Chromatin Accessibility Profiles Research Articles

Abstract PR016: Dynamic chromatin landscapes of colorectal cancer development and cell-free DNA fragmentation

Abstract The stepwise progression of colorectal cancer (CRC) from healthy epithelium, to premalignant adenoma, to cancer is accompanied by genome-wide epigenetic reprogramming which may be reflected in circulating cell-free DNA (cfDNA). However, current analyses of these processes have been hampered by complex mixtures of cells in normal, adenoma and cancer tissue samples, as well as absence of suitable adenoma model systems. Cultured colorectal organoids, comprised of pure epithelial cells, could enable insights on the epigenetic changes leading to progression from healthy tissue to cancer. Such features could be instrumental in the early cancer detection using genome-wide analyses of cfDNA fragmentation. Using 43 patient-derived colorectal organoids derived from healthy, adenoma, and cancer tissues, as well as 10 freshly collected peripheral blood mononuclear cell (PBMC) control samples, we examined the chromatin landscape of tumor progression using transposase accessible chromatin analyses with next-generation sequencing. We defined a consensus set of nucleosome depleted regions for each disease state and identified loci with differential accessibility between PBMCs, colon healthy, colon adenoma, and colon cancer tissues. We analyzed these differential loci for fragmentation characteristics in the circulating cfDNA of 250 healthy individuals and 51 patients with metastatic CRC. Across all organoid samples, we identified >35 million nucleosome depleted regions that we coalesced into a consensus set of 62,708 regions. As 78% of these regions could be linked to genes, we assessed whether these signatures identify known pathways involved in colorectal cancer. Within these regions, we identified unique colon tumor-related signatures comprising genes of known and novel pathways, including those involved in WNT, hippo, and RAS signaling. While the majority of chromatin accessibility differences were shared between adenomas and cancers, we identified 895 chromatin changes in adenomas that were not present in either healthy or malignant tissues. In healthy individuals, we found that the cfDNA coverage was negatively correlated with accessibility of white blood cell accessibility loci (Spearman ρ= - 0.60) while in individuals with cancer, changes in chromatin accessibility were associated with independent circulating tumor DNA abundance by droplet digital PCR (R2=0.69, p<0.001). Chromatin accessibility profiles were correlated with genome-wide cell-free DNA fragmentation features and could be used to distinguish healthy individuals from those with colorectal cancer (AUC=0.97). These analyses are consistent with dynamic chromatin remodeling during colorectal tumor development and may provide an avenue for evaluating these through genome-wide analyses of cfDNA fragmentation. Citation Format: Nicholas A. Vulpescu, Zachariah H. Foda, Pieter H.A. Wisse, Christopher Cherry, Jaime E. Medina, Vilmos Adleff, Remond J.A. Fijneman, Robert B. Scharpf, Gerrit A. Meijer, Beatriz Carvalho, Victor E. Velculescu. Dynamic chromatin landscapes of colorectal cancer development and cell-free DNA fragmentation [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR016.

