species could assist in cold hardiness breeding. Yet the key transcription factors and their downstream functional genes that confer freezing tolerance are far from clear, hindering the breeding process. Here, we used ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) alongside RNA-seq to investigate the variation in chromatin accessibility and patterns of gene expression in freezing-tolerant CMM5 (S. commersonii), before and after its cold treatment. Our results suggest that after exposure to cold, transcription factors including Dof3, ABF2, PIF4, and MYB4 were predicted to further control the genes active in the synthetic/metabolic pathways of plant hormones, namely abscisic acid, polyamine, and reductive glutathione (among others). This suggests these transcription factors could regulate freezing tolerance of CMM5 leaves. In particular, ScDof3 was proven to regulate the expression of ScproC (pyrroline-5-carboxylate reductase, P5CR) according to dual-LUC assays. Overexpressing ScDof3 in Nicotiana benthamiana leaves led to an increase in both the proline content and expression level of NbproC (homolog of ScproC). These results demonstrate the ScDof3-ScproC module regulates the proline content and thus promotes freezing tolerance in potato. Our research provides valuable genetic resources to further study the molecular mechanisms underpinning cold tolerance in potato.

Single-cell RNA sequencing and ATAC sequencing identify novel biomarkers for bicuspid aortic valve-associated thoracic aortic aneurysm.

Bicuspid aortic valve (BAV) is the most prevalent congenital cardiovascular defect and known to cause thoracic aortic aneurysms (TAAs). To improve our understanding of BAV pathogenesis, we characterized the cellular composition of BAV tissues and identified molecular changes in each cell population. Tissue samples from two patients with BAV and two heart transplant donors were analyzed using single-cell RNA sequencing, assay for transposase-accessible chromatin using sequencing, and weighted gene coexpression network analysis for differential gene analysis. TAA-related changes were evaluated by comparing the proportion of each cell type and gene expression profiles between TAA and control tissues. Further, by combining our single-cell RNA sequencing data with publicly available data from genome-wide association studies, we determined critical genes for BAV. We found 20 cell subpopulations in TAA tissues, including multiple subtypes of smooth muscle cells, fibroblasts, macrophages, and T lymphocytes. This result suggested that these cells play multiple functional roles in BAV development. Several differentially expressed genes, including CD9, FHL1y, HSP90AA1, GAS6, PALLD, and ACTA2, were identified. We believe that this comprehensive assessment of the cellular composition of TAA tissues and the insights into altered gene expression patterns can facilitate identification of novel diagnostic biomarkers and therapeutic targets for BAV-associated TAA.

Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

Iterative assay for transposase-accessible chromatin by sequencing to isolate functionally relevant neuronal subtypes.

The Drosophila brain contains tens of thousands of distinct cell types. Thousands of different transgenic lines reproducibly target specific neuron subsets, yet most still express in several cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we developed an iterative assay for transposase-accessible chromatin (ATAC) approach. Open chromatin regions (OCRs) enriched in neurons, compared to whole bodies, drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCR2s that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression, and we used it to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

Cardiac GR Mediates the Diurnal Rhythm in Ventricular Arrhythmia Susceptibility.

Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.

Abstract 5500: Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain metastasis

Abstract Introduction: Brain metastasis (BM) remains incurable with a dismal 2-year survival of less than 2%. BM are surrounded by a complex tumor microenvironment (TME) comprised of a meshwork of extracellular matrix (ECM) proteins and an assemblage of cell types including cancer-associated fibroblasts (CAFs), the most prominent cell type within the TME. We have previously shown that patient-derived BM CAFs can either support or inhibit tumor growth in vivo, suggesting that BM CAFs are heterogenous. Yet, there remains a dearth of data on the functional and mechanistic underpinnings of CAFs in the BM TME. Methods: To explore both the molecular profiles of CAFs and their heterogeneity in the BM TME, we performed a single cell Multiome (RNA and Assay for Transposase-Accessible Chromatin (ATAC)) sequencing (scMultiome-seq) analysis of 9 human BM tissue samples and 4 BM patient-derived CAF cell lines, from both lung and breast primary cancers. Next, we investigated the tumor supportive or inhibitory capabilities of BM CAF cell lines on the growth of BM tumor cells in vitro. Results: ScMultiome-seq analysis of BM tissue samples identified 4 distinct CAF subpopulations, that we termed desmoplastic, immune, contractile, and neural CAFs, in the BM TME. These CAF subpopulations are respectively involved in either regulating the ECM organization, orchestrating the neuroimmune response, triggering tissue contraction or regulating neural components upon BM growth. CellPhoneDB analysis revealed that CAFs had more cell-cell interactions with other cells than any other cell types in the BM TME. Pseudotime analysis demonstrated the potential cell of origin of each BM CAF subpopulation and the cell state transition they undergo. We observed that the end cell state of the desmoplastic CAFs expressed high levels of immunoglobulin superfamily containing leucine rich repeat (ISLR). ISLR was also observed to be highly expressed in a BM patient-derived CAF cell line which was previously reported by our lab to restrain tumor growth in vivo, suggesting that desmoplastic CAFs with high ISLR expression (ISLR-CAFs) might be tumor inhibitory. ScMultiome-seq analysis of BM CAF cell lines validated that tumor inhibitory CAF cell lines were primarily of the desmoplastic ISLR-CAF subpopulation. Conditioned media obtained from ISLR-CAFs containing high levels of ISLR inhibited tumor cell viability in vitro compared to controls. Conclusion: Our study is the first to describe the heterogeneity of CAFs and their potential central roles in the BM TME. We also identify that CAF subpopulations are dynamic and can transition to tumor inhibitory cell states, such as ISLR-CAFs. These data have potential to help design therapeutic strategies that selectively target tumor supportive CAFs whilst protecting tumor inhibitory ones. Citation Format: Thomas Simon, David N. Buckley, Ben Y. Tew, Matthew Salomon, Zeyi Yang, Gerald C. Gooden, Steven A. Toms, Gabriel Zada, Bodour Salhia. Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5500.

