Assay For Transposase-accessible Chromatin Sequencing Research Articles

Chromatin accessibility regulates chemotherapy-induced dormancy and reactivation

Cisplatin-based chemotherapy remains the standard care for non-small cell lung cancer (NSCLC) patients. Relapse after chemotherapy-induced dormancy affects the overall survival of patients. The evolution of cancer cells under chemotherapy stress is regulated by transcription factors (TFs) with binding sites initially buried deep within inaccessible chromatin. The transcription machinery and dynamic epigenetic alterations during the process of dormancy-reactivation of lung cancer cells after chemotherapy need to be investigated. Here, we investigated the chromatin accessibility of lung cancer cells after cisplatin treatment, using an assay for transposase-accessible chromatin sequencing (ATAC-seq). We observed that global chromatin accessibility was extensively improved. Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining (TRRUST) v.2 was used to elucidate TF-target interaction during the process of dormancy and reactivation. Enhancer regions and motifs specific to key TFs including JUN, MYC, SMAD3, E2F1, SP1, CTCF, SMAD4, STAT3, NFKB1, and KLF4 were enriched in differential loci ATAC-seq peaks of dormant and reactivated cancer cells induced by chemotherapy. The findings suggest that these key TFs regulated gene expressions during the process of dormancy and reactivation of cancer cells through altering promoter accessibility of target genes. Our study helps advance understanding of how cancer cells adapt to the stress induced by chemotherapy through TF binding motif accessibility.

PDX Models Recapitulate Genetics and Epigenetics of Pediatric T-Cell Leukemia

Patient-derived xenografts (PDX) emerged as important tools to study leukemia biology and potential therapeutic regimens. Here, we compared cellular, molecular and developmental features of PDX-derived blasts with primary pediatric precursor T-cell acute leukemia (T-ALL) cells. The cells were obtained at the times of initial diagnosis and relapse. We analyzed the samples at the genomic level (whole genome sequencing (WGS), whole exome sequencing (WES), multiplex ligation probe amplification (MLPA), targeted sequencing) and the epigenetic level (DNA methylation, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq)).Altogether 75% of our samples engrafted successfully. Of these 12 were matched pairs of initial diagnosis and relapse as well as corresponding PDX. Of further 12 matched pairs either initial diagnosis (2/12) or relapse (7/12) or neither (3/12) engrafted indicating that relapse samples tend to engraft more successfully (p= 0.1824 (McNemar's test)), which is in agreement with observations in other cancer entities.In 11 out of 12 matched pairs (initial and relapse) of primary and PDX genomic stability of the PDXs was assessed using WGS and WES. Ninety-two % of the mutations with an allele frequencies (AF) >30% were conserved. Of altogether 609 SNVs and InDels (AF>10%) detected in primary samples 68 % (417) were also detected in the corresponding PDXs. In some PDXs, mutations not identified by targeted sequencing in the primary sample (NOTCH1, PHF6, DNA11, USP9X) occured at a high AF (22-59%), which may have been the result of selection of a subclone or of an acquisition of the mutation in the process of PDX generation.Thirty of 35 (86%) large CNA assessed by WGS were preserved in corresponding PDX models indicating that CNAs are likely earlier events in the leukemogenesis that are present at high AF in the primary samples. CNA analysis with MLPA confirmed that PDXs retained 96% (80/83) of the deletions and amplifications of primary leukemia.In 18 out of the matched 22 PDXs all clonal mutations (AF >30%) detected in the primary sample have been preserved in the PDX. Seventeen out of 22 contained more than 1 clone. Fifteen out of these 17 retained the complexity of primary sample with different subclones successfully engrafted.When considering the 11 matched pairs, we observed 4 different engraftment types of initial disease. Type 1 engrafted in a similar way in which they relapsed preserving all or nearly all mutations found in patients' sample (4 samples). Type 2: the relapsing clone that was not detected in initial diagnosis was present in PDX derived of initial diagnosis (PDX_INI) but it was not dominant (3). Type 3: the relapsing clone that was present at initial diagnosis did not engraft into PDX_INI (2). Type 4 preserved the relapsing clone as well as the mutations specific for initial diagnosis (2). Out of 11 PDXs of relapse 9 preserved all or nearly all mutations detected in primary sample. In 2/11 PDXs of relapse one of the clones preexisting in primary relapse was selected for, while the other one was lost.Differential methylation analysis by EPIC array of 21,711 promoters of primary samples and PDXs revealed that the average methylation in primary samples (β=0.43) did not differ from that in PDXs (β=0.45). Principal Component Analysis and unsupervised hierarchical clustering showed that matched pairs, both of the primary and the PDX samples cluster together (ρ=0.9912). Similarly, analysis of chromatin accessibility by ATAC-seq showed that more than 92% of peaks of a height > 20 and more than 75% of the peaks of a height of > 10 were preserved between primary samples and corresponding PDXs.In conclusion, PDX models largely reflect the heterogeneity and the complexity of the primary leukemic sample at the genomic and epigenomic level, although some clonal and particularly subclonal mutations can be lost. Differences in engraftment and various engraftment scenarios of initial diagnosis in contrary to relapse samples suggest that relapse samples are more preselected and stable in engraftment. Altogether, this overall genomic and epigenomic stability of the primary T-ALL in the PDX renders the models as a suitable source for material when primary samples are limiting and also to reduce uncontrollable pre-analytical changes in primary samples. Further, these models are suitable for the preclinical development of new treatment strategies for pediatric T-ALL. DisclosuresSchrappe:Baxalta: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding.

