Identification of gene specific cis-regulatory elements during differentiation of mouse embryonic stem cells: An integrative approach using high-throughput datasets
Fig 4
Validation of predicted enhancers.
(A) Overlap of genomic regions between published sets of enhancers (vertical axis) and the CREs/coCREs chosen for genes in the three gene sets (horizontal axis). The dot plot indicates the significance (-log10(p) with p adjusted for multiple testing) of the pairwise overlaps (red/large size = high significance, orange/small size = low significance). The absence of a dot signifies p > 0.05. The right panel (Enrich) shows a negative control of overlaps with candidate CREs that were not chosen as predictive by our method but with H3K27ac enrichment level similar to the chosen (co)CREs. (B) Expression of Sptbn1 across stages of haematopoietic and cardiac differentiation shown as a blue line chart. The inset plot shows the chromatin activity profile (CAP) for a CRE predicted to be associated with this gene. (C) A UCSC browser snapshot of the predicted CRE within the Sptbn1 gene body. The snapshot shows this region shaded in blue, illustrating the dynamics of the active chromatin mark, H3K27ac (top), and chromatin accessibility (bottom) across the cell types. The coordinate considered for further validation is chr11:30166167–30166587 highlighted in transparent cyan box. (D) A 5-day time course of haematopoietic differentiation, tracking the expression of a YFP reporter gene driven by the predicted CRE. Expression peaks on day 5 (D5), which is equivalent to the haemogenic endothelium (HE). The controls are the ESC line HM1 (black) and HM1 cells targeted with the reporter construct containing the minimal promoter (MP) only (grey).