Chromatin Accessibility Patterns Research Articles

ScCancerExplorer: a comprehensive database for interactively exploring single-cell multi-omics data of human pan-cancer.

Genomic, epigenomicand transcriptomic alterations are hallmarks of cancer cells, and are closely connected. Especially, epigenetic regulation plays a critical role in tumorigenesis and progression. The growing single-cell epigenome data in cancer research provide new opportunities for data mining from a more comprehensive perspective. However, there is still a lack of databases designed for interactively exploring the single-cell multi-omics data of human pan-cancer, especially for the single-cell epigenome data. To fill in the gap, we developed scCancerExplorer, a comprehensive and user-friendly database to facilitate the exploration of the single-cell genome, epigenome (chromatin accessibility and DNA methylation), and transcriptome data of 50 cancer types. Five major modules were provided to explore those data interactively, including 'Integrated multi-omics analysis', 'Single-cell transcriptome', 'Single-cell epigenome', 'Single-cell genome' and 'TCGA analysis'. By simple clicking, users can easily investigate gene expression features, chromatin accessibility patterns, transcription factor activities, DNA methylation states, copy number variationsand TCGA survival analysis results. Taken together, scCancerExplorer is distinguished from previous databases with rich and interactive functions for exploring the single-cell multi-omics data of human pan-cancer. It bridges the gap between single-cell multi-omics data and the end-users, and will facilitate progress in the field of cancer research. scCancerExplorer is freely accessible via https://bianlab.cn/scCancerExplorer.

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

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

Chronic interferon-stimulated gene transcription promotes oncogene-induced breast cancer.

The MRE11 complex (comprising MRE11, RAD50, and NBS1) is integral to the maintenance of genome stability. We previously showed that a hypomorphic Mre11 mutant mouse strain (Mre11 ATLD1/ATLD1 ) was highly susceptible to oncogene-induced breast cancer. Here we used a mammary organoid system to examine which MRE11-dependent responses are tumor-suppressive. We found that Mre11 ATLD1/ATLD1 organoids exhibited an elevated interferon-stimulated gene (ISG) signature and sustained changes in chromatin accessibility. This Mre11 ATLD1/ATLD1 phenotype depended on DNA binding of a nuclear innate immune sensor, IFI205. Ablation of Ifi205 in Mre11 ATLD1/ATLD1 organoids restored baseline and oncogene-induced chromatin accessibility patterns to those observed in WT. Implantation of Mre11 ATLD1/ATLD1 organoids and activation of the oncogene led to aggressive metastatic breast cancer. This outcome was reversed in implanted Ifi205 -/- Mre11 ATLD1/ATLD1 organoids. These data reveal a connection between innate immune signaling and tumor development in the mammary epithelium. Given the abundance of aberrant DNA structures that arise in the context of genome instability syndromes, the data further suggest that cancer predisposition in those contexts may be partially attributable to chronic innate immune transcriptional programs.

Abstract C089: Characterization of the breast tumor immune microenvironment in women of African descent using single-nucleus RNA- and ATAC-sequencing

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts, relative to EA). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. CD45+, EPCAM- cells were further extracted, clustered, and manually annotated for subpopulations based on known marker genes. We detected 26 distinct immune subpopulations across our samples, including 2 inflammatory macrophage, 6 immunosuppressive macrophage, 1 fibronectin+/thrombospondin+ monocyte, 6 T-cell, 1 natural killer cell, 2 B-cell, 1 mast cell, and 4 dendritic cell subpopulations. Differential gene expression analyses of each immune cell subpopulation, adjusted for age, BMI, and tumor subtype, revealed ancestry-specific gene expression patterns, which could provide novel insights into functional differences that may impact tumor immune response. Current efforts focus on in-depth molecular characterization of ancestry-related differences in the tumor immune environment and distinct signatures present in lethal disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany D. Jenkins, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Characterization of the breast tumor immune microenvironment in women of African descent using single-nucleus RNA- and ATAC-sequencing [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C089.