Integrative Oncogenomic Analysis Combining Whole Genome, Transcriptome and Epigenome Identifies Altered Chromatin Accessibility Landscape in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by clinical and genomic heterogeneity. Genomic investigations have identified few recurrently mutated protein-coding genes and various aberrations at the transcriptomic level. However, these changes do not accurately reflect the disease behavior. With accumulation of data in large patient population, it is becoming clear that analysis integrating various genomic correlates will be required to understand chromatin architecture that drives the disease behavior.To gain further insight into MM biology, we integrated whole-genome sequencing, transcriptome sequencing, and epigenome profile using various assays (including the assay for transposase-accessible chromatin sequencing [ATAC-seq] to detect open chromatin regions, multiplexed, ChIP-seq and indexed T7 ChIP-seq [Mint-ChIP] to identify active enhancers and super-enhancers) to characterize the chromatin state of MM. We have analyzed 33 samples including primary MM cells from 18 newly diagnosed and 5 relapsed patients, 5 normal plasma cells from healthy donors and 5 Myeloma cell lines.We first investigated the open chromatin landscape in myeloma cells, identifying over one million open chromatin regions (OCR) in our cohort, with an average of over 60,000 regions per sample. Despite a high heterogeneity between myeloma samples, unsupervised cluster analysis clearly separates primary myeloma samples, normal plasma cells and MM cell lines suggesting pathogenetic relevance. Although there is clear link between transcribed genes and OCR, we observe a significant number of OCR in the intergenic region with an as yet unclear biological significance. Furthermore, ATAC-seq data predicted several copy number abnormalities including the distinction of hyperdiploid from hypodiploid samples. We next characterized enhancers and super-enhancers in myeloma cells and their assigned genes using H3K27ac ChIP-Seq to identify important genes in MM biology. Despite heterogeneity between myeloma samples and myeloma cell lines, we could identify common SE-assigned genes as compared with normal plasma cells. These genes correspond to both known myeloma specific dependencies such as MYC, IRF4, CCND1, and TNSFR17 (BCMA) as well as new therapeutic targets that are currently under investigation.Next, we used WGS data from 10 samples matching with ATAC and RNA sequencing to evaluate the mutational landscape of open chromatin regions and the consequences of the mutational processes targeting myeloma epigenome. Of 53,798 single nucleotide substitutions (SNV) (average of 5380 per sample), 2.7% of SNVs involve OCR (total of 1470 SNV within ATAC peaks, median - 104 mutations involving OCR per sample). We evaluated the presence of mutations within OCR targeting known binding motifs for transcription factors (TFs). Out of 1470 mutations affecting OCR, we identified 547 (37%) SNV targeting a known binding motif for transcription factors such as Sp1, NFkB1 or YY1. Using RNA seq data, we filtered the mutations affecting expressed TFs and assigned genes and observed that the presence of these mutations could significantly impact the assigned gene expression level. Indeed, these mutations generate either a de novo binding motif or alter the binding motif of a given TFs and are potentially responsible for TF hi-jacking to modulate the expression of number of genes important for MM biology. Finally, using non-negative matrix factorization (NMF), we evaluated the mutational signatures affecting the OCR and observed that a single predominant mutational signature affects open chromatin regions, as opposed to other processes such as APOBEC, which targets other regions of the genome.In conclusion, global analysis of primary patient myeloma cells combining whole genome, transcriptome as well as epigenomic characterization reveals dysregulated chromatin landscape in myeloma that may be significantly influenced by specific mutational processes. Such integrated oncogenomic analysis provides an early view on perturbed genomic landscape with a view to understand the biology and specific targetable dependencies. DisclosuresAnderson:C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees.

