Assay For Transposase-accessible Chromatin Research Articles

Single‐nucleus open chromatin accessibility landscape of Pick’s and Alzheimer’s disease

AbstractBackgroundPick’s disease (PiD), a behavioral variant of frontotemporal dementia, is one of the common neurodegenerative dementia that is characterized by tau lesions. Despite the distinct tau aggregation observed in Pick’s disease compared with the tauopathy of Alzheimer’s disease (AD), the similarity in cognitive and behavioral impairments during the progression of the disease make their diagnosis challenging. What determines the differences and similarities between PiD and AD progression, especially at the epigenetic level, are largely unknown. We believe that recent advancement in single‐cell epigenomic profiling methods, Assay for Transposase‐Accessible Chromatin (ATAC) combined with high throughput sequencing, will enable us to map the chromatin‐regulatory landscapes of disease brains at a single‐cell resolution and enable us to understand underlying molecular changes in these diseases.MethodIn the present study, we have isolated single nuclei from the frontal cortex region and performed single‐nucleus ATAC sequencing (snATAC‐seq) of 198,722 nuclei to generate cell‐type specific chromatin accessibility profiles of postmortem brains of PiD and AD patients and to uncover their cellular heterogeneity and similarity. We used cellranger to process the paired‐end FastQ reads of each sample to obtain its chromatin accessibility count matrix. Next, we utilized ArchR and Signac pipelines to generate 501‐bp fixed‐width peak regions and performed downstream differential accessible regions analysis. Quality control was also performed to remove the contribution of low‐quality nuclei.ResultWe constructed chromatin accessibility profiles from 19,690 nuclei from PiD patients, 59,148 nuclei from AD patients, and 119,884 nuclei from healthy controls. We applied UMAP dimensionality reduction and clustered nuclei through open chromatin region to the batch‐corrected epigenomic datasets. We identified seven distinct cell types, including astrocyte, excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, OPC, and pericyte/endothelial cells, and we applied pseudotime trajectory analysis to characterize disease‐associated cell state in both AD and PiD at the epigenomics level.ConclusionOur data identified the cell‐type‐specific open chromatin accessible regions in AD and PiD brains. Although the causative molecular mechanisms of AD and PiD remain unknown, our work helps to identify shared epigenomic changes in AD and PiD, especially in regards to cell‐type‐specific genomic loci with disease risk.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Inflammatory Cytokines That Enhance Antigen Responsiveness of Naïve CD8+ T Lymphocytes Modulate Chromatin Accessibility of Genes Impacted by Antigen Stimulation.

Naïve CD8+ T lymphocytes exposed to certain inflammatory cytokines undergo proliferation and display increased sensitivity to antigens. Such 'cytokine priming' can promote the activation of potentially autoreactive and antitumor CD8+ T cells by weak tissue antigens and tumor antigens. To elucidate the molecular mechanisms of cytokine priming, naïve PMEL-1 TCR transgenic CD8+ T lymphocytes were stimulated with IL-15 and IL-21, and chromatin accessibility was assessed using the assay for transposase-accessible chromatin (ATAC) sequencing. PMEL-1 cells stimulated by the cognate antigenic peptide mgp10025-33 served as controls. Cytokine-primed cells showed a limited number of opening and closing chromatin accessibility peaks compared to antigen-stimulated cells. However, the ATACseq peaks in cytokine-primed cells substantially overlapped with those of antigen-stimulated cells and mapped to several genes implicated in T cell signaling, activation, effector differentiation, negative regulation and exhaustion. Nonetheless, the expression of most of these genes was remarkably different between cytokine-primed and antigen-stimulated cells. In addition, cytokine priming impacted the expression of several genes following antigen stimulation in a synergistic or antagonistic manner. Our findings indicate that chromatin accessibility changes in cytokine-primed naïve CD8+ T cells not only underlie their increased antigen responsiveness but may also enhance their functional fitness by reducing exhaustion without compromising regulatory controls.

Mapping open chromatin by ATAC-seq in bread wheat.