EPCO-01. FROM HIERARCHY TO GEOGRAPHY: MULTI-CLONES DRIVE THE CELLULAR DIVERSITY AND EVOLUTION OF HUMAN MEDULLOBLASTOMA

Abstract We’ve curated a comprehensive multi-omics database of medulloblastoma (MB) serving as a resource to understand spatiotemporal organization and guide therapeutic strategies. Herein, our analysis encompasses single-nucleus transcriptome (n = 51), chromatin accessibility (n = 46), and spatial transcriptomic profiles (n = 31) from human MB samples and patient-derived xenograft lines spanning four subgroups, augmented by bulk RNA (n = 322), whole-genome short-sequencing (n = 279), and bisulfite sequencing (n = 300) from matched samples. We delineate the malignant cellular hierarchy, identifying MB cancer stem-like cells (MB-CSCs), cycling cells, and more differentiated populations. Gene signatures of different cell subpopulations strongly correlate to clinical outcomes, while spatial character of patient-derived materials emphasizes the geographically heterogeneous nature of MB. Examination of somatic copy number variants (CNVs) and transcriptional signatures at single-nucleus resolution reveals geographically defined subclones harboring distinct CNVs, suggesting a competitive agglomeration of co-existing multi-clones for MB. Alongside the trajectorial transition, we observe distinct chromosomal alterations in apical MB-CSCs compared to more differentiated cells, indicative of extensive subclone-primed spatiotemporal evolution. Further analysis of reagent-gene interactions of subclonal lineages contributes to predicting the druggable candidates for treatment optimization. Integration of spatial architecture data highlights the recapitulation of subclone-determined niches through colocation with identified malignant lineages and their own inflammatory or fibrotic adaptions. Taken together, this comprehensive database, comprising five or six kinds of datatypes on the same MB cases, facilitates combinatorial analyses across different genomic modalities, offering genetic and geographic insights into therapeutic advancements.

Analysis of cis-regulatory changes underlying phenotype divergence shaped by domestication in pigs

BackgroundCis-regulatory elements (CREs) are regions of DNA that regulate the expression of nearby genes. Changes in these elements can lead to phenotypic variations and adaptations in different populations. However, the regulatory dynamics underlying the local adaptation of traits remain poorly understood in Chinese and Western pigs. By comparing the chromatin accessibility profiles of skeletal muscle, liver, and fat between these two pig populations, we aimed to identify key regulatory elements that could explain phenotypic differences observed between the two groups.ResultsOur results revealed that the genome-wide chromatin accessibility profiles were largely similar at a qualitative level within tissues. However, we also identified local regions that exhibited quantitative differences, most of which occurred in liver tissue. Interestingly, we found that most of the increased chromatin accessibility in the livers of Chinese pigs was associated with tissue-specific openness. Furthermore, we observed a positive correlation between the ATAC-seq signal at the transcript start site (TSS) and the expression levels of nearby genes. Motif enrichment analysis revealed NR2F1 as a key regulator in Chinese pigs. Differentially expressed genes (DEGs) in Chinese pigs showed enrichment for NR2F1 response targets. One of the genes regulated by NR2F1 in Chinese pigs, NPC1, harbored a high alternative allelic frequency in the intron region.ConclusionOverall, our study provides valuable insights into the regulatory dynamics underlying phenotypic variation in pigs. By elucidating the role of CREs in driving phenotypic variation, we can better understand the genetic basis of complex traits and potentially identify targets for genetic improvement in livestock breeding programs.

Application of computational algorithms for single-cell RNA-seq and ATAC-seq in neurodegenerative diseases.

Recent advancements in single-cell technologies, including single-cell RNA sequencing (scRNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), have greatly improved our insight into the epigenomic landscapes across various biological contexts and diseases. This paper reviews key computational tools and machine learning approaches that integrate scRNA-seq and scATAC-seq data to facilitate the alignment of transcriptomic data with chromatin accessibility profiles. Applying these integrated single-cell technologies in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, reveals how changes in chromatin accessibility and gene expression can illuminate pathogenic mechanisms and identify potential therapeutic targets. Despite facing challenges like data sparsity and computational demands, ongoing enhancements in scATAC-seq and scRNA-seq technologies, along with better analytical methods, continue to expand their applications. These advancements promise to revolutionize our approach to medical research and clinical diagnostics, offering a comprehensive view of cellular function and disease pathology.

Integrative analysis based on ATAC-seq and RNA-seq reveals a novel oncogene PRPF3 in hepatocellular carcinoma.