A unified atlas of CD8 T cell dysfunctional states in cancer and infection

CD8 Tcells play an essential role in defense against viral and bacterial infections and in tumor immunity. Deciphering Tcell loss of functionality is complicated by the conspicuous heterogeneity of CD8 Tcell states described across experimental and clinical settings. By carrying out a unified analysis of over 300 assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) experiments from 12 studies of CD8 Tcells in cancer and infection, we defined a shared differentiation trajectory toward dysfunction and its underlying transcriptional drivers and revealed a universal early bifurcation of functional and dysfunctional Tcell states across models. Experimental dissection of acute and chronic viral infection using single-cell ATAC (scATAC)-seq and allele-specific single-cell RNA (scRNA)-seq identified state-specific drivers and captured the emergence of similar TCF1+ progenitor-like populations at an early branch point, at which functional and dysfunctional Tcells diverge. Our atlas of CD8 Tcell states will facilitate mechanistic studies of Tcell immunity and translational efforts.

Integrative Bulk and Single-Cell Profiling of Premanufacture T-cell Populations Reveals Factors Mediating Long-Term Persistence of CAR T-cell Therapy

The adoptive transfer of chimeric antigen receptor (CAR) T cells represents a breakthrough in clinical oncology, yet both between- and within-patient differences in autologously derived T cells are a major contributor to therapy failure. To interrogate the molecular determinants of clinical CAR T-cell persistence, we extensively characterized the premanufacture T cells of 71 patients with B-cell malignancies on trial to receive anti-CD19 CAR T-cell therapy. We performed RNA-sequencing analysis on sorted T-cell subsets from all 71 patients, followed by paired Cellular Indexing of Transcriptomes and Epitopes (CITE) sequencing and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on T cells from six of these patients. We found that chronic IFN signaling regulated by IRF7 was associated with poor CAR T-cell persistence across T-cell subsets, and that the TCF7 regulon not only associates with the favorable naïve T-cell state, but is maintained in effector T cells among patients with long-term CAR T-cell persistence. These findings provide key insights into the underlying molecular determinants of clinical CAR T-cell function. SIGNIFICANCE: To improve clinical outcomes for CAR T-cell therapy, there is a need to understand the molecular determinants of CAR T-cell persistence. These data represent the largest clinically annotated molecular atlas in CAR T-cell therapy to date, and significantly advance our understanding of the mechanisms underlying therapeutic efficacy.This article is highlighted in the In This Issue feature, p. 2113.

