Fig. 4: The risk allele G of rs73613962 enhances the binding ability of transcription factor HNF4A to promote PRMT7 expression.

a The result of the competitive EMSA. The binding affinity of protein to DNA oligos is demonstrated by adding unlabeled-G or unlabeled-T to the reaction. The light blue arrow indicates the protein-biotin-G-oligo complexes. This experiment is replicated two times at least and similar results are observed. b The enrichment of HNF4A on the rs73613962-containing region examined by ChIP. The left panel shows the diagram for HNF4A-ChIP. The antibody of HNF4A recognizes and binds to HNF4A; thus, the DNA sequence bound by HNF4A is pulled down from the fragmented chromatin. Then, the enriched DNA sequence is detected by the following qPCR as shown in the right panel. P = 0.0001 in HNF4A compared to IgG. c ChIP-PCR results. The agarose gel electrophoresis of PCR products for Input and ChIP, respectively, in the above panel. Light blue arrows indicate the marker bands. Source data are provided as a Source Data file. The relative gray value of each PCR band is analyzed using ImageJ in the bottom panel. P = 0.7769 and P < 0.0001 in HNF4A compared to IgG in Input and ChIP, respectively. d The dual-luciferase assay of enhancer activity for the enhancer-T and enhancer-G before and after the downregulation of HNF4A. P = 0.0046 in Enhancer-G compared to Enhancer-T without HNF4A knockdown, and P = 0.1493 and P = 0.0002 in Enhancer-T or Enhancer-G with HNF4A knockdown to Enhancer-T or Enhancer-G without HNF4A knockdown, respectively. e The result of the blocking EMSA of HNF4A. The amount of anti-HNF4A antibody in lane 3 and lane 4 is 1 μg and 2 μg, respectively. The IgG amount in lane 5 is 2 μg. The light blue arrow indicates the HNF4A-biotin-G-oligo complexes. We repeat this experiment two times at least and always observe the similar results. f qPCR detection of HNF4A and PRMT7 expressions after the downregulation of HNF4A in cells through two siRNAs. For detection of HNF4A expression level, P = 0.0017 and 0.0037 in HNF4A_small interfering RNA (HNF4A_si)1 or HNF4A_si2 compared to small interfering RNA negative control (siNC), respectively. For detection of PRMT7 expression level, P = 0.0001 and P = 0.0002 in HNF4A_si1 or HNF4A_si2 compared to siNC, respectively. All the above experiments were conducted in the QGY-7703 cell line (a–f). g–i The correlation of PRMT7 and HNF4A expressions showed in all HCC subjects (g), and subjects with the rs73613962 TT homozygote (h), or rs73613962 G allele carriers (i) in TCGA LIHC. TPM, transcripts per million. Values are expressed as the mean ± SD, n = 3 in b–d, and f. ‘ns’ means not significant; ** and *** mean P values less than 0.01, and 0.001, respectively (two-sided student’ t-test).