Assay of Transposase Accessible Chromatin Sequencing (ATAC-seq) is a high-throughput sequencing technique that detects open chromatin regions across the genome. These regions are critical in facilitating transcription factor binding and subsequent gene expression. Herein, we utilized ATAC-seq to identify key molecular targets regulating the development and progression of hepatocellular carcinoma (HCC) and elucidate the underlying mechanisms. We first compared chromatin accessibility profiles between HCC and normal tissues. Subsequently, RNA-seq data was employed to identify differentially expressed genes (DEGs). Integrating ATAC-seq and RNA-seq data allowed the identification of transcription factors and their putative target genes associated with differentially accessible regions (DARs). Finally, functional experiments were conducted to investigate the effects of the identified regulatory factors and corresponding targets on HCC cell proliferation and migration. Enrichment analysis of DARs between HCC and adjacent normal tissues revealed distinct signaling pathways and regulatory factors. Upregulated DARs in HCC were enriched in genes related to the MAPK and FoxO signaling pathways and associated with transcription factor families like ETS and AP-1. Conversely, downregulated DARs were associated with the TGF-β, cAMP, and p53 signaling pathways and the CTCF family. Integration of the datasets revealed a positive correlation between specific DARs and DEGs. Notably, PRPF3 emerged as a gene associated with DARs in HCC, and functional assays demonstrated its ability to promote HCC cell proliferation and migration. To the best of our knowledge, this is the first report highlighting the oncogenic role of PRPF3 in HCC. Furthermore, ZNF93 expression positively correlated with PRPF3, and ChIP-seq data indicated its potential role as a transcription factor regulating PRPF3 by binding to its promoter region. This study provides a comprehensive analysis of the epigenetic landscape in HCC, encompassing both chromatin accessibility and the transcriptome. Our findings reveal that ZNF93 promotes the proliferation and motility of HCC cells through transcriptional regulation of a novel oncogene, PRPF3.

Comparison of chromatin accessibility remodeling of granulosa cells in patients with endometrioma or pelvic/tubal infertility.

To elucidatethe epigenetic alteration associated with impaired oogenesis in endometrioma using multi-omic approaches. ATAC-seq was performed on the granulosa cells (GCs) of 6 patients (3 with endometrioma and 3 without). Follicular samples from another 20 patients (10 with endometrioma and 10 without) were collected for mRNA-seq analysis of GCs and extracellular vesicles (EVs) of follicular fluid. qRT-PCR validated candidate genes in GCs from 44 newly enrolled patients (19 with endometrioma and 25 without). mRNA abundance was compared with the Mann-Whitney test. Pearson's correlation analyzed relationships between candidate genes and oocyte parameters. Chromatin accessibility and gene expression profiles of GCs from endometrioma patients differed significantly from the pelvic/tubal infertility group. RNA-seq revealed most differentially expressed genes were downregulated (6216/7325) and enriched in the cellular localization pathway. Multi-omics analyses identified 22 significantly downregulated genes in the GCs of endometrioma patients, including PPIF (P < 0.0001) and VEGFA (P = 0.0148). Both genes were further confirmed by qRT-PCR. PPIF (r = 0.46, p = 0.043) and VEGFA (r = 0.45, p = 0.048) correlated with the total number of retrieved oocytes. GC chromatin remodeling may disrupt GC and EV transcriptomes, interfering with somatic cell-oocyte communication and leading to compromised oogenesis in endometrioma patients.

Deciphering Mechanisms of Adipocyte Differentiation in Abdominal Fat of Broilers.

