Fig. 4: Candidate enhancer elements are native transcriptional enhancers with erythroid temporal activity.

a Enhancer activity kinetics of the 42 elements as measured by GFP expression with flow cytometry during ex vivo erythroid differentiation from mobilized adult human CD34⁺ cells. Y-axis depicts MFI z-score across all elements within each time point measured. An early active (L196, teal) and a late active (L223, magenta) element were selected for further study. b Correlation (Pearson’s r) between the temporal profiles of enhancer activity (MFI) of the 42 elements and their in situ DNase I accessibility profile during ex vivo erythropoiesis. Median Pearson’s r is denoted with a dashed red line. c Comparison between the enhancer activity (MFI, light color) and in situ DNase I accessibility (dark color) for the L196 vector (left) and L223 (right). d The enhancer element in the L196 vector is derived from a DHS intronic to the PVT1 lncRNA and the element in L223 is an exonic DHS in the PPARA locus. Signal density tracks of the DNase I accessibility during erythroid differentiation, GATA1 CUT&RUN occupancy and H3K27Ac CUT&RUN enrichment in HUDEP-2 cells is shown for the PVT1 locus (left) and PPARA locus (right). Bottom track shows consensus footprinted motifs overlapping with each element. e Genetic deletion experiments of PVT1 DHS and PPARA DHS in HUDEP-2 cells result in significant repression of PVT1 and PPARA expression, respectively. Browser tracks of the DNase I accessibility in WT HUDEP-2 and mutant (ΔHS) are shown. Barplots show the mean ± SE of normalized gene counts from n = 4 experiments. Source data for all relevant panels are provided within the Source Data file.