CASB: a concanavalin A‐based sample barcoding strategy for single‐cell sequencing

Sample multiplexing facilitates single‐cell sequencing by reducing costs, revealing subtle difference between similar samples, and identifying artifacts such as cell doublets. However, universal and cost‐effective strategies are rather limited. Here, we reported a concanavalin A‐based sample barcoding strategy (CASB), which could be followed by both single‐cell mRNA and ATAC (assay for transposase‐accessible chromatin) sequencing techniques. The method involves minimal sample processing, thereby preserving intact transcriptomic or epigenomic patterns. We demonstrated its high labeling efficiency, high accuracy in assigning cells/nuclei to samples regardless of cell type and genetic background, and high sensitivity in detecting doublets by three applications: 1) CASB followed by scRNA‐seq to track the transcriptomic dynamics of a cancer cell line perturbed by multiple drugs, which revealed compound‐specific heterogeneous response; 2) CASB together with both snATAC‐seq and scRNA‐seq to illustrate the IFN‐γ‐mediated dynamic changes on epigenome and transcriptome profile, which identified the transcription factor underlying heterogeneous IFN‐γ response; and 3) combinatorial indexing by CASB, which demonstrated its high scalability.

ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma.

More than 60% of supratentorial ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.

SimATAC: a single-cell ATAC-seq simulation framework

Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) identifies regulated chromatin accessibility modules at the single-cell resolution. Robust evaluation is critical to the development of scATAC-seq pipelines, which calls for reproducible datasets for benchmarking. We hereby present the simATAC framework, an R package that generates scATAC-seq count matrices that highly resemble real scATAC-seq datasets in library size, sparsity, and chromatin accessibility signals. simATAC deploys statistical models derived from analyzing 90 real scATAC-seq cell groups. simATAC provides a robust and systematic approach to generate in silico scATAC-seq samples with known cell labels for assessing analytical pipelines.

Venetoclax sensitivity in multiple myeloma is associated with B-cell gene expression

AbstractVenetoclax is a highly potent, selective BCL2 inhibitor capable of inducing apoptosis in cells dependent on BCL2 for survival. Most myeloma is MCL1-dependent; however, a subset of myeloma enriched for translocation t(11;14) is codependent on BCL2 and thus sensitive to venetoclax. The biology underlying this heterogeneity remains poorly understood. We show that knockdown of cyclin D1 does not induce resistance to venetoclax, arguing against a direct role for cyclin D1 in venetoclax sensitivity. To identify other factors contributing to venetoclax response, we studied a panel of 31 myeloma cell lines and 25 patient samples tested for venetoclax sensitivity. In cell lines, we corroborated our previous observation that BIM binding to BCL2 correlates with venetoclax response and further showed that knockout of BIM results in decreased venetoclax sensitivity. RNA-sequencing analysis identified expression of B-cell genes as enriched in venetoclax-sensitive myeloma, although no single gene consistently delineated sensitive and resistant cells. However, a panel of cell surface makers correlated well with ex vivo prediction of venetoclax response in 21 patient samples and may serve as a biomarker independent of t(11;14). Assay for transposase-accessible chromatin sequencing of myeloma cell lines also identified an epigenetic program in venetoclax-sensitive cells that was more similar to B cells than that of venetoclax-resistant cells, as well as enrichment for basic leucine zipper domain–binding motifs such as BATF. Together, these data indicate that remnants of B-cell biology are associated with BCL2 dependency and point to novel biomarkers of venetoclax-sensitive myeloma independent of t(11;14).

Epigenomic features revealed by ATAC‐seq impact transgene expression in CHO cells

Different regions of a mammalian genome have different accessibilities to transcriptional machinery. The integration site of a transgene affects how actively it is transcribed. Highly accessible genomic regions called super-enhancers have beenrecently described as strong regulatory elements that shape cell identity. Super-enhancers have beenidentified in Chinese hamster ovary (CHO) cells using the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq). Genes near super-enhancer regions had high transcript levels and were enriched for oncogenic signaling and proliferation functions, consistent with an immortalized phenotype. Inaccessible regions in the genome with low ATAC signal also had low transcriptional activity. Genes in inaccessible regions were enriched for remote tissue functions such as taste, smell, and neuronal activation. A lentiviral reporter integration assay showed integration into super-enhancer regions conferred higher reporter expression than insertion into inaccessible regions. Targeted integration of an IgG vector into the Plec super-enhancer region yielded clones that expressed the immunoglobulin light chain gene mostly in the top 20% of all transcripts with the majority in the top 5%. The results suggest the epigenomic landscape of CHO cells can guide the selection of integration sites in the development of cell lines for therapeutic protein production.

