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Using Chromatin Accessibility to Delineate Therapeutic Subtypes in Pancreatic Cancer Patient-Derived Cell Lines.

ABSTRACT: Disrupted chromatin regulatory processes contribute to the development of cancer, in particular pancreatic ductal adenocarcinoma. The assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) is typically used to study chromatin organization. Here, we present a revised ATAC-seq protocol to study chromatin accessibility in adherent patient-derived cell lines. We provide details on how to calculate the library molarity using Agilent's Bioanalyzer and an analysis pipeline for peak calling and transcription factor mapping. For complete details on the use and execution of this protocol, please refer to Brunton et al. (2020).

SUBMITTER: Brunton H

PROVIDER: S-EPMC7580199 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Using Chromatin Accessibility to Delineate Therapeutic Subtypes in Pancreatic Cancer Patient-Derived Cell Lines.

Brunton Holly H Garner Ian M IM Bailey Ulla-Maja UM Upstill-Goddard Rosie R Bailey Peter J PJ

STAR protocols 20200804 2

Disrupted chromatin regulatory processes contribute to the development of cancer, in particular pancreatic ductal adenocarcinoma. The assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) is typically used to study chromatin organization. Here, we present a revised ATAC-seq protocol to study chromatin accessibility in adherent patient-derived cell lines. We provide details on how to calculate the library molarity using Agilent's Bioanalyzer and an analysis pipelin ...[more]

PMID: 33111113

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Project description:African American (AA) women have an excessive risk of developing triple-negative breast cancer (TNBC). We employed Assay for Transposase-Accessible Chromatin using sequencing to characterize differences in chromatin accessibility between nine commonly used TNBC cell lines derived from patients of European and African ancestry. Principal component and chromosome mapping analyses of accessibility peaks with the most variance revealed separation of chromatin profiles by patient group. Motif enrichment and footprinting analyses of disparate open chromatin regions revealed differences in transcription factor activity, identifying 79 with ancestry-associated binding patterns (FDR < 0.01). AA TNBC cell lines exhibited increased accessibility for 62 transcription factors associated with epithelial-to-mesenchymal transition, cancer stemness/chemotherapeutic resistance, proliferation, and aberrant p53 regulation, as well as KAISO, which has been previously linked to aggressive tumor characteristics in AA patients with cancer. Differential Assay for Transposase-Accessible Chromatin signal analysis identified 1,596 genes located within promoters of differentially open chromatin regions in AA-derived TNBC, identifying DNA methyltransferase 1 as the top upregulated gene associated with African ancestry. Pathway analyses with these genes revealed enrichment in several pathways, including hypoxia. Culturing cells under hypoxia showed ancestry-specific stress responses that led to the identification of a core set of AA-associated transcription factors, which included members of the Kruppel-like factor and Sp subfamilies, as well as KAISO, and identified ZDHHC1, a gene previously implicated in immunity and STING activation, as the top upregulated AA-specific gene under hypoxia. Together, these data reveal a differential chromatin landscape in TNBC associated with donor ancestry. The open chromatin structure of AA TNBC may contribute to a more lethal disease.SignificanceWe identify an ancestry-associated open chromatin landscape and related transcription factors that may contribute to aggressive TNBC in AA women. Furthermore, this study advocates for the inclusion of diversely sourced cell lines in experimental in vitro studies to advance health equity at all levels of scientific research.

Project description:BackgroundRecent research of clear cell renal cell carcinoma (ccRCC) is focused on the tumor immune microenvironment (TIME). Chromatin accessibility is critical for regulation of gene expression. However, its role in different immunological subtypes of ccRCC based on immune cell infiltration has not been systematically studied.MethodsFive hundred thirty patient data from The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) were adopted to estimate immune cell infiltration. Twenty-four types of immune cells were evaluated with single-sample Gene Set Enrichment Analysis (ssGSEA). Patients were divided into two clusters based on immune cell infiltration. Systematic chromatin accessibility analysis was conducted based on the two clusters.ResultsWe compared the relative expression of the immune gene signatures among 530 patients of TCGA-KIRC using ssGSEA. Overall survival (OS) analysis revealed 10 types of immune cells were significantly associated with prognosis. Patients were divided into two clusters based on 24 types of immune cell infiltration. Immune cell signals as well as PD-1/PD-L1 signal were higher in cluster 1. Among the two clusters, 2,400 differential peaks were found in TCGA-KIRC Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) data. The distribution of differential peaks and prognosis-related immune cells in 23 chromosomes are essentially the same. There is no peak distribution downstream. The proportion of peaks upstream of the 5' transcription start site decreases, and both sides of binding regions of the TSS 0.1-1 kb becomes smaller. Enrichment analysis of GO and KEGG of these differential peaks showed that they are remarkably related to the immune regulation in tumor microenvironment. Known motifs and de novo motifs were found by linking motif annotations to different peaks. Survival analysis of related motif transcription factors were prognostic. The GSEA enrichment analysis showed that high SP1 expression positively correlates with TGF-beta signaling and inflammatory response, while negatively correlates with TNF-alpha signaling via NFKB. High KLF12 expression negatively correlates with interferon gamma response, IL2-STAT5 signaling, TNF-alpha signaling via NFKB, IL6-JAK-STAT3 signaling.ConclusionThe abnormality of chromatin accessibility may play an important regulatory role in ccRCC immunity.

Dataset Information

Using Chromatin Accessibility to Delineate Therapeutic Subtypes in Pancreatic Cancer Patient-Derived Cell Lines.

Publications

Using Chromatin Accessibility to Delineate Therapeutic Subtypes in Pancreatic Cancer Patient-Derived Cell Lines.

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