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

<p>Proximity labeling identifies histone acetylase SIRT1 as a direct KDM4C nuclear interactor and a novel KDM4C–SIRT1–DUSP2 axis regulating ERK activity in PDAC. <b>A,</b> 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>B,</b> Cytoscape analysis of the biotinylated nuclear proteins by KDM4C-miniTurboID, with annotated biological processes. <b>C,</b> 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”. <b>D,</b> 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. <b>E,</b> 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. <b>F,</b> 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. <b>G,</b> Venn diagram illustrating high degree of correlation between the genes with increased H3K36me3 and H3K27Ac peaks at the promoter region in <i>KDM4C</i> KO cells. Integrating ChIP-seq with the RNA-seq data results in a list of 16 genes, including the protein phosphatase <i>DUSP2</i>, which targets ERK phosphorylation. <b>H,</b> RT-qPCR of <i>DUSP</i> 1, 2, 4, 5, and 6 in AsPC1 control cells and <i>KDM4C</i> KO shows <i>DUSP2</i> has the highest FC increase in mRNA expression, *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.01, via unpaired <i>t</i> test. <b>I,</b> Western blot shows increased DUSP2 protein levels in <i>KDM4C</i>-null clones.</p>