Abstract C056: Characterization of chromatin accessibility patterns in the human pancreas and early pancreatic neoplastic lesions using snATAC-seq

Abstract Study of early pancreas neoplasia in humans had previously been limited by lack of available tissue. Recent work by our group on deceased donor pancreata identified pancreatic intraepithelial neoplasia (PanIN) lesions in non-diseased organs and defined a transcriptomic signature for the microenvironment of these pre-cancerous lesions. Prevalence of PanINs in healthy human tissue was higher than expected and established a novel model to expand understanding of the complex biology of these precursor lesions. Epigenetic changes in chromatin structure and the subsequent effects on gene expression are essential processes in neoplasia that have yet to be described at the single cell level in the human pancreas. We hypothesized that previously defined gene signatures could identify acinar, ductal, and PanIN cells in donor pancreata based on chromatin accessibility profiling by single nuclear transposase-accessible chromatin preparation followed by high-throughput sequencing (snATAC-seq). Comparison between the accessibility patterns, transcription factor motifs, and differences in accessibility versus expression profiles can then be used to expand prior knowledge of early pancreatic neoplasia and identify novel targets for further study and therapy selection. To test this hypothesis, single nuclei from seventeen donor pancreata (twenty total samples) were isolated for snATAC-seq using the 10x Genomics platform. Data analysis was conducted using CellRanger, Seurat, Signac, AUCell, and other programs to identify and label chromatin peaks in individual nuclei corresponding to acinar, ductal, and PanIN origin according to gene signatures identified by scRNA-seq and special transcriptomics on matched donor pancreata. Using this method, cells corresponding to these gene signatures, including PanIN- like cells, were indeed identifiable with differentially accessible regions of the genome then analyzed for transcription factor motif enrichment between these cell types. Motifs enriched in cells identified as PanINs compared to either ductal or acinar cells included several transcription factors implicated in tumorigenesis. These results are similar to previously demonstrated essential factors in pancreatic neoplasia, suggesting this strategy is a relevant method for data analysis and discovery of novel factors implicated in tumorigenesis. Ongoing studies include validation of these findings in vitro using human pancreatic normal epithelial cells, human tumor cell lines, and organoid models derived from human normal and tumor samples. Together, this study demonstrates that PanINs can be identified in human pancreas tissue by snATAC-seq and that differentially expressed motifs can be identified, providing opportunity for further integration with transcriptomic information to elucidate detailed understanding of early neoplasia in the pancreas for future drug development and targeted therapeutics studies. Citation Format: Jamie N Mills, Aaron Dendekker, Joyce K Thompson, Hannah Watkoske, Simone Benitz, Padma Kadiyala, Ahmed Elhossiny, Howard Crawford, Eileen Carpenter, Marina Pasca di Magliano, Filip Bednar. Characterization of chromatin accessibility patterns in the human pancreas and early pancreatic neoplastic lesions using snATAC-seq [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C056.

Loss of cold tolerance is conferred by absence of the WRKY34 promoter fragment during tomato evolution

Natural evolution has resulted in reduced cold tolerance in cultivated tomato (Solanum lycopersicum). Herein, we perform a combined analysis of ATAC-Seq and RNA-Seq in cold-sensitive cultivated tomato and cold-tolerant wild tomato (S. habrochaites). We identify that WRKY34 has the most significant association with differential chromatin accessibility and expression patterns under cold stress. We find that a 60 bp InDel in the WRKY34 promoter causes differences in its transcription and cold tolerance among 376 tomato accessions. This 60 bp fragment contains a GATA cis-regulatory element that binds to SWIBs and GATA29, which synergistically suppress WRKY34 expression under cold stress. Moreover, WRKY34 interferes with the CBF cold response pathway through regulating transcription and protein levels. Our findings emphasize the importance of polymorphisms in cis-regulatory regions and their effects on chromatin structure and gene expression during crop evolution.

A single-cell chromatin accessibility dataset of human primed and naïve pluripotent stem cell-derived teratoma

Teratoma, due to its remarkable ability to differentiate into multiple cell lineages, is a valuable model for studying human embryonic development. The similarity of the gene expression and chromatin accessibility patterns in these cells to those observed in vivo further underscores its potential as a research tool. Notably, teratomas derived from human naïve (pre-implantation epiblast-like) pluripotent stem cells (PSCs) have larger embryonic cell diversity and contain extraembryonic lineages, making them more suitable to study developmental processes. However, the cell type-specific epigenetic profiles of naïve PSC teratomas have not been yet characterized. Using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we analyzed 66,384 cell profiles from five teratomas derived from human naïve PSCs and their post-implantation epiblast-like (primed) counterparts. We observed 17 distinct cell types from both embryonic and extraembryonic lineages, resembling the corresponding cell types in human fetal tissues. Additionally, we identified key transcription factors specific to different cell types. Our dataset provides a resource for investigating gene regulatory programs in a relevant model of human embryonic development.