Gene transcription is largely regulated by cis-regulatory elements. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) is an emerging technology that can accurately map cis-regulatory elements in animals and plants. However, the presence of cell walls and chloroplasts in plants hinders the extraction of high-quality nuclei, thereby affects the quality of ATAC-seq data. Meanwhile, it is tricky to perform ATAC-seq with different tissue types, especially for those with limited size and amount. Moreover, with rapid growth of ATAC-seq datasets from plants, powerful and easy-to-use data analysis pipelines for ATAC-seq, especially for wheat is lacking. Here, we provided an all-in-one solution for mapping open chromatin in wheat including both experimental and data analysis procedure. We efficiently obtained nuclei with less cell debris from various wheat tissues. High-quality ATAC-seq data from young spike and ovary, which are hard to harvest were generated. We determined that the saturation sequencing depth of wheat ATAC-seq is about 16 Gb. Particularly, we developed a powerful and easy-to-use online pipeline to analyze the wheat ATAC-seq data and this pipeline can be easily extended to other plant species. The method developed here will facilitate plant regulatory genome study not only for wheat but also for other plant species.

Validation of a Novel Rac1 Inhibitor As Potent Secdrug Against Activating Ras Mutant and Drug-Resistant Multiple Myeloma

Multiple myeloma is the second-most common hematopoietic malignancy in the United States. Although several drugs have so far been approved by the FDA, myeloma still remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs). Furthermore, activating Ras gene (NRAS, KRAS, and HRAS) mutations are known to occur in >50% of myeloma patients, which are also associated with greater tumor burden, cancer aggressiveness, high recurrence rates, and poor survival. We have recently established a novel drug development/re-purposing pipeline called secDrug that incorporates a pharmacogenomics data-driven computational algorithm to introduce several new drug candidates (secDrugs), including EHT1864 (a Rac1 (Ras superfamily GTPase) inhibitor), CCT018159 (Hsp90 inhibitor), and CP466722 (a reversible ATM inhibitor) as synergistic partners of primary drugs against drug-resistant myeloma. The overall objective of this study is to validate EHT1864 using in vitro and ex vivo models of relapsed and refractory myeloma (RRMM) patients. First, we used single-cell RNA sequencing (scRNAseq) as a novel screening tool to demonstrate that EHT1864 is potentially effective against myeloma subclones based on the enrichment of target genes (Figure 1). For in vitro validation, we used a human myeloma cell line (HMCL) panel representing drug-sensitive (FLAM76), innate/refractory resistance (LP1), and acquired/relapsed resistance (parental and clonally derived PI-resistant and IMiD-resistant HMCL pairs U266 P/VR, RPMI P/VR, MM1S P/VR/LenR). FLAM76 KRas (FLAM76-K12) and FLAM76 Nras (FLAM76-N12) cell lines were generated using Adeno-associated viral (AAV) vector-mediated delivery of CRISPR-Cas9 for genome editing in humans. Ras mutations were confirmed using Sanger DNA sequencing method. Cell viability measured using CellTiter-Glo luminescent cell viability assay showed the potency of EHT1864 as a single-agent (IC50 range 15-40µM) as well as in combination with PIs (represented by Ixazomib) and IMiDs (represented by Lenalidomide). Combination index (CI) values calculated using Chou-Talalay's CI theorem were consistently less than 0.9, while all the four synergy scores (HSA, Bliss, Loewe, and ZIP) were higher than 5, demonstrating high synergy. Cell death due to apoptosis was confirmed using flow cytometry (Annexin-V-Propidium Iodide staining), Caspase 3/7 activity assay, and immunoblotting. Interestingly, EHT1864 was most potent against drug-resistant and Ras-mutant HMCLs. Further, we showed that EHT1864 treatment also affects ROS generation and mitochondrial membrane potential, indicating that EHT1864 induces apoptosis via mitochondria-mediated pathway. Finally, we performed pre-vs-post-treatment genome-wide transcriptome analysis (tumor mRNAseq) and single-cell multi-ome analysis (single-cell RNA sequencing/scRNAseq + single-cell Assay of Transposase Accessible Chromatin sequencing/scATACseq) analysis to derive molecular signatures and pathways representing the basis of (on-target and off-target) efficacy of EHT1864 and drug synergy at the bulk tumor and subclonal levels. Finally, we will perform single-cell proteomics (high-dimensional immunophenotyping using mass cytometry/CyTOF/Cytometry time of flight) in CD138+ bone marrow cells derived from newly-diagnosed and relapsed myeloma patients (ex vivo) to demonstrate efficacy in RRMM. Thus, our work lays a framework to establish EHT1864 as a clinical trial-ready therapeutic option for the management of drug-resistant myeloma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genotyping of Multiple Genomic Loci with Chromatin Accessibility Profiling in Single Cells Links Clonal Hierarchy with Epigenetic Variation in Acute Myeloid Leukemia

