Project description:The many modifications involved in transcription are extensively coupled, and levels of histone modifications, insulators, and RNA polymerase II could be measured using genomic tiling microarrays. After 0, 30, 60 min of stimulation of HUVEC with TNF alpha, we crosslinked cells and fixed, then immunoprecipitated to prepare DNA using antibodies for modified histone, insulators and RNA polymerase. We apply samples collected every 7.5 min to arrays and monitor the growth of several long transcripts. Keywords: time course Using tumor necrosis factor a (TNF-alpha) to switch on transcription of selected human genes rapidly and synchronously, we apply samples collected every 30 min to arrays
Project description:The many modifications involved in transcription are extensively coupled, and levels of histone modifications, insulators, and RNA polymerase II could be measured using genomic tiling microarrays. After 0, 30, 60 min of stimulation of HUVEC with TNF alpha, we crosslinked cells and fixed, then immunoprecipitated to prepare DNA using antibodies for modified histone, insulators and RNA polymerase. We apply samples collected every 7.5 min to arrays and monitor the growth of several long transcripts. Keywords: time course
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.
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.
Project description:Unraveling the complexity of transcriptional programs coded by different cell types has been one of the central goals of cell biology. Using genome-wide location analysis, we examined how two different cell types generate different responses to the NF-kappaB signaling pathway. We showed that, after tumor necrosis factor-alpha (TNF-alpha) treatment, NF-kappaB p65 subunit binds to distinct genome locations and subsequently induces different subsets of genes in human monocytic THP-1 cells versus HeLa cells . Interestingly, the differential p65 binding in two cell types correlates with pre-existing cell-type specific enhancers prior to TNF-alpha stimulation, marked by histone modifications. We also found that two transcription factors, PU.1 and C/EBPalpha, appear to synergistically mediate enhancer creation and affect NF-kappaB target selection in THP-1 cells. In HeLa cells, co-expression of PU.1 and C/EBPalpha conferred TNF-alpha responsiveness to a subset of THP-1 specific NF-kappaB target genes. These results suggest that the diversity of transcriptional programs in mammalian cells arises, at least in part, from pre-existing enhancers that are established by cell specific transcription factors. We used Affymetrix microarray (GPL570) to obtain gene expression data for THP1 and HeLa cells before and after TNF-alpha treatment.
