Project description:Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) analysis in TNF-alpha-stimulated human umblical vein endothelial cells (HUVECs)
Project description:Lysine 9 di-methylation and lysine 27 tri-methylation of histone H3 (H3K9me2 and H3K27me3) are mostly linked to gene repression. However, functions of repressive histone methylation dynamics during inflammatory responses remain poorly understood. Here, we show that lysine demethylase 7A (KDM7A) and 6A (UTX) are rapidly transported to nuclear factor kappa-B (NF-κB) related elements in human endothelial cells in response to tumor necrosis factor (TNF)-α. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and cooperatively activate NF-κB dependent inflammatory genes. Furthermore, using both in situ Hi-C and other 3C based technology, loops between super enhancers (SEs) are newly formed following TNF-α-stimuli at NF-κB-dependent inflammatory loci where KDM7A- and UTX-recruitment coincide. Collectively, these findings suggest that erasing of repressive histone marks by KDM7A and UTX within NF-κB-related elements might functionally associate with formation of SE-SE three-dimensional interactions and could be a cue signal during inflammatory responses in human endothelial cells.
Project description:HUVECs were stimulated and samples were prepared after 0 and 30 min. Chromatin interaction mediated by active RNA polymerase II was detected by ChIA-PET. Samples were made afrer 0 and 30 min after TNF alpha stimulation.
Project description:Lysine 9 di-methylation and lysine 27 tri-methylation of histone H3 (H3K9me2 and H3K27me3) are mostly linked to gene repression. However, functions of repressive histone methylation dynamics during inflammatory responses remain poorly understood. Here, we show that lysine demethylase 7A (KDM7A) and 6A (UTX) are rapidly transported to nuclear factor kappa-B (NF-κB) related elements in human endothelial cells in response to tumor necrosis factor (TNF)-α. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and cooperatively activate NF-κB dependent inflammatory genes. Furthermore, using both in situ Hi-C and other 3C based technology, loops between super enhancers (SEs) are newly formed following TNF-α-stimuli at NF-κB-dependent inflammatory loci where KDM7A- and UTX-recruitment coincide. Collectively, these findings suggest that erasing of repressive histone marks by KDM7A and UTX within NF-κB-related elements might functionally associate with formation of SE-SE three-dimensional interactions and could be a cue signal during inflammatory responses in human endothelial cells.
Project description:Lysine 9 di-methylation and lysine 27 tri-methylation of histone H3 (H3K9me2 and H3K27me3) are mostly linked to gene repression. However, functions of repressive histone methylation dynamics during inflammatory responses remain poorly understood. Here, we show that lysine demethylase 7A (KDM7A) and 6A (UTX) are rapidly transported to nuclear factor kappa-B (NF-κB) related elements in human endothelial cells in response to tumor necrosis factor (TNF)-α. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and cooperatively activate NF-κB dependent inflammatory genes. Furthermore, using both in situ Hi-C and other 3C based technology, loops between super enhancers (SEs) are newly formed following TNF-α-stimuli at NF-κB-dependent inflammatory loci where KDM7A- and UTX-recruitment coincide. Collectively, these findings suggest that erasing of repressive histone marks by KDM7A and UTX within NF-κB-related elements might functionally associate with formation of SE-SE three-dimensional interactions and could be a cue signal during inflammatory responses in human endothelial cells.
Project description:TNF alpha is one of the inflammatory mediator and induce genes mainly by transcriptional factor, p65, in endothelial cells. This time, we performed a time course study to detect the change of localization of p65 and Pol II. To identify p65 and Pol II binding sites, we used chromatin immunoprecipitation with deep sequencing (ChIP-seq) of HUVECs treated with or without TNF alpha for 30 mins. Cells were starved before stimulation longer than 16 hours. HUVECs were used within the first 6 passages. For crosslinking, 10 mM of EGS in 50% glacial acetic acid was used for 45 min, followed by 20 min of 1% paraformaldehyde treatmet was used.