IRF8 configures enhancer landscape in postnatal microglia and directs microglia specific transcriptional programs.

Microglia are innate immune cells in the brain. Transcription factor IRF8 is highly expressed in microglia. However, its role in postnatal microglia development is unknown. We demonstrate that IRF8 binds stepwise to enhancer regions of postnatal microglia along with Sall1 and PU.1, reaching a maximum after day 14. IRF8 binding correlated with a stepwise increase in chromatin accessibility, which preceded the initiation of microglia-specific transcriptome. Constitutive and postnatal Irf8 deletion led to a loss of microglia identity and gain of disease-associated microglia-like genes. Combined analysis of scRNA-seq and scATAC-seq revealed a correlation between chromatin accessibility and transcriptome at a single-cell level. IRF8 was also required for microglia-specific DNA methylation patterns. Lastly, in the 5xFAD model, constitutive and postnatal Irf8 deletion reduced the interaction of microglia with Aβ plaques and the size of plaques, lessening neuronal loss. Together, IRF8 sets the epigenetic landscape, which is required for postnatal microglia gene expression.

Emerging Approaches to Profile Accessible Chromatin from Formalin-Fixed Paraffin-Embedded Sections.

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurementallowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Abstract 6883: Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics

Abstract Background: Pediatric sarcoma represents the second most common solid tumor in children. Despite advances in multimodal therapies, one-third of sarcoma patients do not survive the disease. Over the past few decades, the development of novel therapeutic strategies for sarcoma has been impeded by an insufficient understanding of the tumor microenvironment (TME). In particular, the potential of immunotherapy remains constrained by the gap of knowledge about the molecular and functional phenotypes of the immune cells in the sarcoma TME. Methods: To address this knowledge gap, we collected treatment-naïve tumor samples from 32 patients with five pediatric sarcoma subtypes. We employed a single-nuclei RNA+ATAC-Seq multi-omics assay to comprehensively characterize the transcriptomic and epigenomic heterogeneity of tumor and immune cells within the sarcoma TME. Results: Our analysis revealed that tumor-associated macrophages (TAMs) were the most abundant immune cell type in the sarcoma TME. We identified seven distinct TAM subpopulations, each exhibiting unique gene expression and chromatin accessibility patterns. Among these, TAM subsets with higher expression of SPP1 and C1QC genes displayed the strongest immunosuppressive potential. Transcriptional regulatory network (TRN) analysis identified transcription factors that were most likely to drive the phenotypes of SPP1+ and C1QC+ TAMs, including ELF2, JUND and RUNX1. Through cell-cell communication analysis, we discovered that SPP1+ TAMs interact with T cells via SPP1-CD44 signaling, while C1QC+ TAMs interact with NK cells through HLA-E-KLRC1 signaling, suggesting the potential involvement of these signaling pathways in TAM-mediated suppression of these cytotoxic lymphocytes. We prioritized ligand-receptor gene pairs, such as SEMA3D-NRP1 and LTBP3-ITGB5, which likely mediate the interaction between malignant sarcoma cells and SPP1+/C1QC+ TAMs based on their dominance across sarcoma subtypes and association with poorer clinical outcomes. On the other hand, our analysis revealed that sarcoma subtype-specific interactions between TAMs and malignant cells are common, highlighting the heterogeneity of signaling events across sarcoma subtypes. Conclusion and Significance: Our study presents a comprehensive single-cell multi-omics characterization of pediatric sarcomas and sheds light on its tumor microenvironment. By identifying unique markers for immunosuppressive TAM subpopulations and elucidating intercellular interactions promoting tumor development, this study proposes potential therapeutic targets for sarcoma treatment based on the role of TAMs in sarcoma TME. Citation Format: Zhan Zhang, Kyung Jin Ahn, Changya Chen, Rumeysa Biyik Sit, Hesham Mohei, Brian Lockhart, Malay Haldar, Kathrin Bernt, Vinodh Pillai, Kai Tan. Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics [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 6883.

Abstract 6108: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. Within cancerous cells, striking intra- and inter-tumoral heterogeneity was observed by genetic ancestry even within molecular subtype groups. Current efforts focus on in-depth molecular characterization of ancestry- and risk factor-related differences in the tumor epithelium and microenvironment and distinct signatures present in lethal disease. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [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 6108.