The excessive deposition of abdominal fat tissue (AFT) in broilers has emerged as a major concern in the poultry industry. Despite some progress in recent years, the molecular mechanisms underlying AFT development remain ambiguous. The current study combined RNA-seq with transposase-accessible chromatin sequencing (ATAC-seq) to map the dynamic profiling of chromatin accessibility and transcriptional reprogramming in AFT adipocyte differentiation in broilers at day 3 (D3) and D14. Our results found that the levels of CDK1 and PCNA were down-regulated at D14, D28, and D42 compared to D3, while the levels of C/EBPα and FABP4 were up-regulated at D14 and D42 compared to D3. Meanwhile, PPARγ was significantly up-regulated at D28 and D42. RNA-seq of AFT identified 1705 up-regulated and 1112 down-regulated differential expression genes (DEGs) between D3 and D14. Pathways based on up-regulated DEGs mainly enriched some pathways related to adipocyte differentiation, while down-regulated DEGs pointed to DNA replication, cell cycle, and gap junction. Gene set enrichment analysis (GSEA) revealed that DNA replication and the cell cycle were down-regulated at D14, while the insulin signaling pathway was up-regulated. In the OA-induced immortalized chicken preadipocyte (ICP2) model, protein dynamic changes were consistent with AFT from D3 to D14. Same pathways were enriched in ICP2. In addition, based on overlapped DEGs from AFT and ICP2, enriched pathways related to adipocyte differentiation or proliferation mentioned above were all involved. A total of 1600 gain and 16727 loss differential peaks (DPs) were identified in ICP2 by ATAC-seq. Predicted genes from DPs at the promoter regions were enriched in glycerophospholipid metabolism, TGF-β signaling, FoxO signaling, and ubiquitin-mediated proteolysis. DNA motifs predicted 159 transcription factors (TFs) based on gain and loss peaks from the promoter regions, where 1 and 10 TFs were overlapped with up or down TFs from DEGs. Overall, this study presents a framework for the comprehension of the epigenetic regulatory mechanisms of adipocyte differentiation and identifies candidate genes and potential TFs involved in AFT adipocyte differentiation in broilers.

Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.

Targeting immune-fibroblast cell communication in heart failure.

Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction1,2. However, the molecular mechanisms driving immune-fibroblast cell communication in human cardiac disease remain unexplored and there are at present no approved treatments that directly target cardiac fibrosis3,4. Here we performed multiomic single-cell gene expression, epitope mapping and chromatin accessibility profiling in 45 healthy donor, acutely infarcted and chronically failing human hearts. We identified a disease-associated fibroblast trajectory that diverged into distinct populations reminiscent of myofibroblasts and matrifibrocytes, the latter expressing fibroblast activator protein (FAP) and periostin (POSTN). Genetic lineage tracing of FAP+ fibroblasts in vivo showed that they contribute to the POSTN lineage but not the myofibroblast lineage. We assessed the applicability of experimental systems to model cardiac fibroblasts and demonstrated that three different in vivo mouse models of cardiac injury were superior compared with cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2(CCR2) macrophages and fibroblasts mediated by interleukin-1β (IL-1β) signalling drove the emergence of FAP/POSTN fibroblasts within spatially defined niches. In vivo, we deleted the IL-1 receptor on fibroblasts and the IL-1β ligand in CCR2+ monocytes and macrophages, and inhibited IL-1β signalling using a monoclonal antibody, and showed reduced FAP/POSTN fibroblasts, diminished myocardial fibrosis and improved cardiac function. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and preserve organ function.

Heat stress causes chromatin accessibility and related gene expression changes in crown tissues of barley (Hordeum vulgare).

Plant responses to stress caused by high temperatures involve changes occurring at the molecular, metabolic, and physiological levels. Understanding the mechanisms by which plants recognize signals to activate this response is a prerequisite for identifying key genes and signaling pathways and for obtaining heat-tolerant plants. We demonstrated the first implementation of an assay for transposase-accessible chromatin to identify open chromatin regions (OCRs) in crown tissues of barley using three genotypes carrying different allelic forms of the sdw1 gene encoding gibberellin 20-oxidase subjected to elevated temperatures. In parallel, we performed gene expression analysis, which allowed us to relate changes in chromatin state to changes in transcriptional activity. The obtained data revealed that the hypersensitive chromatin regions within the genes were more repeatable than those outside the gene intervals. We observed that prolonged exposure to high temperatures increased chromatin accessibility. Genes with OCRs in their regulatory regions were involved in stress signaling and tolerance, including calcium-dependent protein kinase, mitogen-activated protein kinase (MAPK3), receptor-like cytoplasmic kinase (RLK), TIFY domain-containing transcriptional regulator, bZIP transcription factor, and regulatory protein NPR1. The effect of genotype on gene expression was not as pronounced as that of temperature. By combining results from the differential analysis of chromatin accessibility and expression profiles, we identified genes with high temperature-induced changes in chromatin accessibility associated with expression alterations. Importantly, our data revealed a relationship between the loss of chromatin accessibility in response to heat and the downregulation of genes related to gibberellin signaling.

