Fig. 4: Interaction between KDM1A and HNF4A regulates histone methylation. | Nature Communications

Fig. 4: Interaction between KDM1A and HNF4A regulates histone methylation.

From: Transposable elements-mediated recruitment of KDM1A epigenetically silences HNF4A expression to promote hepatocellular carcinoma

Fig. 4

a, b Co-IP assay was performed to test the interaction between endogenous (a) or exogenous (b) KDM1A and HNF4A. Each experiment was repeated twice with similar results. c Co-IP assay was performed in 293T cells overexpressing V5-tagged KDM1A and one of FOXA1-Flag, HNF4A-Flag, FOXA3-Flag, GATA4-Flag, or HNF1A-Flag. Interaction of the bait protein with V5-tagged KDM1A was detected through Western blot using Flag antibody. The experiment was repeated twice with consistent results. d Immunofluorescence staining with KDM1A and HNF4A antibodies was performed in HepG2 cells, and their colocalization was visualized by laser confocal imaging. Bar = 25 μm. The experiment was repeated three times with similar results. e RNA-seq experiment was performed in stable KDM1A knockdown HepG2 cells, and genes significantly upregulated in both sh-KDM1A-1 and sh-KDM1A-2 samples (Foldchange >2, P < 0.05) were defined as KDM1A-repressed genes. HOMER software was used to analyze the transcription factor motifs enriched in the promoter of KDM1A-repressed genes, which utilizes the one-sided hypergeometric test to calculate p-values, Benjamini–Hochberg (BH) method for p-value adjustment. The top 5 significant motifs were displayed. f Conserved recognition motifs of HNF4A were identified using MEME-ChIP software from ChIP-seq data of HepG2 cells. The significance was estimated by a one-sided Fisher’s exact test, with the p-values multiplied by the number of discovered motifs for adjustment. The top three significantly enriched motifs are shown. ChIP-seq intensities of KDM1A, HNF1A, FOXA3, and GATA4 in the ±10 kb region of the HNF4A motifs were shown by ngsplot. g CUT&Tag-seq experiments of H3K4me1, H3K4me2, H3K9me2, and H3K27ac were performed in HepG2 cells with stable KDM1A knockdown or treated with SP2509. Ngsplot displays the sequencing signal intensity distribution in the ±10 kb region of the HNF4A motif for the above experiments. h Heatmap shows the sequencing signal intensity in the HNF4A protein complex common binding region and ±10 kb region for the above sequencing experiments. Source data are provided as a Source Data file.

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