ABSTRACT: SOXF transcription factors are highly relevant for endothelial biology, yet comprehensive binding data of the endogenous proteins in endothelial cells has not previously been published. We used CUT&RUN to determine the binding patterns of endogenous SOX7 and SOX17 in baseline human umbilical vein endothelial cells (HUVEC).
Project description:Endothelial colony-forming cells (ECFCs) have been reported as promising cells for regenerative medicine thanks to their angiorepair properties. Transcription factors are primary determinants of the functional capacity of the cells and TAL1 has been shown as a critical regulator of endothelial lineage in both development and adult life. However, only few (three) TAL1 targets have been identified so far in mouse and human endothelial cells. This ChIP-seq experiment was designed to identify genome binding/occupancy of TAL1 by ChIP and high throughput sequencing in primary human endothelial stem/progenitor cells. TAL1 ChIP and IgG ChIP (negative control) were performed in crosslinked ECFCs derived from human umbilical cord blood.
Project description:Endothelial colony-forming cells (ECFCs) have been reported as promising cells for regenerative medicine thanks to their angiorepair properties. Transcription factors are primary determinants of the functional capacity of the cells and TAL1 has been shown as a critical regulator of endothelial lineage in both development and adult life. However, only few (three) TAL1 targets have been identified so far in mouse and human endothelial cells. This microarray experiment, where TAL1 expression was knocked-down, was designed to identify TAL1-dependent genes in primary human endothelial stem/progenitor cells. ECFCs were isolated from three independent cord blood samples (n=3, biological replicates) and cultured in complete EGM-2 medium. The knockdown of TAL1 was induced by infection with lentiviruses expressing an anti-TAL1 shRNA. A scrambled shRNA was used as a negative control. Cells were then harvested for RNA extraction. DNA-free total RNA was isolated with RNeasy Mini Kit and hybridized to the Affymetrix Human Gene 1.0 ST gene expression microarray.
Project description:Endothelial progenitors represent one of the most promising cell-based strategies for vascular repair of ischemic tissue damage, including limb ischemia, myocardial infarction and stroke. We have shown that the transcription factor TAL1 regulates a transcription program that drives the migration and adhesion of ECFCs. Furthermore, treatment of ECFCs with the HDAC inhibitor TSA increases the expression of TAL1-dependent genes and promotes the migration, chemotaxis and adhesion of ECFCs. Finally, ex vivo treatment with TSA also improves the vascular repair properties of ECFCs in vivo when these cells are transplanted in a mouse model of hindlimb ischemia. The goal of this experiment was to test whether TSA treatment of ECFCs affect TAL1 genomic binding. TAL1 ChIP-sequencing was performed from ECFCs that have been treated or not TSA. As negative controls, we performed Mock-ChIP-seq from the same samples using normal IgG instead of the TAL1 antibody. Overall, we find that there is no change in TAL1 genomic binding in ECFCs upon TSA treatment.
Project description:The study aimed to identify circular RNAs (circRNAs) commonly back-spliced to intronic region of different sets of endothelial cells (human cardiac microvascular endothelial cells (HCMEC), human aortic endothelial cells (HAoEC), human umbilical vein endothelial cells (HUVECs)) and to evaluate their overall expression and their expression compared to their respective host gene. Identified circRNAs were quality controlled by their detection in an additional exonuclease RNase R treated RNA-Seq dataset performed with RNA of HUVECs. CircRNAs were compared for overlapping detection between datasets and filtered by annotation for circRNAs back-spliced to intronic regions. Common endothelial intronic circRNAs candidates were compared to respective murine circRNAs stored in the circATLAS database. The prime candidate cZNF292 was functionally characterized in vivo and in vitro.