Acquisition of multiple somatic mutations and epigenetic changes can result in malignant transformation, intra-tumoral heterogeneity, therapy resistance and disease relapse. However, our current knowledge of how cooperating driver genetic and epigenetic events shape gene regulation and chromatin landscapes in patient samples is still limited. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), a novel method for high efficiency capture of open chromatin with parallel amplification of multiple genomic targets. This provides genetic clonal identity and epigenetic state at single-cell resolution (Fig. 1). We first tested this method in leukemia cell lines. GTAC successfully detected 8 genomic loci encoding leukemic variants in 96-100% of single cells. Low allelic dropout rates (1.5% - 11%) allowed high-confidence mutation calling for heterozygous loci in 90-99% of cells. Corresponding scATAC-seq libraries were of high quality, independent of the number of genotyped loci, with >105 unique fragments/cell on average and unperturbed signal-to-noise ratio. 93.75% of cells had simultaneous detection of all 8 genomic targets and efficient scATAC-seq library generation. Finally, a mixing experiment on three cell lines showed that 97%, 96% and 99% of cells had correct mutation calls for the ASXL1, RUNX1 and NRAS loci, respectively. We next applied GTAC to 3520 single cells from a primary human acute myeloid leukemia (AML) sample harbouring mutations in TET2, ASXL1, RUNX1, and two BCOR loci (Fig. 2). Single-cell genotyping successfully assigned clonal state for 96.8% of cells. This showed that TET2, ASXL1, and RUNX1 mutations were acquired sequentially, followed by branched acquisition of two independent clones with BCOR loss-of-function mutations. Integration of clonal structure and chromatin accessibility revealed that wild type cells were mainly found in T/NK cell clusters, while TET2 and TET2/ASXL1 mutant cells were predominant in the hemopoietic stem and multipotent progenitor clusters, which were most likely preleukemic. In contrast, on acquisition of the RUNX1 mutation, cells were entirely distributed within lympho-myeloid progenitor-like compartments, establishing the importance of the RUNX1 mutation in transformation from preleukemia to leukemia. Global transcription factor binding motif analysis confirmed profound chromatin landscape changes between clones. RUNX1 mutant cells were strongly enriched in motifs of pro-proliferative transcription factors and those central to lympho-myeloid differentiation, such as SPI1, CEBPA, BCL11A/B and FOS/JUN. Further clonal evolution occurred within leukemic cells, resulting in a significant enrichment of BCOR mutant clones within the lymphoid-primed multipotent progenitor (LMPP)-like differentiation state (P<0.0001). Interestingly, this cluster was characterized by higher accessibility of stemness-related HOXA9, MECOM, HLF, FOXO, ERG and ETV6 binding motifs coupled with higher CD34 and lower CD38 accessibility. LMPP-like cells also displayed higher accessibility of leukemia stem cell (LSC)-associated genes (Gentles et al., JAMA, 2010). Moreover, BCOR mutant cells were enriched in MEF2 family motifs linked to AML therapy resistance. This, together with our previous data showing LSC function within LMPP-like cells, suggests a bias towards an LSC-like chromatin state upon BCOR loss. Our dataset uniquely allowed us to examine the impact that BCOR loss-of-function exerts on chromatin accessibility specifically within LMPP-like cells by comparing ATAC profiles of BCOR-mutant and wild type cells within this compartment. BCOR mutant LMPP-like cells had increased accessibility of motifs bound by HOXA/B, NANOG, and NF-kB transcription factors, and a more open INPP4B gene, recently associated to LSC maintenance (Woolley et al., BioRxiv, 2022). This suggests that aberrant PRC1.1 activity, caused by BCOR gene loss, may further confer a fitness advantage to BCOR mutant cells in the TET2/ASXL1/RUNX1-mutant context. In summary, GTAC enables detailed single-cell analysis of chromatin landscapes in normal, pre-malignant and fully transformed subclones within complex clonal hierarchies in primary patient cells, revealing how gene regulation is corrupted as clones evolve. GTAC is a widely applicable tool to study malignant and pre-malignant states. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract B061: Identification of cell identity pathways that cooperate with oncogenic Kras in pancreatic cancer initiation by single-cell epigenomic analysis

