Assay For Transposase-accessible Chromatin Research Articles

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

IntroductionBicuspid 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.MethodsTissue 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.ResultsWe 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.DiscussionWe 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.

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.

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 early 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. The objectives are to investigate the recruitment of transcription factors to time-of-day differentially accessible chromatin that underpins day-night ion channel rhythms and to assess the significance of this for the heart's day-night rhythm in VA susceptibility. Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the inactive (zeitgeber time, time of lights on, start of sleep period) and active (time of lights off, start of awake period [ZT12]) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) binding site 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.

Abstract 6284: Multimoics reveal aberrant pathways and regulation of castration resistant and metastatic prostate cancer

Abstract Prostate cancer is a highly heterogeneous disease and in the end, 30% of patients will experience metastatic and fatal castration resistant prostate cancer (mCRPC). In spite of the vigorous efforts to search the causes of metastatic CRPC (mCRPC), the mechanisms are still largely unresolved. To understand the progression of mCRPC, we utilized scRNA-seq to profile global expression patterns in LNCaP, LNCaP-Abl and PC3 representing castration sensitive and resistant states. ScRNA-seq revealed 335 and 2393 differentially expressed (DE) genes in early and advanced castration resistant states enriched in 43 and 163 aberrant pathways. These included focal adhesion, adherens junction, leukocyte transendothelial migration, ECM-receptor interaction, regulation of actins, gap junction, TGFb, WNT, Hippo, Ephrin and other pathways. Upregulation of these pathways predicted worst disease free/overall survival of The Cancer Genomes Atlas (TCGA) PC cohort using Kaplan-Meir estimation. Further scRNA-seq also indicated most of the pathways were also enriched in ex vivo circulating tumor cells derived from biochemical recurrent or mCRPC patients. Small molecule perturbation verified function of 9 pathways involved in cell proliferation, migration, invasion, and organoid formation of mCRPC cell lines and CTCs. We then delved into how these genes are regulated by the global chromatin landscape through Assay for Transposase-Accessible Chromatin and high throughput DNA-seq (ATAC-seq). In castration sensitive LNCaP, active chromatins are associated with genes but most of them are located in distal intergenic regions. Conversely, the majority (51.9-57.6%) of open chromatins in castration resistant PC cells are situated in promoters that lie within 1kb of transcription start sites. KEGG pathway enrichment analysis uncovered Hippo, Wnt, tight junction, adherens junction, circadian and other pathways that corroborate the scRNA-seq findings. GRO-seq data indicated that nascent transcription supports gene body transcription in castration resistant cells but leads to massive pausing in castration sensitive cells. Citation Format: Aaron M. Horning, Chia-Nung Hung, Brandon Liebermann, Chih-Wei Chou, Che-Kuang Lin, Yao Vickie Wang, Victor X. Jin, Michael A. Liss, Chiou-Miin Wang, Chun- Lin, Meizhen Chen, Tim H-M Huang, Chun-Liang Chen. Multimoics reveal aberrant pathways and regulation of castration resistant and metastatic prostate cancer [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 6284.