Trisomy 21-driven metabolite alterations are linked to cellular injuries in Down syndrome

Down syndrome (DS) arises from a genetic anomaly characterized by an extra copy of chromosome 21 (exCh21). Despite high incidence of congenital diseases among DS patients, direct impacts of exCh21 remain elusive. Here, we established a robust DS model harnessing human-induced pluripotent stem cells (hiPSCs) from mosaic DS patient. These hiPSC lines encompassed both those with standard karyotype and those carrying an extra copy of exCh21, allowing to generate isogenic cell lines with a consistent genetic background. We unraveled that exCh21 inflicted disruption upon the cellular transcriptome, ushering in alterations in metabolic processes and triggering DNA damage. The impact of exCh21 was also manifested in profound modifications in chromatin accessibility patterns. Moreover, we identified two signature metabolites, 5-oxo-ETE and Calcitriol, whose biosynthesis is affected by exCh21. Notably, supplementation with 5-oxo-ETE promoted DNA damage, in stark contrast to the protective effect elicited by Calcitriol against such damage. We also found that exCh21 disrupted cardiogenesis, and that this impairment could be mitigated through supplementation with Calcitriol. Specifically, the deleterious effects of 5-oxo-ETE unfolded in the form of DNA damage induction and the repression of cardiogenesis. On the other hand, Calcitriol emerged as a potent activator of its nuclear receptor VDR, fostering amplified binding to chromatin and subsequent facilitation of gene transcription. Our findings provide a comprehensive understanding of exCh21’s metabolic implications within the context of Down syndrome, offering potential avenues for therapeutic interventions for Down syndrome treatment.

Identification of lineage-specific epigenetic regulators FOXA1 and GRHL2 through chromatin accessibility profiling in breast cancer cell lines.

Breast cancer is a heterogeneous disease, and breast cancer cell lines are invaluable for studying this heterogeneity. However, the epigenetic diversity across these cell lines remains poorly understood. In this study, we performed genome-wide chromatin accessibility analysis on 23 breast cancer cell lines, including 2 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative (ER+/HER2-), 3 ER+/HER2+, 3 HER2+, and 15 triple-negative breast cancer (TNBC) lines. These cell lines were classified into three groups based on their chromatin accessibility: the receptor-positive group (Group-P), TNBC basal group (Group-B), and TNBC mesenchymal group (Group-M). Motif enrichment analysis revealed that only Group-P exhibited coenrichment of forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2) motifs, whereas Group-B was characterized by the presence of the GRHL2 motif without FOXA1. Notably, Group-M did not show enrichment of either FOXA1 or GRHL2 motifs. Furthermore, gene ontology analysis suggested that group-specific accessible regions were associated with their unique lineage characteristics. To investigate the epigenetic landscape regulatory roles of FOXA1 and GRHL2, we performed knockdown experiments targeting FOXA1 and GRHL2, followed by assay for transposase-accessible chromatin sequencing analysis. The findings revealed that FOXA1 maintains Group-P-specific regions while suppressing Group-B-specific regions in Group-P cells. In contrast, GRHL2 preserves commonly accessible regions shared between Group-P and Group-B in Group-B cells, suggesting that FOXA1 and GRHL2 play a pivotal role in preserving distinct chromatin accessibility patterns for each group. Specifically, FOXA1 distinguishes between receptor-positive and TNBC cell lines, whereas GRHL2 distinguishes between basal-like and mesenchymal subtypes in TNBC lines.

Chromatin accessibility and epigenetic deoxyribose nucleic acid (DNA) modifications in chronic kidney disease (CKD) osteoblasts: a study of bone and osteoblasts from pediatric patients with CKD.

Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and MethylationEpic in primary osteoblasts from nine adolescent and young adult dialysis patients with end-stage kidney disease and three healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from the iliac crest and evaluated by proteomics. Gene set enrichment analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of nuclear factor of activated T cells (NFAT) signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. Gene set enrichment analysis identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified 13 potential ligand-receptor pairs. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix, which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.

