Fig. 5: Perturbation of regulatory elements in DENND1A impacts testosterone levels.

a Perturbation loci: We targeted four candidate regulatory elements (Elements 1-4, also Figure S23) and the promoter regions of DENND1A. We designed between 5&7 guides per region. The STARR-seq activity (purple) and chromatin accessibility (grey) for these regions are shown. b H295R cells that stably express dCas9-p300 or dCas9-KRAB were transduced with lentiviral pools of guide RNAs for each regulatory element. The cell media supernatant was collected 2- and 4-days post transduction for measuring testosterone concentration produced by the cells. RNA was harvested from the cells 4-days post transduction to measure changes in gene expression. Created using BioRender. c, d Log-fold change of DENND1A expression (GAPDH as control) for H295R-dCas9-p300 (c) and H295R-dCas9-KRAB (d) cells with 10 μM forskolin or DMSO control. A set of 5 non-targeting control guides were designed to not target any part of the human genome as control cell population. Each individual data point is a biological replicate (n = 4); and represent data from 3 technical replicates. e, f Testosterone concentration (ng/ml) measured in the cell media 4 days post transduction in H295R-dCas9-p300 (e) and H295R-dCas9-KRAB (f) cells targeted with the specific guide RNA. Cells were cultured in the presence or absence of 10 μM forskolin. For each sample, the respective drug-treated scrambled gRNA sample was used as the control. Each individual data point is a biological replicate (n = 4); and represent data from 2 technical replicates. For (c–f); the box ranges from the 25th to the 75th percentile, and the median value (middle line). The whiskers extend from each quartile to the minimum and maximum values within 1.5 × interquartile range (IQR) of the box. The individual data points are also plotted. Significance of perturbations was done using two-sided t-test by comparing each perturbation to the negative control for the respective DMSO/Forskolin conditions. If the p-value is less 0.05, it is specified on the plot. Source data are provided as a Source Data file.