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Fig. 4 | Molecular Cancer

Fig. 4

From: m6A modification-mediated CBX8 induction regulates stemness and chemosensitivity of colon cancer via upregulation of LGR5

Fig. 4

CBX8 maintains the H3K4me3 status at the LGR5 promoter by binding to KMT2b. a, Overview of the LGR5 promoter region with ChIP-seq data from ENCODE for H3K27me3, H3K4me3 and H3K27Ac in HCT-116 cells and for KMT2b in HepG2 cells. b, ChIP-qPCR analysis identified enrichment of H3K3me3 in the CBS1-CBS4 regions after CBX8 knockdown in DLD-1 cells. ChIP-qPCR with IgG was performed as the control. c, ChIP-qPCR analysis identified enrichment of H3K3me3 in the CBS1-CBS4 regions after CBX8 overexpression in DLD-1 cells. ChIP-qPCR with IgG was performed as the control. d, The RNAi screen of KMT2s (KMT2A-G) showed that KMT2b siRNA significantly downregulated LGR5 mRNA levels in DLD-1 cells. e, Western blot analysis of LGR5 expression in KMT2b knockdown DLD-1 cells overexpressing CBX8. f, CoIP showed the interaction between the CBX8 and KMT2b proteins in DLD-1 cells. g, HEK293 cells were transfected with Flag-tagged CBX8 with or without HA-tagged KMT2b. Lysates were subjected to IP using anti-Flag and anti-HA antibodies. h, ChIP-qPCR analysis performed to determine the binding affinity of KMT2b to the seven LGR5 promoter regions in DLD-1 cells showed that CBX8 bound to the KBS1-KBS3 regions in the LGR5 promoter. ChIP-qPCR with IgG was performed as the control. i, ChIP-qPCR analysis was used to determine the binding affinity of KMT2b to the KBS1-KBS3 regions after CBX8 knockdown. j, ChIP-qPCR analysis showed the enrichment of CBX8 and H3K4me3 in the CBS1-CBS3 regions after KMT2b knockdown in DLD-1 cells. k, Schematics of the luciferase reporter gene constructs. l, The luciferase reporter gene constructs were cotransfected with shCBX8 or shCtrl into DLD-1 cells, and reporter gene activity was measured after 48 h by a dual luciferase assay. The relative value in DLD-1 cells cotransfected with pGL3 (− 1224/317) and shCtrl was set to 100%. m, The luciferase reporter gene constructs were cotransfected with the CBX8 overexpression plasmid or empty vector into DLD-1 cells, and reporter gene activity was measured after 48 h by dual luciferase assay. n, Schematic of the five Flag-CBX8 recombinant proteins (CB1-CB5). o, Plasmids encoding a Flag-tagged, CBX8 truncation mutant were transfected into DLD-1 cells, anti-Flag antibody was used to immunoprecipitate the bound proteins, and the KMT2b level in the immunoprecipitates was determined by Western blot. Data are shown as the mean ± SD of three replicates (*, P < 0.05; **, P < 0.01)

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