Abstract Background: The major genetic driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, a probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation in mouse models. With the concomitant loss of transcription factors that regulate acinar cell differentiation, such as Pdx1 (Pancreatic and Duodenal Homeobox 1), acinar cells undergo a rapid cell identity switch, known as acinar-to-ductal metaplasia (ADM). Consequently, KrasG12D;Pdx1f/f (Pdx1 knockout) mice present with massively accelerated tumor formation. How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear. Methods: To elucidate mechanisms responsible for the accelerated cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) from frozen pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis (two and ten weeks after CreERT-induced recombination) and compared to those of KrasG12D and CreERT control animals. Changes in the epigenome were correlated to RNA-seq data. Expression of differentially regulated genes was validated by RNAscope and immunohistochemistry staining. The role of identified target genes was studied in pancreatic cancer cell lines. Results: Single-cell ATAC-seq proved as a very powerful and robust tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. By analyzing tissue of KrasG12D;Pdx1f/f mice at different time points, we captured the complete metaplastic conversion of acinar cells into ADM and different subsets of precancerous lesions. Despite initially appearing phenotypically normal, the chromatin profiles of Pdx1 knockout acinar cells showed a striking similarity to ADM cells. Interestingly, acinar and ADM cells of KrasG12D;Pdx1f/f animals as well as ADM lesions of KrasG12D mice showed elevated accessibility and expression of the Ror2 gene. As a receptor protein tyrosine kinase, Ror2 regulates essential signaling pathways, such as Ras-MAPK signaling. Immunostaining of pancreatic cancer tissues from KrasG12D;p53mut (KPC) mice and human PDAC specimens also revealed Ror2 expression in a subset of neoplasms and cancer cells. Knockdown of Ror2 in pancreatic cancer cell lines with a basal-like subtype significantly decreased cell proliferation and expression of cell cycle regulators, while ectopic overexpression in PDAC lines with a classical differentiation induced a dramatic change in cell identity and massively increased cell proliferation. Conclusions: Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 lead to vast alterations of acinar cell identity and significantly accelerated transformation. We identified de-regulation of the receptor kinase Ror2, which cooperates with KrasG12D to induce and promote pancreatic carcinogenesis and proliferation. Citation Format: Simone Benitz, Ian Loveless, Malak Nasser, Hui-Ju Wen, Daniel Long, Erick Davis, Jacee Moore, Tobias Straub, Ivonne Regel, Filip Bednar, Howard Crawford. Identification of cell identity pathways that cooperate with oncogenic Kras in pancreatic cancer initiation by single-cell epigenomic analysis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B061.

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chromatin Accessibility Landscapes of B-Cell Acute Lymphoblastic Leukemia Identify Extensive Epigenomic Reprogramming and Heterogeneity Among Subtypes

Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients and represents about a quarter of all pediatric malignancies. Past work has characterized B-cell lineage ALL (B-ALL) into molecular subtypes spanning a range of aberrant chromosomal rearrangements and oncogene chimeric fusions driving malignancy. While transcriptional and DNA methylome profiling of these subtypes has been extensively examined, the accompanying chromatin landscape and corresponding gene regulatory repertoire are not well characterized for many B-ALL subtypes. To better profile the B-ALL epigenome and gene regulatory networks we examined chromatin accessibility of 11 distinct molecular subtypes (BCR-ABL1, DUX4/ERG, ETV6-RUNX1, Hyperdiploid, Low hypodiploid, KMT2A-rearranged, Ph-like, PAX5-altered, TCF3-PBX1, ZNF384-rearranged and B-other) using 154 primary patient samples from across the United States with assay for transposase-accessible chromatin and high-throughput sequencing (ATAC-seq). We mapped extensive chromatin reprogramming when comparing B-ALL and normal B-cells that drive oncogenic signaling pathways and inhibit normal B-cell functions. Strikingly, we also uncovered chromatin accessibility sites in B-ALL (42,457 sites) with distinct subtype-specific enrichment. A classification model utilizing subtype distinct loci was able to accurately predict B-ALL subtypes with 87% cross-validation accuracy. Transcription factor (TF) footprint profiling and transcriptomic information further identified candidate subtype-enriched TFs, such as DUX4, ZNF384, HOXA9, MEIS1, GATA3 and RARA, along with subtype distinct gene regulatory networks. In connecting these findings with genetic variation, we identified 9080 inherited DNA sequence variants that act as ATAC-seq chromatin accessibility quantitative trait loci (ATAC-QTLs) and contribute to variability in chromatin accessibility among patient samples. Nearly one-third of ATAC-QTLs map to TF footprints and commonly altered DNA consensus motifs impacting chromatin accessibility include ETS family TFs (PU.1, SPI-B, ELF3, EHF), ZNF family (ZNF263, ZNF460) and CTCF. Collectively, using the largest B-ALL chromatin accessibility dataset to date, our investigation offers a unique window into the gene regulatory landscape that highlights the complexity and heterogeneity of chromatin accessibility among B-ALL molecular subtypes.

OR16-3 Analysis of Testis-Enriched Genes in Estrogen-Dependent Genome Regulation in Breast Cancer

Abstract Emerging studies show that testis-enriched genes play critical roles in several biological processes. The expression of these genes is tightly regulated and limited to immune-privileged organs; however, many of them escape regulation and are aberrantly expressed in several diseases such as cancer. Intriguingly, our genomic analysis revealed that many of them are transcribed from previously considered gene deserts. We also found that a significant number of them are robustly regulated by estrogen and other peptide hormones such as Tumor Necrosis Factor-alpha (TNF alpha). However, their direct role in estrogen-dependent transcriptional regulation and biological outcomes remains unstudied. In this regard, our integrated genomic approach, including RNA with 4-thiouridine (4sU) coupled with RNA-sequencing and CRISPR-based genetic screening, has identified key estrogen-regulated testis-enriched genes that may modulate estrogen-signaling. Through a series of molecular and phenotypic assays, we selected a potential but understudied transcript, which is predominantly localized to the nucleus. Furthermore, in the current study, we have interrogated the functional mechanisms by which selected transcript controls estrogen-dependent gene regulation in estrogen receptor-positive (ER+) breast cancer. We found that the transcript controls estrogen-dependent cell and tumor growth. Additionally, we have manipulated the levels of the transcript in estrogen-receptor-positive (ER+) breast cancer cells employing gain- and loss-of-function studies and performed 4sU-RNA-seq. Interestingly, the transcript modulates the expression of estrogen-driven transcripts that are transcribed from ER-alpha (ERa) binding sites. Mechanistically, the transcript is localized in the nucleus and interacts with chromatin-associated proteins. In addition, our integrated genomic analyses comprising 'Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq)' and 'chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing ChIP-seq' (H3K27ac, H3K27me3) identified a critical role for the transcript in controlling the epigenetic landscape at target gene promoters and enhancers in ER+ breast cancer. Collectively, our data support a functional role for testis-enriched genes in enhancer transcription and chromatin organization. This study sheds light on the molecular functions of testis-enriched genes and presents an opportunity to identify new diagnostic and develop treatment strategies in ER+ breast cancer. S.S.G. is supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT; RR170020). S.S.G. is also supported by a grant from Lizanell and Colbert Coldwell foundation. Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.