A single-cell atlas of mouse olfactory bulb chromatin accessibility

Olfaction, the sense of smell, is a fundamental trait crucial to many species. The olfactory bulb (OB) plays pivotal roles in processing and transmitting odor information from the environment to the brain. The cellular heterogeneity of the mouse OB has been studied using single-cell RNA sequencing. However, the epigenetic landscape of the mOB remains mostly unexplored. Herein, we apply single-cell assay for transposase-accessible chromatin sequencing to profile the genome-wide chromatin accessibility of 9,549 single cells from the mOB. Based on single-cell epigenetic signatures, mOB cells are classified into 21 clusters corresponding to 11 cell types. We identify distinct sets of putative regulatory elements specific to each cell cluster from which putative target genes and enriched potential functions are inferred. In addition, the transcription factor motifs enriched in each cell cluster are determined to indicate the developmental fate of each cell lineage. Our study provides a valuable epigenetic data set for the mOB at single-cell resolution, and the results can enhance our understanding of regulatory circuits and the therapeutic capacity of the OB at the single-cell level.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 can arise in the embryonic thymus, from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 migrated to mucosal tissues with colonization of the intestinal lamina propria. RORα expression repressed T cell development, while promoting ILC2 in the thymus. From RNA-seq, ATAC-seq and ChIP-seq data we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 from common progenitors in the thymus. When Notch signaling is present, Bcl11b dampens Nfil3/Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the Nfil3/Id2 repression, allowing Id2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

Downregulated METTL14 accumulates BPTF that reinforces super-enhancers and distal lung metastasis via glycolytic reprogramming in renal cell carcinoma.

Background: Methyltransferase-like 14 (METTL14) participates in tumorigenesis in several malignancies, but how METTL14 mediates the metastasis of renal cell carcinoma (RCC) has never been reported.Methods: Western blotting, quantitative real-time PCR, and immunohistochemistry were used to determine the mRNA and protein levels of relevant genes. Methylated RNA immunoprecipitation sequencing and RNA sequencing were utilized to screen potential targets of METTL14. Chromatin immunoprecipitation sequencing and assay for transposase-accessible chromatin sequencing were performed to investigate epigenetic alterations. The biological roles and mechanisms of METTL14/BPTF in promoting lung metastasis were confirmed in vitro and in vivo using cell lines, patient samples, xenograft models, and organoids.Results: Utilizing the TCGA-KIRC and Ruijin-RCC datasets, we found low expression of METTL14 in mRCC samples, which predicted poor prognosis. METTL14 deficiency promoted RCC metastasis in vitro and in vivo. Mechanistically, METTL14-mediated m6A modification negatively regulated the mRNA stability of bromodomain PHD finger transcription factor (BPTF) and depended on BPTF to drive lung metastasis. Accumulated BPTF in METTL14-deficient cells remodeled the enhancer landscape to reinforce several oncogenic crosstalk. Particularly, BPTF constituted super-enhancers that activate downstream targets like enolase 2 and SRC proto-oncogene nonreceptor tyrosine kinase, leading to glycolytic reprogramming of METTL14-/- cells. Finally, we determined the efficacy of the BPTF inhibitor AU1 in suppressing mRCC of patient-derived cells, mRCC-derived organoids (MDOs), and orthotopic xenograft models.Conclusions: Our study is the first to investigate the essential role of m6A modification and the METTL14/BPTF axis in the epigenetic and metabolic remodeling of mRCC, highlighting AU1 as a vital therapeutic candidate.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis.

SummaryRegulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.

NEUROD1 Intrinsically Initiates Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells.