Abstract 6225: Tumor evolution through selection by ECM stiffness

Abstract In this study, we used experimental evolution to investigate the extent to which the mechanical stiffness of the tumor extracellular matrix (ECM) exerts selection pressure on a genetically diverse population of breast cancer cells. Polyacrylamide hydrogels were fabricated and conjugated with collagen type I to create the ECM with different stiffness. MDA-MB-231 cells were continuously cultured on the hydrogels of the same corresponding stiffness. Growth rate was determined at certain time points by harvesting and counting the cells. Abundance of clonal MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection was identified with sequencing of DNA barcode tags. Cellular phenotypes were characterized through immunofluorescence staining and confocal microscopy. 3D migration of the cancer cells was examined using Boyden chamber assay. Cellular contractility was measured through traction force microscopy. Whole genome transcriptome sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing, and reduced representation bisulfite sequencing were performed for transcriptomic and epigenetic analysis. Sustained culture of MDA-MB-231 breast cancer cells overall three months on soft ECM resulted in an increased proliferation rate and beta1 integrin-dependent spreading area of the cells. Such changes did not occur in clonal cells with low genetic variation and did not occur in ancestral cells on stiff substrates. Barcode analysis revealed two distinct surviving populations post-genetic selection, with one demonstrating robust proliferation on both soft and stiff ECM, and the other exhibiting preferential adaptation to the soft matrix. The soft-selected populations exhibited nuclear localization of the transcriptional regulator yes-associated protein and less wrinkled nuclei compared with ancestral populations when both populations were grown on soft ECM. Aggressive migration was observed in the soft-selected populations, indicating a more malignant selected phenotype. The soft-selected cells showed increased RhoA activity and contractile tension on a soft substrate compared to the ancestral cells. RNA-seq analysis revealed systematic differences between ancestral, stiff-selected, and soft-selected populations corresponding to enriched biological processes related to cell adhesion and ECM organization. Significant differences in DNA methylation and chromatin accessibility were observed in soft selected populations which were correlated with changes in RNA expression. ECM stiffness exerts selection pressure on genetically variable cancer cell populations, selecting specific genetic variants in a cell population optimally adapted for that stiffness. Selected variants on soft ECM tend to be highly migratory, proliferative and generate high traction even on soft ECM. Our results highlight the importance of ECM stiffness-driven evolution in cancer development. Citation Format: Ting-Ching Wang, Suchitaa Sawhney, Daylin Morgan, Richa Rashmi, Marcos R. Estecio, Amy Brock, Irtisha Singh, Richard L. Bennett, Charles F. Baer, Jonathan D. Licht, Tanmay P. Lele. Tumor evolution through selection by ECM stiffness [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 6225.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase(Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and isthen released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Primary osteoarthritis chondrocyte map of chromatin conformation reveals novel candidate effector genes

ObjectivesOsteoarthritis is a complex disease with a huge public health burden. Genome-wide association studies (GWAS) have identified hundreds of osteoarthritis-associated sequence variants, but the effector genes underpinning these signals remain...

Deconvoluting multi-person biological mixtures and accurate characterization and identification of separated contributors using non-targeted single-cell DNA sequencing

The genetic characterization and identification of individuals who contributed to biological mixtures are complex and mostly unresolved tasks. These tasks are relevant in various fields, particularly in forensic investigations, which frequently encounters crime scene stains generated by more than one person. Currently, forensic mixture deconvolution is mostly performed subsequent to forensic DNA profiling at the level of the mixed DNA profiles, which comes with several limitations. Some previous studies attempted at separating single cells prior to forensic DNA profiling. However, these approaches are biased at selection of the cells and, due to their targeted DNA analysis on low template DNA, provide incomplete and unreliable forensic DNA profiles. We recently demonstrated the feasibility of performing mixture deconvolution prior to forensic DNA profiling through the utilization of a non-targeted single-cell transcriptome sequencing (scRNA-seq). In addition to individual-specific mixture deconvolution, this approach also allowed accurate characterisation of biological sex, biogeographic ancestry and individual identification of the separated mixture contributors. However, RNA has the forensic disadvantage of being prone to degradation, and sequencing RNA - focussing on coding regions - limits the number of single nucleotide polymorphisms (SNPs) utilized for genetic mixture deconvolution, characterization, and identification. These limitations can be overcome by performing single-cell sequencing on the level of DNA instead of RNA. Here, for the first time, we applied non-targeted single-cell DNA sequencing (scDNA-seq) by applying the scATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) technique to address the challenges of mixture deconvolution in the forensic context. We demonstrated that scATAC-seq, together with our recently developed De-goulash data analysis pipeline, is capable of deconvoluting complex blood mixtures of five individuals from both sexes with varying biogeographic ancestries. We further showed that our approach achieved correct genetic characterization of the biological sex and the biogeographic ancestry of each of the separated mixture contributors and established their identity. Furthermore, by analysing in-silico generated scATAC-seq data mixtures, we demonstrated successful individual-specific mixture deconvolution of i) highly complex mixtures of 11 individuals, ii) balanced mixtures containing as few as 20 cells (10 per each individual), and iii) imbalanced mixtures with a ratio as low as 1:80. Overall, our proof-of-principle study demonstrates the general feasibility of scDNA-seq in general, and scATAC-seq in particular, for mixture deconvolution, genetic characterization and individual identification of the separated mixture contributors. Furthermore, it shows that compared to scRNA-seq, scDNA-seq detects more SNPs from fewer cells, providing higher sensitivity, that is valuable in forensic genetics.