Normalization benchmark of ATAC-seq datasets shows the importance of accounting for GC-content effects

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) allows the study of epigenetic regulation of gene expression by assessing chromatin configuration for an entire genome. Despite its popularity, there have been limited studies investigating the analytical challenges related to ATAC-seq data, with most studies leveraging tools developed for bulk transcriptome sequencing. Here, we show that GC-content effects are omnipresent in ATAC-seq datasets. Since the GC-content effects are sample specific, they can bias downstream analyses such as clustering and differential accessibility analysis. We introduce a normalization method based on smooth-quantile normalization within GC-content bins and evaluate it together with 11 different normalization procedures on 8 public ATAC-seq datasets. Accounting for GC-content effects in the normalization is crucial for common downstream ATAC-seq data analyses, improving accuracy and interpretability. Through case studies, we show that exploratory data analysis is essential to guide the choice of an appropriate normalization method for a given dataset.

Neutrophil Nanovesicle Protects against Experimental Autoimmune Encephalomyelitis through Enhancing Myelin Clearance by Microglia.

Timely clearance of myelin debris is the premise of neuroinflammation termination and tissue regeneration in multiple sclerosis (MS). Microglia are the main scavengers of myelin debris in MS lesions, but its phagocytic capability is limited in MS patients. Here, we develop neutrophil-derived nanovesicles (NNVs) to enhance the efficiency of myelin debris clearance in microglia for MS therapy. RNA sequencing (RNAseq) results demonstrate that NNVs treatment ameliorates lesional neuroinflammation of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Consequently, EAE mice exhibit favorable neurological functions and white matter integrity after NNVs treatment. Specifically, NNVs treatment upregulates the expression of nuclear factor E2-related factor 2 (NRF2) in microglia, as revealed by Assay for Transposase Accessible Chromatin using sequencing (ATACseq). We also demonstrate that NRF2 can activate the transcription of RUBCN (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein), which in turn enhances LC3-associated phagocytosis (LAP) in microglia. As a result, myelin debris engulfed by microglia can be efficiently catabolized in NNVs-treated EAE mice without obvious side effects. Together, this study proves that NNVs can modulate neuroinflammation by clearing myelin debris and is a promising MS treatment strategy.

G9a Modulates Lipid Metabolism in CD4 T Cells to Regulate Intestinal Inflammation

Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. Human naive T cells were isolated from peripheral blood and differentiated invitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The invivo role of G9a was assessed using 3 murine models. We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) invitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion invivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development invitro and invivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

Telomere-to-telomere genome assembly of bitter melon (Momordica charantia L. var. abbreviata Ser.) reveals fruit development, composition and ripening genetic characteristics.

Momordica charantia L. var. abbreviata Ser. (Mca), known as bitter gourd or bitter melon, is a Momordica variety with medicinal value and belongs to the Cucurbitaceae family. In view of the lack of genomic information on bitter gourd and other Momordica species and to promote Mca genomic research, we assembled a 295.6-Mb telomere-to-telomere (T2T) high-quality Mca genome with six gap-free chromosomes after Hi-C correction. This genome is anchored to 11 chromosomes, which is consistent with the karyotype information, and comprises 98 contigs (N50 of 25.4Mb) and 95 scaffolds (N50 of 25.4Mb). The Mca genome harbors 19 895 protein-coding genes, of which 45.59% constitute predicted repeat sequences. Synteny analysis revealed variations involved in fruit quality during the divergence of bitter gourd. In addition, assay for transposase-accessible chromatin by high-throughput sequencing and metabolic analysis showed that momordicosides and other substances are characteristic of Mca fruit pulp. A combined transcriptomic and metabolomic analysis revealed the mechanisms of pigment accumulation and cucurbitacin biosynthesis in Mca fruit peels, providing fundamental molecular information for further research on Mca fruit ripening. This report provides a new genetic resource for Momordica genomic studies and contributes additional insights into Cucurbitaceae phylogeny.

Integration of ATAC-seq and RNA-seq analysis identifies key genes affecting intramuscular fat content in pigs.