Abstract 6248: Multilayered molecular interactions underlying glioblastoma pathogenesis through multi-omics analysis

Abstract Glioblastoma, IDH-wild type (GBM) is the most common malignant and treatment-refractory brain tumor with a dismal prognosis. Despite the identification of various driver genes in previous large-scale genomic analyses, there has been limited progress in the development of novel treatments. Acquiring an extensive understanding of the molecular mechanisms underlying GBM pathogenesis is crucial to improve prognostic outcomes. We analyzed 289 whole-genome sequencing data (WGS) including 159 unpublished deep WGS (≥ ×120 coverage) along with RNA-seq, DNA methylation array, and assay for transposase-accessible chromatin with sequencing (ATAC-seq) to uncover the molecular mechanisms regulating development and progression of GBM. Our deep WGS enables us to delineate a fine view of clonal architecture, where mutational signature varies between clonal and subclonal mutations, suggesting that different mutational processes contribute to GBM pathogenesis depending on its developmental stage. The differentiation status of tumor cells detected by transcriptional deconvolution analysis is associated with multi-layer profiles including genomic alterations, expression subtypes, DNA methylation, and open chromatin status. Tumors predominantly comprised of differentiated cells display genetic and epigenetic profiles that align with the classical subtype, whereas tumors predominantly composed of stem-like cells exhibit profiles consistent with the proneural subtype. ATAC-seq unveils genome-wide chromatin accessibility features associated with gene expression subtypes. Motif enrichment analysis of differentially accessible sites among the subtypes identified specific transcription factors. The proneural subtype is enriched for the SOX10 binding motif, which is associated with regulating cell states, and exhibits higher expression of SOX10 compared to other subtypes. Conversely, the mesenchymal and classical subtypes are enriched for the CREB1 binding motif, which promotes cell proliferation and angiogenesis through TGFβ2 upregulation, wherein TGFβ2 expression is significantly elevated. These findings support a model in which the difference in chromatin structure also regulates the progression of GBM.Our analysis reveals the multi-layer molecular interactions underlying the progression of GBM enhancing our understanding of the pathogenesis. Citation Format: Takuma Nakashima, Yusuke Funakoshi, Ryo Yamamoto, Yuriko Sugihara, Shohei Nambu, Yoshiki Arakawa, Shota Tanaka, Joji Ishida, Ryuta Saito, Ryosuke Hanaya, Koji Yoshimoto, Yoshitaka Narita, Hiromichi Suzuki. Multilayered molecular interactions underlying glioblastoma pathogenesis through multi-omics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6248.

Abstract 1246: Erg-driven prostate cancer emerges from a basal subset of cells with luminal transcriptomic features

Abstract Translocations of the ETS family transcription factor ERG are found in nearly 150,000 newly diagnosed prostate cancers (PCas) each year in the United States underscoring the importance of defining the mechanism by which ERG promotes prostate epithelial transformation. A consequence of nearly all ERG translocations is the aberrant expression of ERG in prostate luminal cells under transcriptional control of TMPRSS2. Multiple lines of evidence implicate ERG translocations as an initiating event in prostate cancer. ERG expression is seen in prostatic intraepithelial neoplasia (PIN) and proliferative inflammatory atrophy; expression in cancers is typically uniform; and, in the context of PI3K pathway activation, ERG is sufficient to induce cancers in mice. Yet, despite extensive research the mechanism by which ERG initiates prostate cancer is unclear. To gain insight into how ERG translocations cause prostate cancer, we performed both transcriptional profiling (scRNA-seq) and chromatin accessibility via Assay for Transposase-Accessible Chromatin using sequencing (snATAC-seq) in single cells of an autochthonous mouse model at an early stage of disease initiation. Surprisingly, we observed in prostate epithelial cells broadly expressing ERG enhanced proliferation primarily in a hybrid subpopulation of cells having basal identity but with luminal morphology and cytokeratin expression. Through a series of lineage tracing and primary prostate epithelial transplantation experiments, we confirmed tumor initiating activity resided within a subpopulation of basal cells expressing luminal genes (e.g., Tmprss2 and Nkx3-1). A trajectory analysis from single-cell Chromatin accessibility indicated Erg+ cells began to lose basal (Trp63) accessibility and gained in STAT/NFAT TFs while retaining the ability to rapidly differentiate into luminal cells that reflect the lineage of ERG-positive cancers. These findings help resolve a preexisting debate about the cell of origin of ERG-driven prostate cancer and narrow the focus for future mechanistic studies to a basal-luminal subpopulation of tumor initiating cells. Citation Format: Erik Ladewig, Weiran Feng, Christina Leslie, Charles Sawyers. Erg-driven prostate cancer emerges from a basal subset of cells with luminal transcriptomic features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1246.