Fig. 5: Epigenetic reprogramming validates putative regulatory elements.
From: Endothelial gene regulatory elements associated with cardiopharyngeal lineage differentiation
a Top: Schematic overview of CRISPR-dCas9:LSD1 system: the fusion protein dCas9:LSD1 is able to bind DNA target and LSD1 can demethylate histone H3 lysine 4 (H3K4me1 and me2) near the putative enhancer region to decommission the enhancer. Bottom: cartoon of the experimental plan. mESC #B1 dCas9-LSD1 were transfected with fluorescent gRNAs. Fluorescent-sorted cells were differentiated into ECs from day 0 to day 8. Samples were collected on day 4, day 6, and day 8 to analyze the gene expression. b Quantitative real-time PCR (qPCR) analysis of Kdr; Cdh5; Eng; Notch1; Flt1, and Pecam1 mRNA expression level in cells of clone #B1 dCas9-LSD1 transfected with gRNAs targeted (in red) or control (in black) during EC differentiation. X-axis denotes the three time points (d4-d6–d8); y-axis indicates the expression level, evaluated using the 2−ΔCt method. Gapdh expression was used as a normalizer. Values are the average of four (n = 4) biological replicates \(\pm\) standard deviation (SD). p value (*) <0.05 and p value (**) <0.01 are considered significant; ns no statistical significance (parametric paired t-test, one-tailed).