10064-GGE-6 A MULTI-OMIC LANDSCAPE OF GLIOBLASTOMA, IDH-WILD TYPE

Abstract Glioblastoma (GBM) is the most common and highly resistant malignant brain tumor. Although the previous large-scale genomic analyses identified numerous driver genes, limited progress has been achieved in the development of novel treatments. To obtain further insights into the molecular mechanisms underlying the development of GBM, an integrated analysis including epigenetic and transcriptomic analyses, known to regulate malignant tumor progression, is imperative. We analyzed 289 whole-genome sequencing (WGS) including 159 unpublished deep WGS (≥ ×120 coverage) along with RNA-seq, DNA methylation array, whole-genome bisulfite sequencing, and assay for transposase-accessible chromatin with sequencing (ATAC-seq).Mutational analysis identified known driver alterations exhibiting inter-tumoral heterogeneity. Deep WGS enables us to delineate a fine view of clonal architecture demonstrating distinct mutational signatures between clonal and subclonal mutations, suggesting different mutational processes contribute to GBM pathogenesis depending on the developmental stage. Genetic alterations are strongly associated with gene expression subtypes and DNA methylation patterns. Transcriptional deconvolution analysis reveals the heterogeneous proportion of differentiated and stem-like cell states among cases. 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. Genome-wide chromatin accessibility patterns are well associated with expression subtypes of GBM. Motif enrichment analysis of open chromatin sites identified specific transcription factor binding sites, such as the SOX10 motif in the proneural subtype, known to regulate cell states, and the CREB motif in the mesenchymal and classical subtypes, which promote cell proliferation and angiogenesis through TGF-beta regulation. These findings support a model in which the difference in chromatin structure also regulates the progression of GBM.Our analysis encompassing multilayer molecular mechanisms reveals that GBM evolves through harboring genetic alterations and epigenetic modifications depending on the tumor initiation stage and cellular differentiation status.

Mapping enhancer and chromatin accessibility landscapes charts the regulatory network of Alzheimer's disease

BackgroundEnhancers are regulatory elements that target and modulate gene expression and play a role in human health and disease. However, the roles of enhancer regulatory circuit abnormalities driven by epigenetic alterations in Alzheimer's disease (AD) are unclear. MethodsIn this study, a multiomic integrative analysis was performed to map enhancer and chromatin accessibility landscapes and identify regulatory network abnormalities in AD. We identified differentially methylated enhancers and constructed regulatory networks across brain regions using AD brain tissue samples. Through the integration of snATAC-seq and snRNA-seq datasets, we mapped enhancers with DNA methylation alterations (DMA) and chromatin accessibility landscapes. Core regulatory triplets that contributed to AD neuropathology in specific cell types were further prioritized. ResultsWe revealed widespread DNA methylation alterations (DMA) in the enhancers of AD patients across different brain regions. In addition, the genome-wide transcription factor (TF) binding profiles showed that enhancers with DMA are pervasively regulated by TFs. The TF-enhancer-gene regulatory network analysis identified core regulatory triplets that are associated with brain and immune cell proportions and play important roles in AD pathogenesis. Enhancer regulatory circuits with DMA exhibited distinct chromatin accessibility patterns, which were further characterized at single-cell resolutions. ConclusionsOur study comprehensively investigated DNA methylation-mediated regulatory circuit abnormalities and provided novel insights into the potential pathogenesis of AD.

Abstract C044: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=32), Kenyan (n=18), and European American (EA, n=20) women in relation to ancestry and risk factor exposure. In an initial pilot in 10 Kenyan women, we successfully isolated intact, high-quality single nuclei from frozen breast tumor tissue through an optimized combination of enzymatic digestion and an automated tissue homogenizer. We performed multiome sequencing on a total of 36525 nuclei across these 10 pilot tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Seurat-v4 for snRNA; Harmony for snATAC), we characterized tumor, stromal, and immune cell populations (totaling 10 distinct cell types). We observed striking intra- and inter-tumoral heterogeneity across Kenyan women, with increased abundance of immune subpopulations (myeloid, T-cells, B-cells) in triple-negative subtypes. After demonstrating technical feasibility and success in this sample subset, we are now performing integration, cell type annotation, and downstream analyses in our larger cohort to characterize differences in both tumor biology and the tumor microenvironment in European American, African American, and Kenyan women at the single cell level. To date, 70 tumors spanning breast cancer subtypes have been sequenced. We are currently performing downstream analyses in this cohort to characterize ancestry- and risk factor-related differences in the tumor epithelium and microenvironment at the single-cell level. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C044.

Revisiting chromatin packaging in mouse sperm.

Mammalian sperm show an unusual and heavily compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, nucleosome localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis, we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, and appropriate lysis conditions, are together required to reveal a sperm chromatin state distinct from most previous reports. Using ATAC-seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with early development and transcriptional control. Histone modification and chromosome folding profiles also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies show that our knowledge of chromosome packaging in mammalian sperm remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.

