Figure 6 from The Lysine Demethylase KDM4C Is an Oncogenic Driver and Regulates ERK Activity in KRAS-Mutant Pancreatic Ductal Adenocarcinoma

Proximity labeling identifies histone acetylase SIRT1 as a direct KDM4C nuclear interactor and a novel KDM4C–SIRT1–DUSP2 axis regulating ERK activity in PDAC. A, Validating biotinylation activity by KDM4C-miniTurboID. AsPC1 cells were transfected with KDM4C-miniTurboID and then treated with 50 μmol/L biotin 24 hours after transfection for 30 minutes. Cells were then lysed and the whole lysates probed for biotin by streptavidin–HRP along with “untransfected” and “KDM4C-miniTurboID without biotin” as controls. B, Cytoscape analysis of the biotinylated nuclear proteins by KDM4C-miniTurboID, with annotated biological processes. C, List of high-confidence nuclear KDM4C interactors based on proximity labeling experiment. The values shown are the abundance ratio between “KDM4C-miniTurboID + biotin” over “KDM4C-miniTurboID without biotin”. D, KDM4C co-immunoprecipitation confirms binding to SIRT1 in PDAC cells but not to PARP1 or ZNF148. IgG isotype antibody was used as a control for nonspecific binding. E, Graphical illustration of KDM4C domain-specific deletion constructs: FL = full-length KDM4C construct, ΔTudor = Tudor domain–deleted KDM4C, ΔPHD = PHD-deleted KDM4C, and ΔJMJ = JMJ domain–deleted KDM4C construct. All constructs are HA-tagged for immunoprecipitation. F, The Tudor domain is responsible for KDM4C binding to SIRT1. HEK293 cells were cotransfected with Flag-tagged SIRT1 construct and either FL KDM4C or a KDM4C domain–deleted construct, and then the cells were lysed, and the HA-enriched eluates for respective samples were probed for SIRT1 using anti-Flag. All constructs could pull down SIRT1 except ΔTudor. G, Venn diagram illustrating high degree of correlation between the genes with increased H3K36me3 and H3K27Ac peaks at the promoter region in KDM4C KO cells. Integrating ChIP-seq with the RNA-seq data results in a list of 16 genes, including the protein phosphatase DUSP2, which targets ERK phosphorylation. H, RT-qPCR of DUSP 1, 2, 4, 5, and 6 in AsPC1 control cells and KDM4C KO shows DUSP2 has the highest FC increase in mRNA expression, *, P ≤ 0.05; **, P ≤ 0.01, via unpaired t test. I, Western blot shows increased DUSP2 protein levels in KDM4C-null clones.