Meat quality is one of the most important economic traits in pig breeding and production, and intramuscular fat (IMF) content is the major factor in improving meat quality. The IMF deposition in pigs is influenced by transcriptional regulation, which is dependent on chromatin accessibility. However, how chromatin accessibility plays a regulatory role in IMF deposition in pigs has not been reported. Xidu black is a composite pig breed with excellent meat quality, which is an ideal research object of this study. In this study, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analysis to identify the accessible chromatin regions and key genes affecting IMF content in Xidu black pig breed with extremely high and low IMF content. First, we identified 21,960 differential accessible chromatin peaks and 297 differentially expressed genes. The motif analysis of differential peaks revealed several potential cis-regulatory elements containing binding sites for transcription factors with potential roles in fat deposition, including Mef2c, CEBP, Fra1, and AP-1. Then, by integrating the ATAC-seq and RNA-seq analysis results, we found 47 genes in the extremely high IMF (IMF_H) group compared with the extremely low IMF (IMF_L) group. For these genes, we observed a significant positive correlation between the differential gene expression and differential ATAC-seq signal (r2 = 0.42). This suggests a causative relationship between chromatin remodeling and the resulting gene expression. We identified several candidate genes (PVALB, THRSP, HOXA9, EEPD1, HOXA10, and PDE4B) that might be associated with fat deposition. Through the PPI analysis, we found that PVALB gene was the top hub gene. In addition, some pathways that might regulate fat cell differentiation and lipid metabolism, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and calcium signaling pathway, were significantly enriched in the ATAC-seq and RNA-seq analysis. To the best of our knowledge, our study is the first to use ATAC-seq and RNA-seq to examine the mechanism of IMF deposition from a new perspective. Our results provide valuable information for understanding the regulation mechanism of IMF deposition and an important foundation for improving the quality of pork.

Genome-Wide Mapping of Active Regulatory Elements Using ATAC-seq.

Active cis-regulatory elements (cREs) in eukaryotes are characterized by nucleosomal depletion and, accordingly, higher accessibility. This property has turned out to be immensely useful for identifying cREs genome-wide and tracking their dynamics across different cellular states and is the basis of numerous methods taking advantage of the preferential enzymatic cleavage/labeling of accessible DNA. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has emerged as the most versatile and widely adaptable method and has been widely adopted as the standard tool for mapping open chromatin regions. Here, we discuss the current optimal practices and important considerations for carrying out ATAC-seq experiments, primarily in the context of mammalian systems.

Single-Cell Chromatin Accessibility Data Combined with GWAS Improves Detection of Relevant Cell Types in 59 Complex Phenotypes.

Several disease risk variants reside on non-coding regions of DNA, particularly in open chromatin regions of specific cell types. Identifying the cell types relevant to complex traits through the integration of chromatin accessibility data and genome-wide association studies (GWAS) data can help to elucidate the mechanisms of these traits. In this study, we created a collection of associations between the combinations of chromatin accessibility data (bulk and single-cell) with an array of 201 complex phenotypes. We integrated the GWAS data of these 201 phenotypes with bulk chromatin accessibility data from 137 cell types measured by DNase-I hypersensitive sequencing and found significant results (FDR adjusted p-value ≤ 0.05) for at least one cell type in 21 complex phenotypes, such as atopic dermatitis, Graves’ disease, and body mass index. With the integration of single-cell chromatin accessibility data measured by an assay for transposase-accessible chromatin with high-throughput sequencing (scATAC-seq), taken from 111 adult and 111 fetal cell types, the resolution of association was magnified, enabling the identification of further cell types. This resulted in the identification of significant correlations (FDR adjusted p-value ≤ 0.05) between 15 categories of single-cell subtypes and 59 phenotypes ranging from autoimmune diseases like Graves’ disease to cardiovascular traits like diastolic/systolic blood pressure.