The MaNAP1-MaMADS1 transcription factor module mediates ethylene-regulated peel softening and ripening in banana.

The banana (Musa spp.) peel undergoes rapid softening during ripening, leading to finger drop and a shortened shelf life. The regulatory mechanism behind this process remains to be elucidated. In this study, we confirmed the role of peel softening in banana finger drop and uncovered the underlying transcriptional regulatory network. Cell wall-related (CWR) genes were substantially upregulated in both the peel and finger drop zone during ethylene-induced ripening. Transcriptome analysis and genome-wide profiling of chromatin accessibility and transcription factor (TF) binding revealed that two key regulators of fruit ripening, Musa acuminata NAC-like, Activated by apetala3/Pistillata1 (MaNAP1) and MaMADS1, regulate CWR genes by directly binding to their promoters or by targeting other ripening-related TFs to form a hierarchical regulatory network. Notably, MaNAP1 and MaMADS1 were directly targeted by ETHYLENE INSENSITIVE3 (MaEIN3), and MaNAP1 and MaMADS1 associated with tissue-specific histone modifications, enabling them to integrate MaEIN3-mediated ethylene signaling and undergo epigenetic regulation. Overexpression of MaNAP1, MaMADS1 or other identified regulatory TFs upregulated CWR genes and promoted peel softening. Our findings unveil a MaNAP1-MaMADS1-centered regulatory cascade governing banana peel softening and finger drop, offering potential targets for enhancing banana texture and shelf life.

Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are multipotent stem cells that are under investigation for use in clinical trials because they are capable of self-renewal and differentiating into different cell types under defined conditions. Nonetheless, the therapeutic effects of MSCs have been constrained by low engraftment rates, cell fusion, and cell survival. Various strategies have been explored to improve the therapeutic efficacy of MSCs, with platelet-derived growth factor (PDGF)-BB emerging as a promising candidate. To enhance our comprehension of the impact of PDGF-BB on the gene expression profile and chromosomal accessibility of MSCs, RNA-sequencing and analysis of chromatin accessibility profiles were conducted on three human primary MSCs in culture, both with and without stimulation by PDGF-BB.ResultsIntegrative analysis of gene expression and chromatin accessibility demonstrated that PDGF-BB treatment modified the chromatin accessibility landscape, marking regions for activation or repression through the AP-1 family transcription factors TEAD, CEBP, and RUNX2. These changes in AP-1 transcription factor expression, in turn, led to cell proliferation and differentiation potential towards osteoblasts, adipocytes, or chondrocytes. The degree of MSC differentiation varies among cells isolated from different donors. The presence of an enrichment of exosome-related genes is also noted among all the differentially expressed genes.ConclusionsIn conclusion, the observed changes in AP-1 transcription factor expression not only induced cellular proliferation and differentiation, but also revealed variations in the degree of MSC differentiation based on donor-specific differences. Moreover, the enrichment of exosome-related genes among differentially expressed genes suggests a potential significant role for PDGF-BB in facilitating intercellular communication.

CCL2-mediated endothelial injury drives cardiac dysfunction in long COVID.