Cell type specification is a delicate biological event in which every step is under tight regulation. From a molecular point of view, cell fate commitment begins with chromatin alteration, which kickstarts lineage-determining factors to initiate a series of genes required for cell specification. Several important neuronal differentiation factors have been identified from ectopic over-expression studies. However, there is scarce information on which DNA regions are modified during induced pluripotent stem cell (iPSC) to neuronal progenitor cell (NPC) differentiation, the cis regulatory factors that attach to these accessible regions, or the genes that are initially expressed. In this study, we identified the DNA accessible regions of iPSCs and NPCs via the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). We identified which chromatin regions were modified after neuronal differentiation and found that the enhancer regions had more active histone modification changes than the promoters. Through motif enrichment analysis, we found that NEUROD1 controls iPSC differentiation to NPC by binding to the accessible regions of enhancers in cooperation with other factors such as the Hox proteins. Finally, by using Hi-C data, we categorized the genes that directly interacted with the enhancers under the control of NEUROD1 during iPSC to NPC differentiation.

The Transition from Quiescent to Activated States in Human Hematopoietic Stem Cells Is Governed by Dynamic 3D Genome Reorganization.

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy

Allergen-specific immunotherapy is a disease-modifying treatment that induces long-term T-cell tolerance. We sought to evaluate the role of circulating CXCR5+PD-1+ T follicular helper (cTFH) and T follicular regulatory (TFR) cells following grass pollen subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) and the accompanying changes in their chromatin landscape. Phenotype and function of cTFH cells were initially evaluated in the grass pollen-allergic (GPA) group (n= 28) and nonatopic healthy controls (NAC, n= 13) by mathematical algorithms developed to manage high-dimensional data and cell culture, respectively. cTFH and TFR cells were further enumerated in NAC (n= 12), GPA (n= 14), SCIT- (n= 10), and SLIT- (n= 8) treated groups. Chromatin accessibility in cTFH and TFR cells was assessed by assay for transposase-accessible chromatin sequencing (ATAC-seq) to investigate epigenetic mechanisms underlying the differences between NAC, GPA, SCIT, and SLIT groups. cTFH cells were shown to be distinct from TH2- and TH2A-cell subsets, capable of secreting IL-4 and IL-21. Both cytokines synergistically promoted B-cell class switching to IgE and plasma cell differentiation. Grass pollen allergen induced cTFH-cell proliferation in the GPA group but not in the NAC group (P< .05). cTFH cells were higher in the GPA group compared with the NAC group and were lower in the SCIT and SLIT groups (P< .01). Time-dependent induction of IL-4, IL-21, and IL-6 was observed in nasal mucosa following intranasal allergen challenge in the GPA group but not in SCIT and SLIT groups. TFR and IL-10+ cTFH cells were induced in SCIT and SLIT groups (all, P< .01). ATAC-seq analyses revealed differentially accessible chromatin regions in all groups. For the first time, we showed dysregulation of cTFH cells in the GPA group compared to NAC, SCIT, and SLIT groups and induction of TFR and IL-10+ cTFH cells following SCIT and SLIT. Changes in the chromatin landscape were observed following allergen-specific immunotherapy in cTFH and TFR cells.

Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction.

Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. Collagen1α1-GFP (green fluorescent protein)-positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor-β signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.

Abstract LB-263: Pancreatic cancer prognosis is predicted by chromatin accessibility microarray