Dominance reversals: the resolution of genetic conflict and maintenance of genetic variation.

Beneficial reversals of dominance reduce the costs of genetic trade-offs and can enable selection to maintain genetic variation for fitness. Beneficial dominance reversals are characterized by the beneficial allele for a given context (e.g. habitat, developmental stage, trait or sex) being dominant in that context but recessive where deleterious. This contextdependence at least partially mitigates the fitness consequence of heterozygotes carrying one non-beneficial allele for their context and can result in balancing selection that maintains alternative alleles. Dominance reversals are theoretically plausible and are supported by mounting empirical evidence. Here, we highlight the importance of beneficial dominance reversals as a mechanism for the mitigation of genetic conflict and review the theory and empirical evidence for them. We identify some areas in need of further research and development and outline three methods that could facilitate the identification of antagonistic genetic variation(dominance ordination, allele-specific expression and allele-specific ATAC-Seq(assay for transposase-accessible chromatin with sequencing)). There is ample scope for the development of new empirical methods as well as reanalysis of existing data through the lens of dominance reversals. A greater focus on this topic will expand our understanding of the mechanisms that resolve genetic conflict and whether they maintain genetic variation.

The adverse effects of developmental exposure to polystyrene nanoparticles on cognitive function in weaning rats and the protective role of trihydroxy phenolacetone

Polystyrene nanoplastic(PS-NP) can originate from sources such as plastic waste and industrial wastewater and have been shown to have deleterious effects on abnormal neurobehaviors. However, evidence regarding the health impacts, biological mechanisms, and treatment strategies underlying developmental exposure to low dose PS-NP is still lacking. This study aimed to fill this knowledge gap by administering low doses of PS-NP(50 and 100 μg/L) to weaning rats for 4 consecutive weeks. Behavioral and morphological experiments were performed to evaluate hippocampal damage, and transcriptomics and Assay for Transposase Accessible Chromatin with hight-throughput sequencing(ATAC) analyses were conducted to identify potential key targets. Additionally, Connectivity Map(CMap) database, Limited proteolysis-mass spectrometry(LiP-SMap), and molecular-protein docking were used to examine potential phytochemicals with therapeutic effects on key targets. The results indicated that developmental exposure to PS-NP can induce hippocampal impairment and aberrant neurobehaviors in adulthood. Multi-omics analyses consistently showed that apoptosis-related signaling pathways were sensitive to PS-NP exposure, and mitogen-activated protein kinase 3(Mapk3) was identified as the core gene by the gene network, which was further validated in vitro experiments. The CMap database provided a series of phytochemicals that might regulate Mapk3 expression, and trihydroxy-phenolacetone(THP) was found to have directly binding sites with Mapk3 through LiP-SMap and molecular docking analysis. Furthermore, THP administration could significantly alleviate apoptosis induced by PS-NP exposure in primary hippocampal cells through down-regulation of Mapk3. These findings suggested that developmental exposure to PS-NP has adverse effects on cognitive function and that THP can alleviate these effects by directly binding to Mapk3.

