Fig. 1

High-throughput siRNA and chemical screens identify RRM2 depletion and resveratrol as mediators of XCR. a Schematic of the X chromosomes in female reporter MEFs carrying the luciferase reporter transgene in the Hprt locus specifically on the Xi. The Xist deletion on one of the chromosomes skews X-inactivation to the wild-type Xist-bearing X chromosome. b Diagram of the screening workflow. siRNAs from the mouse genome-wide library and selected chemical libraries were assayed in 384-well plates containing a column of positive and negative controls. Xi-luciferase reporter MEFs were added and incubated for 72 h in the presence of 5-aza-2′-dC (0.2 μM) prior to a luciferase assay. c Gene activity distribution plot ranked by the –log of the p-value obtained with the redundant siRNA activity (RSA) assay from duplicate genome-wide siRNA screens following transformation of the luminescence activity values into robust z-scores. The top validated hits, Dnmt1, Atf7ip, and Rrm2, are labeled. d (i) Graph depicting Xi-luciferase reporter reactivation upon knockdown of Rrm2 with the three siRNAs (A, B, C) obtained from the genome-wide library in the presence or absence of 5-aza-2′-dC (0.2 μM) in the 12-well format. Luminescence was measured 72 h after the start of the treatment. Error bars indicate standard deviation of luminescence unit values from three individual wells with a given treatment in one experiment. (ii) RT-qPCR for RNA levels of Rrm2 normalized to siGFP control and Gapdh expression. RNA was harvested in parallel to luciferase assays shown in (i). Error bars indicate standard deviation from three measurements in one experiment. e Activity of chemicals in the chemical screen in the presence of 5-aza-2′-dC (0.2 μM), ranked by luminescence unit with the value corresponding to resveratrol designated. f Xi-luciferase reporter assay as described in (di) titrating the resveratrol concentration with or without 5-aza-2′-dC (0.2 μM). g Xi-luciferase reporter assay as in (di) titrating hydroxyurea (HU) with or without (untreated) 5-aza-2′-dC (0.2 μM). The result for resveratrol treatment in the same experiment is given for comparison. h (i) Xi-luciferase reporter assay as in (di) comparing the consequences of 0.2 μM 5-aza-2′-dC treatment and siRNA-mediated knockdown of Dnmt1 to elicit reporter reactivation by 20 μM resveratrol. (ii) RT-qPCR for Dnmt1 RNA levels normalized to siGFP control and Gapdh expression in the same experiment as (i)