Fig. 6

G4 status is not involved in the hemin-BACH1-NRF2-mediated enhancer activation process. A, B Volcano plot (A) and heatmap (B) showing gene expression changes in HEK293T cells in response to hemin treatment for 6 h. C Network enrichment analysis with Metascape [67] of upregulated and downregulated genes by hemin. Each cluster is represented by different colors and each enriched term is denoted by a circle node. D, E Analysis of hemin binding signals, G4-CUT&Tag signals, Pol II, H3K4me3, and H3K27ac ChIP-Rx signals at promoters of the downregulated genes by hemin (N=262). Track examples at the ID4 and ID2 loci (D), heatmaps and metaplots (E) with 2-kb windows are shown. Hemin treatment induced G4 formation, reduced Pol II occupancy, and altered Histone 3 modifications at hemin-bound promoters, which eventually perturbed gene expression. F, G UCSC genome browser snapshots of G4-CUT&Tag signals, BACH1, NRF2, H3K27ac, P300, and Pol II ChIP-Rx signals and qPRO-seq signals at NRF2-induced HMOX1 (F) and FTH1 (G) loci. Hemin-induced HMOX1 and FTH1 expression was mediated by the release of BACH1 and binding of NRF2 at enhancer regions, which were accompanied by increased P300 occupancy and H3K27ac level. Interestingly, G4 is not involved in the enhancer activation process. H Motif analysis of BACH1 and NRF2 binding sites. I, J Heatmap (I) and boxplot (J) analyses demonstrate that hemin induced BACH1 release and NRF2 binding, without major alteration of G4-CUT&Tag signals