Molecular Profiling of Response and Resistance to Venetoclax-Decitabine Therapy in Acute Myeloid Leukemia

The hypomethylating agent decitabine and the BCL-2 inhibitor venetoclax combination (DEC-VEN) is now standard of care in AML, yet a subset of patients remains refractory, and factors that reduce efficacy remain to be fully elucidated. This study aimed to systematically identify molecular determinants of resistance via single-cell (sc)RNA-seq and scATAC-seq analysis of bone marrow (BM) samples from patients participating in a prospective clinical trial of DEC-VEN (NCT03404193). For scRNA-seq, 17 BM samples (total 39,607 cells) from 12 AML patients (median age 67; 8 responders; 4 non-responders; 5 pre- and post-treatment pairs) and 3 healthy BM controls were integrated and annotated into 23 transcriptional cell clusters. In the Uniform Manifold Approximation and Projection (UMAP) plot, C1 overlapped with healthy hematopoietic stem cells (HSCs), C2 appeared only in AML patients, and C3 overlapped with progenitor cells. C1 was characteristic of refractory AML and persisted after DEC-VEN treatment. (Figure 1) C1 cells showed significantly higher expression of FLT3, BCL2, MCL1 and transcription factors MYB, ETV6, RUNX1, LMO2, JUND compared to HSCs from healthy donors. DEC-VEN treatment markedly increased ribosomal protein ( RP ) genes and mitochondrial electron transfer COX family genes and increased expression of the leukemia stem cell (LSC) marker CD74 in C1 of non-responders. In C2 cells, high expression of LSC marker genes FLT3, PIM1, MYC and transforming master regulator genes MYB, ETV6, RUNX2, LMO2, STAT3, NOTCH1, BCL11A was observed. DEC-VEN significantly upregulated RP genes and CD74 in C2 in non-responders, as in C1. In responders, hemoglobin family genes were significantly increased in C2. C3 cells were characterized by highly expressed iron metabolism genes, which were further increased by DEC-VEN in responders. GSEA analysis demonstrated that DEC-VEN upregulated ATP metabolic processes and ribosomal biosynthesis and downregulated stem cell differentiation genes in refractory C1 and C2. In responders' AML clusters, DEC-VEN upregulated cellular oxidant detoxification and iron ion homeostasis and downregulated myeloid leukocyte activation. Next, we extended the AML transcriptome analysis by profiling accessible chromatin by scATAC-seq using BM from pre- and post- DEC -VEN treatment of 2 AML patients (1 refractory, 1 relapsed after remission with incomplete count recovery [CRi]), and 2 healthy controls (total 41,062 cells). Leukemia-like cluster (LLC) cells showed significantly higher chromatin accessibility of CD74, RUNX1, RUNX2, NOTCH1, LMO2, ETV6, JUND, STAT3, BCL2, BCL11A and enriched binding motifs of RUNX2, ETV6, MYB, JUN, JUND, STAT3 compared to other clusters, which was consistent with gene expression findings of C1 and C2 by scRNA-seq. DEC-VEN treatment induced further increased motif enrichment of STAT3 in LLC in CRi AML, and STAT3 RUNX2, IKZF1 in refractory AML. To interpret T cell kinetics in response to DEC-VEN in AML, T cells from scRNA-seq profiling were subclassified based on canonical gene expression. The results showed that the CD3+ and CD3+CD8+ cell signatures were significantly lower in non-responders compared to responders post-treatment (p<0.005). Notably, the ratio of exhausted CD8+ cell clusters was significantly higher in non-responders than in responders in DEC-VEN post-treatment (p<0.05), with downregulation of CD8+ effector marker GZMK, and regulator of CD8+ cytotoxic T cell development TOX. In summary, we identified a subpopulation of AML cells that overlap with HSCs in UMAP plots and are associated with DEC-VEN resistance. LSC-related genes were enriched in resistant AML clusters, while hemoglobin-related genes were increased in sensitive clusters. DEC-VEN induced mitochondrial metabolism genes in resistant cells and further enhanced erythrocytic differentiation in sensitive cells. In addition, scATAC-seq revealed LSC-specific chromatin accessibility patterns, and depletion of CD8+ T cells was observed in non-responders. These findings shed light on the molecular mechanisms of DEC-VEN resistance.