8 results found
Brunton H, Garner IM, Bailey U-M, et al., 2020, Using Chromatin Accessibility to Delineate Therapeutic Subtypes in Pancreatic Cancer Patient-Derived Cell Lines., STAR Protoc, Vol: 1
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).
Brunton H, Caligiuri G, Cunningham R, et al., 2020, HNF4A and GATA6 Loss Reveals Therapeutically Actionable Subtypes in Pancreatic Cancer, CELL REPORTS, Vol: 31, ISSN: 2211-1247
Barnes JL, Evans IC, Garner IM, et al., 2017, Role and regulation of PRDM8 in pulmonary fibrosis associated with systemic sclerosis, Autumn Meeting of the British-Society-for-Matrix-Biology (BSMB), Publisher: WILEY, Pages: A8-A8, ISSN: 0959-9673
Evans IC, Barnes JL, Garner IM, et al., 2016, Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis, Clinical Science, Vol: 130, Pages: 575-586, ISSN: 1470-8736
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.
Garner IM, Evans IC, Barnes JL, et al., 2014, Hypomethylation Of The Tnxb Gene Contributes To Increased Expression And Deposition Of Tenascin-X In Idiopathic Pulmonary Fibrosis, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 189, ISSN: 1073-449X
Evans IC, Barnes JL, Garner IM, et al., 2014, Epigenetic Regulation Of Cyclooxygenase-2 By C8orf4 In Pulmonary Fibrosis, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 189, ISSN: 1073-449X
Garner IM, Evans IC, Maher TM, et al., 2013, Genome-wide analysis identifies multiple genes with altered methylation and expression in IPF and SSc lung fibroblasts, Spring Meeting of the British-Society-for-Matrix-Biology, Publisher: WILEY-BLACKWELL, Pages: A15-A16, ISSN: 0959-9673
Garner IM, Evans IC, Barnes JL, et al., 2013, Hypomethylation of the TNXB gene contributes to increased expression and deposition of tenascin X in idiopathic pulmonary fibrosis, Spring Meeting of the British-Society-for-Matrix-Biology, Publisher: WILEY-BLACKWELL, Pages: A15-A15, ISSN: 0959-9673
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