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Multi-species RELA, JUN, and histone modification ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation

ABSTRACT: Multi-species RELA, JUN, and histone modification ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation

PROVIDER: PRJEB32196 | ENA |

REPOSITORIES: ENA

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Multi-species RELA ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation

Project description:TNFα has an evolutionary conserved role in mediating inflammation via activation of the transcription factor NF-κB. The functions of individual NF-κB binding sites are not well understood. To identify conserved and functionally important NF-κB binding sites in mammals, we performed ChIP-seq to map the genome-wide binding of RELA and select histone modifications in primary vascular endothelial cells (ECs) isolated from the aortas of human (HAEC), mouse (MAEC) and cow (BAEC), before and after TNFα. The conserved RELA binding sites show strong epigenetic changes in response to TNFα and enrich near genes controlling vascular development and pro-inflammatory responses. Our method identifies novel modes of RELA-chromatin interactions that are conserved in mammals and shared between multiple cell-types. Particularly, genomic regions bound by RELA prior to stimulation are important responders during TNFα stimulation. We use CRISPR/Cas9 genome editing to validate the roles of the conserved RELA pre-bound sites near pro-inflammatory genes such as CCL2 and PLK2. Our evolutionary approach describes new aspects of mammalian NF-κB biology including its role within super-enhancers and relevance in inflammatory disorders.

2020-10-03 | E-MTAB-7889 | biostudies-arrayexpress

4C-seq of CCL2 promoter contacts in primary human aortic endothelial cells and TeloHAEC cell line before and after TNFα stimulation

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RNA-seq of primary aortic human endothelial cells before and after TNFα stimulation

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Multi-species RELA ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation

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ChRO-seq on TeloHAEC cells before and after TNFα stimulation

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Effect of TNFα and RELA inhibition on gene expression in human PDAC cells

Project description:The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. Here, we characterised previously unknown endogenous RELA-GFP dynamics in PDAC cell lines by live single cell imaging, which revealed rapid, sustained and non-oscillatory nuclear RELA following TNFα stimulation. Using Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. Using RNA-seq, we identified the actin regulators NUAK2 and ARHGAP31 as transcriptionally regulated by RELA. In turn, NUAK2 or ARHGAP31 siRNA depletion downregulates TNFα-stimulated RELA nuclear localisation in PDAC cells, establishing a novel negative feedback loop regulating RELA activation by TNFα. We identify an additional actin-independent feedback loop involving RELB, which suppresses TNFα-mediated RELA nuclear localisation following RELA mediated upregulation of RELB. Taken together, we provide computational and experimental support for interdependence between the F-actin network and RELA translocation dynamics in PDAC.

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ATAC-seq of primary mammalian aortic endothelial cells before and after TNFα stimulation

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ATAC-seq of primary mammalian aortic endothelial cells before and after TNFα stimulation

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RNA-seq of primary aortic human endothelial cells before and after TNFα stimulation

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