Fig. 6: ZMYM3 is required for the epigenetic regulatory role of KDM1A in liver cancer cells.

(a) Screening KDM1A interacting proteins using LC-MS. b KDM1A negatively regulated genes were screened from RNA-seq data of HepG2 cells with KDM1A knockdown (sh-KDM1A-1 vs. sh-control and sh-KDM1A-2 vs. sh-control, Foldchange >2 and P < 0.05), and then genes bound by KDM1A were selected based on HepG2 cell KDM1A-ChIP-seq data. Finally, the transcriptional regulatory regions of genes directly negatively regulated by KDM1A were analyzed for transcriptional regulator (TR) enrichment using LISA software. The TRs that significantly enriched for KDM1A bound and negatively regulated genes were ranked by −Log P values and shown. LISA utilizes a one-sided Wilcoxon rank sum test to calculate p-values without adjustment. c Co-IP assay was performed in HepG2 and PLC cells using endogenous KDM1A or ZMYM3 as bait protein to verify the interaction between ZMYM3 and HNF4A. Each experiment was repeated three times with similar results. d The 3D plot shows the correlation among the ChIP-seq signal intensities of KDM1A, ZMYM3, and HNF4A at the gene transcription regulatory regions (TSS ± 10 kb). The Spearman correlation coefficients between each pair of these proteins were shown in Supplementary Fig. 6b. Red and blue points represent the TRR region with and without liver-TE, and the comparisons between peak scores in these two types of TRR were shown in Supplementary Fig. 6c. e, f CUT&Tag-seq analysis reveals the effects of ZMYM3 knockdown on KDM1A binding and H3K4me1 modification. CUT&Tag-seq experiments were performed using KDM1A and H3K4me1 antibodies in three groups of 97H cells: Vector control + sh-control, KDM1A + sh-control, and KDM1A + sh-ZMYM3. Ngsplot was used to display the changes in KDM1A binding strength and H3K4me1 modification within ±10 kb regions surrounding KDM1A binding sites, ZMYM3 motifs (e), and liver-TEs (f). The results showed that the knockdown of ZMYM3 decreased the binding of KDM1A to its target regions and increased the corresponding H3K4me1 modifications. g Colony formation ability was assessed in HepG2, PLC, and 97H cells across three groups: Vector control + sh-control, KDM1A + sh-control, and KDM1A + sh-ZMYM3, n = 3 biological replicates. Significance was examined by a two-sided t-test, and mean ± SD was shown. Source data are provided as a Source Data file.