Evidence linking the endothelium to cardiac injury in long coronavirus disease (COVID) is well documented, but the underlying mechanisms remain unknown. Here we show that cytokines released by endothelial cells (ECs) contribute to long-COVID-associated cardiac dysfunction. Using thrombotic vascular tissues from patients with long COVID and induced pluripotent stem cell-derived ECs (iPSC-ECs), we modeled endotheliitis and observed similar dysfunction and cytokine upregulation, notably CCL2. Cardiac organoids comprising iPSC-ECs and iPSC-derived cardiomyocytes showed cardiac dysfunction after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, driven by CCL2. Profiling of chromatin accessibility and gene expression at a single-cell resolution linked CCL2 to 'phenotype switching' and cardiac dysfunction, validated by high-throughput proteomics. Disease modeling of cardiac organoids and exposure of human ACE2 transgenic mice to SARS-CoV-2 spike proteins revealed that CCL2-induced oxidative stress promoted post-translational modification of cardiac proteins, leading to cardiac dysfunction. These findings suggest that EC-released cytokines contribute to cardiac dysfunction in long COVID, highlighting the importance of early vascular health monitoring in patients with long COVID.

Single-Cell Detection of Somatic Copy Number Alterations in Human Kidney by Chromatin Accessibility Profiling

Single-Cell Detection of Somatic Copy Number Alterations in Human Kidney by Chromatin Accessibility Profiling

The chromatin landscape of high-grade serous ovarian cancer metastasis identifies regulatory drivers in post-chemotherapy residual tumour cells

Disease recurrence following chemotherapy is a major clinical challenge in ovarian cancer (OC), but little is known regarding how the tumour epigenome regulates transcriptional programs underpinning chemoresistance. We determine the single cell chromatin accessibility landscape of omental OC metastasis from treatment-naïve and neoadjuvant chemotherapy-treated patients and define the chromatin accessibility profiles of epithelial, fibroblast, myeloid and lymphoid cells. Epithelial tumour cells display open chromatin regions enriched with motifs for the oncogenic transcription factors MEIS and PBX. Post chemotherapy microenvironments show profound tumour heterogeneity and selection for cells with accessible chromatin enriched for TP53, TP63, TWIST1 and resistance-pathway-activating transcription factor binding motifs. An OC chemoresistant tumour subpopulation known to be present prior to treatment, and characterised by stress-associated gene expression, is enriched post chemotherapy. Nuclear receptors RORa, NR2F6 and HNF4G are uncovered as candidate transcriptional drivers of these cells whilst closure of binding sites for E2F2 and E2F4 indicate post-treated tumour having low proliferative capacity. Delineation of the gene regulatory landscape of ovarian cancer cells surviving chemotherapy treatment therefore reveals potential core transcriptional regulators of chemoresistance, suggesting novel therapeutic targets for improving clinical outcome.

ScMMO-atlas: a single cell multimodal omics atlas and portal for exploring fine cell heterogeneity and cell dynamics.

Single-cell multimodal sequencing parallelly captures multiple modalities of the same cell, providing unparalleled insights into cell heterogeneity and cell dynamics. For example, joint profiling of chromatin accessibility and transcriptome from the same single cell (scATAC+RNA) identified new cell subsets within the well-defined clusters. However, lack of single-cell multimodal omics (scMMO) database has led to data fragmentation, seriously hindering access, utilization and mining of scMMO data. Here, we constructed a scMMO atlas by collecting and integrating various scMMO data, then constructed scMMO database and portal called scMMO-atlas (https://www.biosino.org/scMMO-atlas/). scMMO-atlas includes scATAC+RNA (ISSAAS-seq, SNARE-seq, paired-seq, sci-CAR, scCARE-seq, 10X Multiome and so on), scRNA+protein, scATAC+proteinand scTri-modal omics data, with 3168824 cells from 27 cell tissues/organs. scMMO-atlas offered an interactive portal for visualization and featured analysis for each modality and the integrated data. Integrated analysis of scATAC+RNA data of mouse cerebral cortex in scMMO-atlas identified more cell subsets compared with unimodal omics data. Among these new cell subsets, there is an early astrocyte subset highly expressed Grm3, called Astro-Grm3. Furthermore, we identified Ex-L6-Tle4-Nrf1, a progenitor of Ex-L6-Tle4, indicating the statistical power provided by the big data in scMMO-atlas. In summary, scMMO-atlas offers cell atlas, database and portal to facilitate data utilization and biological insight.