Abstract Personalized therapy is the future of cancer care. Almost half of the cancer patients do not respond to chemotherapy. For instance, pancreatic ductal adenocarcinoma (PDAC) patients with the limited local disease and with no detectable metastasis typically have their primary tumor surgically resected, but the disease recurs in approximately 50% of cases within 1 year of surgery, in spite of adjuvant chemotherapy. Although gene expression signatures correlating prognosis have been described in PDAC, the therapeutic utility of these signatures has been limited based in part on a large number of genes displaying an altered expression. On the other hand, regulatory regions common to these genes might be amenable to collective epigenetic reprogramming using epigenetic drugs. We interrogated genome-wide chromatin accessibility using Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) on EpCAM+ PDAC malignant cells sorted from a cohort of 54 treatment-naïve resected tumors, in hopes of defining a tumor-intrinsic chromatin signature associated with recurrence. We discovered a signature of 1092 loci that were differentially accessible between recurrent (disease-free survival (DFS) &amp;lt; 1 year) and non-recurrent patients (DFS &amp;gt; 1 year). Through transcription factor (TF) binding motif analysis, we identified candidate TFs whose accessible motifs were differentially associated with recurrence. Nuclear localization of two such TFs, ZKSCAN1 and HNF1b, were assessed by immunostaining on tissue microarrays (TMA) representing 40 out of 54 patients. Nuclear staining of HNF1b was strong in tumor tissue from non-recurrent patients and weak or absent in recurrent patients, but ZKSCAN1 staining patterns were not significantly associated with recurrence. In a TMA representing an independent PDAC cohort (n=97) preselected for 52 long (OS 6 years)- and 45 short (OS 6 months)- term survivors, the number of nuclear positive cells for HNF1b was 52-fold higher in the long-term compared to the short-term survivors and that for ZKSCAN1 was 5.3-fold higher in the short-term compared to the long-term survivors. We further validated the 1092 chromatin accessibility signature by a novel microarray-based platform technology that we termed “ATAC-Array”, where the differentially accessible regions from the signatures were probed on a glass slide and then hybridized with fluorescent-labeled ATAC-libraries. This is a cost-effective, easy-to-use platform technology avoiding the time and cost of next-generation ATAC library sequencing. ATAC-array is the only microarray that reads chromatin accessibility. We have compared ATAC-array side-by-side with ATAC-seq (n=30) and found significant correlation (Pearson's median r= 0.64, range= 0.50- 0.77). By performing ATAC-array on the PDAC cohort (n=38), we have independently re-classified the patients who recurred early and the ones who did not (Gehan-Breslow-Wilcoxon test p=0.0076). ATAC-array, as the technology itself, has enormous potential for a wide range of applications, and we propose to develop it as a clinically validated theragnostic tool to predict and stratify cancer patients for epigenetic therapy. Citation Format: Surajit Dhara, Sagar Chhangawala, Himanshu Chintalapudi, Alexandra L. Massa, Victoria Aveson, Gokce Askan, Liguo Zhang, Remy Nicolle, Alvin P. Makohon-Moore, Smrita Sinha, Jiang Gui, Richard Moffitt, Kenneth H. Yu, Vinod Balachandran, Rohit Chandwani, Christina Leslie, Steven D. Leach. Pancreatic cancer prognosis is predicted by chromatin accessibility microarray [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-263.

Developmental trajectory of prehematopoietic stem cell formation from endothelium

AbstractHematopoietic stem and progenitor cells (HSPCs) in the bone marrow are derived from a small population of hemogenic endothelial (HE) cells located in the major arteries of the mammalian embryo. HE cells undergo an endothelial to hematopoietic cell transition, giving rise to HSPCs that accumulate in intra-arterial clusters (IAC) before colonizing the fetal liver. To examine the cell and molecular transitions between endothelial (E), HE, and IAC cells, and the heterogeneity of HSPCs within IACs, we profiled ∼40 000 cells from the caudal arteries (dorsal aorta, umbilical, vitelline) of 9.5 days post coitus (dpc) to 11.5 dpc mouse embryos by single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing. We identified a continuous developmental trajectory from E to HE to IAC cells, with identifiable intermediate stages. The intermediate stage most proximal to HE, which we term pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD motifs. A developmental bottleneck separates pre-HE from HE, with RUNX1 dosage regulating the efficiency of the pre-HE to HE transition. A distal candidate Runx1 enhancer exhibits high chromatin accessibility specifically in pre-HE cells at the bottleneck, but loses accessibility thereafter. Distinct developmental trajectories within IAC cells result in 2 populations of CD45+ HSPCs; an initial wave of lymphomyeloid-biased progenitors, followed by precursors of hematopoietic stem cells (pre-HSCs). This multiomics single-cell atlas significantly expands our understanding of pre-HSC ontogeny.