Highly sensitive single-cell chromatin accessibility assay and transcriptome coassay with METATAC

Recent advances in single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) and its coassays have transformed the field of single-cell epigenomics and transcriptomics. However, the low detection efficiency of current methods has limited our understanding of the true complexity of chromatin accessibility and its relationship with gene expression in single cells. Here, we report a high-sensitivity scATAC-seq method, termed multiplexed end-tagging amplification of transposase accessible chromatin (METATAC), which detects a large number of accessible sites per cell and is compatible with automation. Our high detectability and statistical framework allowed precise linking of enhancers to promoters without merging single cells. We systematically investigated allele-specific accessibility in the mouse cerebral cortex, revealing allele-specific accessibility of promotors of certain imprinted genes but biallelic accessibility of their enhancers. Finally, we combined METATAC with our high-sensitivity single-cell RNA sequencing (scRNA-seq) method, multiple annealing and looping based amplification cycles for digital transcriptomics (MALBAC-DT), to develop a joint ATAC-RNA assay, termed METATAC and MALBAC-DT coassay by sequencing (M2C-seq). M2C-seq achieved significant improvements for both ATAC and RNA compared with previous methods, with consistent performance across cell lines and early mouse embryos.

Dynamics of transcriptome and chromatin accessibility revealed sequential regulation of potential transcription factors during the brown adipose tissue whitening in rabbits.

Brown adipose tissue (BAT) represents a valuable target for treating obesity in humans. BAT losses of thermogenic capacity and gains a “white adipose tissue-like (WAT-like)” phenotype (BAT whitening) under thermoneutral environments, which could lead to potential low therapy responsiveness in BAT-based obesity treatments. However, the epigenetic mechanisms of BAT whitening remain largely unknown. In this study, BATs were collected from rabbits at day0 (D0), D15, D85, and 2 years (Y2). RNA-sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed to investigate transcriptome and chromatin accessibility of BATs at the four whitening stages, respectively. Our data showed that many genes and chromatin accessible regions (refer to as “peaks”) were identified as significantly changed during BAT whitening in rabbits. The BAT-selective genes downregulated while WAT-selective genes upregulated from D0 to Y2, and the de novo lipogenesis-related genes reached the highest expression levels at D85. Both the highly expressed genes and accessible regions in Y2 were significantly enriched in immune response-related signal pathways. Analysis of different relationships between peaks and their nearby genes found an increased proportion of the synchronous changes between chromatin accessibility and gene expression during BAT whitening. The synergistic changes between the chromatin accessibility of promoter and the gene expression were found in the key adipose genes. The upregulated genes which contained increased peaks were significantly enriched in the PI3K-Akt signaling pathway, steroid biosynthesis, TGF-beta signaling pathway, osteoclast differentiation, and dilated cardiomyopathy. Moreover, the footprinting analysis suggested that sequential regulation of potential transcription factors (TFs) mediated the loss of thermogenic phenotype and the gain of a WAT-like phenotype of BAT. In conclusion, our study provided the transcriptional and epigenetic frameworks for understanding BAT whitening in rabbits for the first time and might facilitate potential insights into BAT-based obesity treatments.

Integrating ATAC-seq and RNA-seq Reveals the Dynamics of Chromatin Accessibility and Gene Expression in Apple Response to Drought.

Drought resistance in plants is influenced by multiple signaling pathways that involve various transcription factors, many target genes, and multiple types of epigenetic modifications. Studies on epigenetic modifications of drought focus on DNA methylation and histone modifications, with fewer on chromatin remodeling. Changes in chromatin accessibility can play an important role in abiotic stress in plants by affecting RNA polymerase binding and various regulatory factors. However, the changes in chromatin accessibility during drought in apples are not well understood. In this study, the landscape of chromatin accessibility associated with the gene expression of apple (GL3) under drought conditions was analyzed by Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq. Differential analysis between drought treatment and control identified 23,466 peaks of upregulated chromatin accessibility and 2447 peaks of downregulated accessibility. The drought-induced chromatin accessibility changed genes were mainly enriched in metabolism, stimulus, and binding pathways. By combining results from differential analysis of RNA-seq and ATAC-seq, we identified 240 genes with higher chromatin accessibility and increased gene expression under drought conditions that may play important functions in the drought response process. Among them, a total of nine transcription factor genes were identified, including ATHB7, HAT5, and WRKY26. These transcription factor genes are differentially expressed with different chromatin accessibility motif binding loci that may participate in apple response to drought by regulating downstream genes. Our study provides a reference for chromatin accessibility under drought stress in apples and the results will facilitate subsequent studies on chromatin remodelers and transcription factors.