Scalable Hypothalamic Arcuate Neuron Differentiation from Human Pluripotent Stem Cells Suitable for Modeling Metabolic and Reproductive Disorders.

The hypothalamus, composed of several nuclei, is essential for maintaining our body's homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

An Atlas of Accessible Chromatin in Advanced Prostate Cancer Reveals the Epigenetic Evolution during Tumor Progression.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal disease that resists therapy targeting androgen signaling, the primary driver of prostate cancer. mCRPC resists androgen receptor (AR) inhibitors by amplifying AR signaling or by evolving into therapy-resistant subtypes that do not depend on AR. Elucidation of the epigenetic underpinnings of these subtypes could provide important insights into the drivers of therapy resistance. In this study, we produced chromatin accessibility maps linked to the binding of lineage-specific transcription factors (TF) by performing assay for transposase-accessible chromatin sequencing on 70 mCRPC tissue biopsies integrated with transcriptome and whole-genome sequencing. mCRPC had a distinct global chromatin accessibility profile linked to AR function. Analysis of TF occupancy across accessible chromatin revealed 203 TFs associated with mCRPC subtypes. Notably, ZNF263 was identified as a putative prostate cancer TF with a significant impact on gene activity in the double-negative subtype (AR- neuroendocrine-), potentially activating MYC targets. Overall, this analysis of chromatin accessibility in mCRPC provides valuable insights into epigenetic changes that occur during progression to mCRPC. Significance: Integration of a large cohort of transcriptome, whole-genome, and ATAC sequencing characterizes the chromatin accessibility changes in advanced prostate cancer and identifies therapy-resistant prostate cancer subtype-specific transcription factors that modulate oncogenic programs.

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.

Abstract C078: Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignant and stromal cell subtypes

Abstract Pancreatic ductal adenocarcinoma (PDAC) disease progression involves complex cell state transitions in both malignant and non-malignant populations within the tumor microenvironment. To uncover PDAC cell states and epigenetic features associated with clinical outcome, we profiled 40 tumor samples (33 primary tumors and 7 metastases) from 39 patients with single cell ATAC-seq, along with 10 sample-matched single cell RNA-seq profiles. Through topic modeling of high dimensional chromatin data, we uncovered distinct cis-regulatory networks underlying the classical and basal-like molecular subtypes of PDAC, which showed differential activity in basal-classical hybrid cells. Furthermore, delineation of epigenetic alterations by basal, classical, and hybrid subtypes revealed a core set of chromatin regions opened in all PDAC subtypes relative to ductal and acinar cells. These universally open regions showed striking overlap with gastrointestinal enhancers, highlighting a common endoderm lineage infidelity process across pancreatic tumors. Within the stromal compartment, we identified novel regulators of myofibroblastic cancer-associated fibroblasts (myCAFs) and inflammatory CAFs (iCAFs) from enhancer networks active in each CAF subtype. Importantly, our data uncover a highly prognostic gene program active in only one of two distinct myCAF chromatin states found in our data, highlighting a putative tumor-promoting myCAF subpopulation. Our work provides a high-resolution description of epigenetic cell state heterogeneity in PDAC, revealing novel regulators and gene programs associated with PDAC subtypes and clinical outcome. Citation Format: Kevin MacPherson-Hawthorne, Jason M. Link, Patrick Worth, Brett Sheppard, Andrew Fields, Colin Daniel, Andrew Nishida, Carl Pelz, Trent Waugh, Ethan Agritelle, John Muschler, Andrew Adey, Rosalie Sears. Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignant and stromal cell subtypes [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 C078.