Ferroptosis model system by the re-expression of BACH1

Abstract

Ferroptosis is a regulated cell death induced by iron-dependent lipid peroxidation. The heme-responsive transcription factor BTB and CNC homology 1 (BACH1) promotes ferroptosis by repressing the transcription of genes involved in glutathione (GSH) synthesis and intracellular labile iron metabolism, which are key regulatory pathways in ferroptosis. We found that BACH1 re-expression in Bach1-/- immortalized mouse embryonic fibroblasts (iMEFs) can induce ferroptosis upon 2-mercaptoethanol removal, without any ferroptosis inducers. In these iMEFs, GSH synthesis was reduced, and intracellular labile iron levels were increased upon BACH1 re-expression. We used this system to investigate whether the major ferroptosis regulators glutathione peroxidase 4 (Gpx4) and apoptosis-inducing factor mitochondria-associated 2 (Aifm2), the gene for ferroptosis suppressor protein 1, are target genes of BACH1. Neither Gpx4 nor Aifm2 was regulated by BACH1 in the iMEFs. However, we found that BACH1 represses AIFM2 transcription in human pancreatic cancer cells. These results suggest that the ferroptosis regulators targeted by BACH1 may vary across different cell types and animal species. Furthermore, we confirmed that the ferroptosis induced by BACH1 re-expression exhibited a propagating effect. BACH1 re-expression represents a new strategy for inducing ferroptosis after GPX4 or system Xc- suppression and is expected to contribute to future ferroptosis research.

Keywords: BACH1 Abbreviations: AIFM2, apoptosis-inducing factor mitochondria-associated 2; ANOVA, analysis of variance; BACH1, BTB and CNC homology 1; Bach1−/− mice, Bach1 knockout mice; BTB, Broad complex, Tramtrack, Bric-a-brac domain; bZIP, basic leucine zipper; ChIP-seq, chromatin immunoprecipitation sequencing; CNC, Cap‘n’Collar region; DAPI, 4′,6-diamidino-2-phenylindole; DFX, deferasirox; DMSO, dimethyl sulfoxide; EMT, epithelial–mesenchymal transition; Ferr-1, ferrostatin-1; FINs, ferroptosis inducers; FSP1, Ferroptosis suppressor protein 1; Fth1, ferritin heavy chain 1; Ftl, ferritin light chain; GCL, glutamate-cysteine ligase; Gclc, GCL catalytic subunit; Gclm, GCL modifier subunit; GEO, Gene Expression Omnibus; GPX4, glutathione peroxidase 4; GSH, glutathione; HO-1 (Hmox1), heme oxygenase 1; iMEFs, immortalized MEFs; KuO, Kusabira Orange; MAFK, musculoaponeurotic fibrosarcoma oncogene homolog bZIP transcription factor K; mBACH1, Bach1 gene of Mus musculus; 2-ME, 2-mercaptoethanol; MEFs, mouse embryonic fibroblasts; NRF2, nuclear factor-erythroid 2-related factor 2; NSA, necrosulfonamide; PDAC, pancreatic ductal adenocarcinoma; PI, Propidium iodide; Ptgs2, prostaglandin-endoperoxide synthase 2; RSL3, (1S,3R)-RSL3; Slc40a1, solute carrier family 40 member 1; Slc7a11, solute carrier family 7 member 11; TFRC, transferrin receptor 1; Z-VAD.FMK, Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone; extracellular signal; ferroptosis; fibroblasts; transcription.