Extended Data Fig. 8: Sequential ChIP analysis of p53^R175H-BACH1-LSD2 complex and diagram for differential regulation of repression targets and activation targets of BACH1 by p53^R175H and LSD2.

a, Diagram for sequential ChIP analysis. Sequential ChIP analysis was followed the established protocol by Beischlag et al 2018 (Ref. ⁵²). Co-enrichment of BACH1/p53^R175H/LSD2 in gene promoter was analyzed by qPCR. b-e, Single ChIP assay (by BACH1 specific antibody) and sequential ChIP assay (firstly ChIP-ed by BACH1 antibody, secondly by p53 DO-1 antibody and thirdly by LSD2 antibody) for SLC7A11 promoter (b), CEMIP promoter (c), GCLM promoter (d) and FTH1 promoter (e) using Cal-33 cells. n = 3 technical replicates. The experiment was repeated twice with similar results. f. Diagram for differential regulation of repression targets (for example, SLC7A11) and activation targets (for example, CEMIP) of BACH1 by p53^R175H and LSD2. BACH1 has opposite functions in transcription on two different types of target genes: on one hand, it acts as a transcriptional repressor to downregulate a number of targets such as SLC7A11 critically involved in ferroptosis; on the other hand, BACH1 can also function as a transcriptional activator to induce pro-metastatic targets such as CEMIP to promote cancer metastasis. The interaction between LSD2 and BACH1 is very weak and unstable when p53^R175H is absent, but this interaction is significantly enhanced in the presence of p53^R175H expression since p53^R175H can strongly interact with both BACH1 and LSD2. p53^R175H is able to abrogate BACH1-mediated repression of SLC7A11 through the recruitment of LSD2 demethylase. This formation of p53^R175H-BACH1-LSD2 complex modifies the histone methylation status at the promoter of SLC7A11 and subsequently abrogates its transcriptional repression mediated by BACH1. Conversely, the recruitment of LSD2 demethylase by p53^R175H to the promoter of CEMIP results in enhanced transcriptional activation of CEMIP by BACH1. Data represent mean of three technical replicates.