Project description:Human endothelial cellular models are useful to disentangle the pathophysiological role of dysfunctional endothelium in the development of cardiovascular (CV) disease and organ damage in T2D. Here, we performed a RNAseq of human umbilical vein endothelial cells (HUVECs) undergoing replicative senescence and exposed to high glucose (25 mM) to investigate the combined effects of replicative senescence and high glucose on the transcriptional landscape of these cells. To gain insight into the molecular mechanisms driving the acquisition of a senescent phenotype following exposure to HG, we performed a RNA-seq assay on control (Ctr) and replicative senescent (Sen) HUVECs cultivated in presence/absence of HG culture medium (total number of samples = 12; number of samples in each cell type-medium condition group = 3) using the NovaSeq 6000 Illumina system. Differential expression analysis was performed in R environment (version 4.2.2) using the limma and edgeR Bioconductor R packages.
Project description:To investigate lncRNAs interacting with the EZH2 enzyme, the catalytic subcomponent of the polycomb repressive complex 2. We performed nuclear extraction of the INA-6 MM cell line and proceeded do an immunoprecipitation against the EZH2 protein.
Project description:The role of propionate-induced rearrangement of membrane proteins in the formation of the virulent phenotype of Crohn's Disease-associated adherent-invasive Escherichia coli. Adhesive-invasive Escherichia coli (AIEC) were first isolated from the ileal mucosa of a patient with Crohn's disease (CD) which is a severe chronic immune-mediated granulomatous inflammatory disease of the gastrointestinal tract. It turned out that they are able to successfully penetrate the mucin layer, overcome the epithelial barrier, and also survive and multiply inside macrophage. Bacteria with such properties were assigned to a special group of pathobiont adhesive-invasive E. coli. AIEC activity is accompanied by the release of pro-inflammatory cytokines, i.e. surviving and multiplying inside macrophages, they enhance the inflammatory process. The role of AIEC in the onset or chronicity of CD is not well-defined. However, it has been proposed that these bacteria could trigger the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells, and then, due to their survival within macrophages, they could stimulate chronic inflammation and granuloma developmen. Pathogens use a variety of mechanisms, including the induction of inflammation, the direct or indirect destruction of commensal species, and the use of alternative carbon sources to survive. AIEC are shown to be able to utilize ethanolamine and propanediol, which are formed during the catabolism of phospholipids, fucose or rhamnose, propionate and other metabolites. Metabolic plasticity is thought to allow AIEC to act as an opportunistic pathogen in conditions of intestinal inflammation. We have previously shown that passage of AIEC from a CD patient (CD isolate) on M9 minimal medium supplemented with sodium propionate (PA) as a carbon source leads to a strong increase, and passage on M9 medium supplemented with glucose, on the contrary, leads to a significant decrease in adhesive-invasive properties and ability to survive in macrophages. We were able to compare the isogenic CD isolate in two states: virulent with high adhesive-invasive activity and ability to survive in macrophages, and non-virulent, when these properties are lost. In contrast to the CD isolate, passage of the laboratory strain K12 Mg1655 on the M9 medium supplemented with PA did not cause a similar effect. We performed a comparative proteomic analysis of membrane fractions isolated from ZvL2-PA and ZvL2-GLU using LC-MS. The laboratory strain K12 Mg1655 was used as a control.
Project description:Transposon insertion site sequencing (TIS) is a powerful tool that has significantly advanced our knowledge of functional genomics. While providing valuable insights, these applications of TIS focus on (conditional) gene essentiality and neglect possibly interesting but subtle differences in the importance of genes for fitness. Notably, data from TIS experiments can be used for fitness quantification and constructing genetic interaction maps, though this potential is only sporadically exploited. We aimed to develop a method to quantify the fitness of gene disruption mutants using data obtained from the TIS screen SATAY. This dataset was used to determine the reproducibility of the fitness estimates across biological and technical replicates of the same strain of S. cerevisiae. In addition, a mutant bem3∆ strain was utilized to compare the genetic interactions inferred from these fitness estimates with those documented in published databases. The dataset for the wild-type strain was created by transforming strain yWT01 with plasmid pBK549 and picking 4 different colonies from the transformation plate. These 4 biological replicates were then renamed to FD7, FD9, FD11 and FD12.