Fig. 7: HMGA2-FACT-ATM-pH2A.X axis is required to solve R-loops and induce DNA demethylation.

a DNA immunoprecipitation (DIP) based promoter analysis of selected HMGA2 target genes using antibodies specific for double-stranded DNA (dsDNA) or 5-methylcytosine (5mC) and genomic DNA from Hmag2 + /+, Hmag2−/− MEF, as well as Hmga2−/− MEF that were stably transfected with a tetracycline-inducible expression construct (tetOn) for either WT Hmga2-myc-his or the lyase-deficient mutant RΔA Hmga2-myc-his. MEF were treated with doxycycline as indicated. b Schematic representation of the sequential order of events during transcription activation mediated by the HMGA2-FACT-ATM-pH2A.X axis. c ChIP-based promoter analysis of selected HMGA2 target genes using the indicated antibodies and chromatin from MEF treated as in a. In addition, MEF were treated with ATM inhibitor (ATMi; KU-55933) as indicated. d DIP-based promoter analysis as in a, using 5mC-specific antibodies. In addition, MEF were treated with ATMi as indicated. e ChIP-based promoter analysis of selected HMGA2 target genes using the indicated antibodies and chromatin from MEF treated as in a. In addition, MEF were transfected with control (Ctrl) or Gadd45a-specific small interfering RNA (siRNA) as indicated. f DIP-based promoter analysis as in a, using 5mC-specific antibodies. In addition, MEF were transfected with Ctrl or Gadd45a-specific siRNA as indicated. g ChIP-based promoter analysis of selected HMGA2 target genes using the indicated antibodies and chromatin from MEF treated as in a. h WB analysis using antibodies specific for HIS-tag, HA-tag and H3 after DRIP using the antibody S9.6⁴¹ and chromatin isolated from MLE-12 cells that were stably transfected either with a control (scramble, scr) or an Hmga2-specific short hairpin DNA (sh) construct and transiently transfected with WT Hmga2-myc-his or the lyase-deficient mutant RΔA Hmga2-myc-his and Gadd45-HA as indicated. Representative image from two independent experiments. Input (Inp), 5% of IP starting material; immunoglobulin G (IgG), negative control. In all bar plots, data are shown as means ± s.e.m. (n = 3 biologically independent experiments); asterisks, P-values after one-tailed t-test, ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, non-significant. See also Supplementary Fig. 8. Source data are provided as a Source Data files 01 and 02.