Fig. 4

DNA methylation represses hypoxia-induced cryptic transcript activation (a) Heatmaps of HIF1β binding (RPKM) and DNA methylation as determined using SeqCapEpi BS-seq at regions flanking the summit of HIF1β binding peaks (± 5 kb). Shown are HIF1β binding peaks that are shared between vehicle-treated and aza-treated MCF7 cells, or that are specific to aza-treated cells. In total, 12,782 HIF1β binding peak positions were detected across vehicle- and aza-treated MCF7 using a P < 10^− 15 threshold. b Violin plots of methylation detected by SeqCapEpi BS-seq at HIF1β binding peaks that are shared between vehicle-treated and aza-treated MCF7 cells, or that are specific to aza-treated MCF7 cells. c HIF1β binding sites in LINEs, LTRs, and SINEs after 10,000 random permutations and as observed by HIF1β ChIP-seq (actual HIF1β binding) for HIF1β binding peaks that are shared between vehicle- and aza-treated MCF7 cells, or specific to aza-treated MCF7 cells for all HIF1β sites (top panel) and only for distal HIF1β sites (bottom panel). d Distribution of HIF1β binding peaks detected in aza-treated MCF7 cells at retrotransposon families, color-coded by retrotransposon class (green: LTR; violet: LINE; yellow: SINE). e Volcano plots showing differential expression of HIF-bound cryptic transcripts, as determined by CREDENToR in MCF7 cells exposed to vehicle (DMSO) or 5-aza-2′-deoxycytidine (aza; 1 μM) for 4 days, hypoxia (0.5% oxygen, 1 day) or normoxia. Significantly upregulated and downregulated transcripts are highlighted in red and blue, respectively. The associated numbers refer to how many transcripts are up- or downregulated at a 1% FDR and a 0.001% FDR, as indicated by the horizontal line. f dsRNA formation potential of all cryptic transcripts (gray) and of HIF-bound cryptic transcripts (red). Shown are the fraction of all RNAs for which transcription overlaps with a transcript expressed from the complementary strand (“sense-antisense”), and RNAs containing the same retrotransposon repeat element in sense and antisense orientation (“palindromic”). P values from the chi-square test. g, h Expression of HIF-bound (g) and non-HIF-bound (h) cryptic transcripts relative to vehicle-treated controls (vehicle normoxia) in MCF7 cells wild-type (WT) (gupper panels and h) or knockout (KO) (gbottom panels) for HIF1B. Shown are expression changes as assessed using CREDENToR (gleft panels and h) and RepEnrich (gright panels), with error bars indicating geometric mean ± s.e.m. n.s. not significant, ***P < 0.001 by t-test