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1.
STAR Protoc ; 1(2): 100079, 2020 09 18.
Article in English | MEDLINE | ID: mdl-33111113

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).


Subject(s)
Chromatin Immunoprecipitation Sequencing/methods , Chromatin/physiology , Primary Cell Culture/methods , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/genetics , Gene Library , High-Throughput Nucleotide Sequencing/methods , Humans , Nucleosomes , Pancreas/metabolism , Pancreas/pathology , Pancreatic Neoplasms/classification , Pancreatic Neoplasms/physiopathology , Sequence Analysis, DNA/methods , Transcription Factors/metabolism , Transposases/metabolism
2.
Cell Rep ; 31(6): 107625, 2020 05 12.
Article in English | MEDLINE | ID: mdl-32402285

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3ß) a key regulator of glycolysis. Pharmacological inhibition of GSK3ß results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3ß inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.


Subject(s)
Adenocarcinoma/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/genetics , GATA6 Transcription Factor/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Cell Line, Tumor , Humans
3.
Clin Sci (Lond) ; 130(8): 575-86, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26744410

ABSTRACT

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.


Subject(s)
Cyclooxygenase 2/genetics , DNA Methylation , Epigenesis, Genetic , Fibroblasts/enzymology , Lung/enzymology , Neoplasm Proteins/genetics , Pulmonary Fibrosis/genetics , Scleroderma, Systemic/genetics , Aged , Binding Sites , Case-Control Studies , Cell Proliferation , Cells, Cultured , Cyclooxygenase 2/metabolism , DNA Methylation/drug effects , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/metabolism , Dinoprostone/metabolism , Dose-Response Relationship, Drug , Down-Regulation , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic/drug effects , Female , Fibroblasts/drug effects , Fibroblasts/pathology , Gene Expression Regulation, Neoplastic , Genotype , Humans , Lung/drug effects , Lung/pathology , Male , Middle Aged , Neoplasm Proteins/metabolism , Phenotype , Promoter Regions, Genetic , Pulmonary Fibrosis/enzymology , Pulmonary Fibrosis/pathology , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Scleroderma, Systemic/enzymology , Scleroderma, Systemic/pathology , Transcription, Genetic , Transfection
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