Analysis of single nuclear chromatin accessibility reveals unique myeloid populations in human pancreatic ductal adenocarcinoma.

A better understanding of the pancreatic ductal adenocarcinoma (PDAC) immune microenvironment is critical to developing new treatments and improving outcomes. Myeloid cells are of particular importance for PDAC progression; however, the presence of heterogenous subsets with different ontogeny and impact, along with some fluidity between them, (infiltrating monocytes vs. tissue-resident macrophages; M1 vs. M2) makes characterisation of myeloid populations challenging. Recent advances in single cell sequencing technology provide tools for characterisation of immune cell infiltrates, and open chromatin provides source and function data for myeloid cells to assist in more comprehensive characterisation. Thus, we explore single nuclear assay for transposase accessible chromatin (ATAC) sequencing (snATAC-Seq), a method to analyse open gene promoters and transcription factor binding, as an important means for discerning the myeloid composition in human PDAC tumours. Frozen pancreatic tissues (benign or PDAC) were prepared for snATAC-Seq using 10× Chromium technology. Signac was used for preliminary analysis, clustering and differentially accessible chromatin region identification. The genes annotated in promoter regions were used for Gene Ontology (GO) enrichment and cell type annotation. Gene signatures were used for survival analysis with The Cancer Genome Atlas (TCGA)-pancreatic adenocarcinoma (PAAD) dataset. Myeloid cell transcription factor activities were higher in tumour than benign pancreatic samples, enabling us to further stratify tumour myeloid populations. Subcluster analysis revealed eight distinct myeloid populations. GO enrichment demonstrated unique functions for myeloid populations, including interleukin-1b signalling (recruited monocytes) and intracellular protein transport (dendritic cells). The identified gene signature for dendritic cells influenced survival (hazard ratio=.63, p=.03) in the TCGA-PAAD dataset, which was unique to PDAC. These data suggest snATAC-Seq as a method for analysis of frozen human pancreatic tissues to distinguish myeloid populations. An improved understanding of myeloid cell heterogeneity and function is important for developing new treatment targets in PDAC.

Variation of gene expression of fatty acid acyl CoA reductase associated with wax secretion of a scale insect, Ericerus pela, and identification of its regulation factors through the accessible chromatin analyses and yeast one-hybrid.

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 107 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI.

Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors.

Hypoxia in solid tumors is an important source of chemoresistance that can determine poor patient prognosis. Such chemoresistance relies on the presence of cancer stem cells (CSCs), and hypoxia promotes their generation through transcriptional activation by HIF transcription factors. We used ovarian cancer (OC) cell lines, xenograft models, OC patient samples, transcriptional databases, induced pluripotent stem cells (iPSCs) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Here, we show that hypoxia induces CSC formation and chemoresistance in ovarian cancer through transcriptional activation of the PLD2 gene. Mechanistically, HIF-1α activates PLD2 transcription through hypoxia response elements, and both hypoxia and PLD2 overexpression lead to increased accessibility around stemness genes, detected by ATAC-seq, at sites bound by AP-1 transcription factors. This in turn provokes a rewiring of stemness genes, including the overexpression of SOX2, SOX9 or NOTCH1. PLD2 overexpression also leads to decreased patient survival, enhanced tumor growth and CSC formation, and increased iPSCs reprograming, confirming its role in dedifferentiation to a stem-like phenotype. Importantly, hypoxia-induced stemness is dependent on PLD2 expression, demonstrating that PLD2 is a major determinant of de-differentiation of ovarian cancer cells to stem-like cells in hypoxic conditions. Finally, we demonstrate that high PLD2 expression increases chemoresistance to cisplatin and carboplatin treatments, both in vitro and in vivo, while its pharmacological inhibition restores sensitivity. Altogether, our work highlights the importance of the HIF-1α-PLD2 axis for CSC generation and chemoresistance in OC and proposes an alternative treatment for patients with high PLD2 expression.