Fig. 7: HKDC1 was a downstream transcription target of HNF4α.

A Overexpression of HNF4α7, but not HNF4α7(ΔDBD), resulted in an elevation of HKDC1 mRNA expression in HGC-27 and OCUM-1 cells. B Integration of HNF4α ChIP-seq data in seven HNF4α-positive GC cell lines (IM95, Ist1, KATO-III, NUGC4, OCUM-1, SNU16, and YCC3) and histone ChIP-seq data (H3K27ac, H3K4me3, and H3K4me1) in IM95 cell line from the GEO database (GSE114018 and GSE75898) was performed at human HKDC1 gene locus (NR_120648.1). The arrow indicated the direction of transcription. The blue frames denoted potential HNF4α binding regions, namely E1, E2, E3, E4 and E5. C HNF4α promoted the transcription of the potential HNF4α binding region E4. HEK293T cells transiently expressing or HGC-27 cells stably expressing pLV vector or pLV-HNF4α7 were cotransfected with pRL-TK and pGL6-TA constructs containing E1, E2, E3, E4, or E5, as indicated. Twenty-four hours later, cells were harvested, and a dual-luciferase reporter assay was conducted. D Two potential HNF4α response elements (HREs), namely S1 ( + 4323 ~ +4334 bp) and S2 ( + 4365 ~ +4376 bp), were predicted in the potential HNF4α binding region E4 within the human HKDC1 gene locus. The sequence (+4323 ~ +4381 bp, named S3), containing S1 and S2, was employed in subsequent experiments. E Overexpression of HNF4α7 promoted the transcription of S3, but not its mutant with mutations in both S1 and S2 mentioned in (D). HEK293T cells transiently expressing or HGC-27 cells stably expressing pLV vector or pLV-HNF4α7 were cotransfected with pRL-TK and pGL6-TA constructs containing S3 or its mutant, as indicated. F–H The binding of HNF4α to S3 within the human HKDC1 gene locus was detected by EMSA (F) and ChIP (H) assay. For EMSA, recombinant GST-HNF4α7 or its mutant GST-HNF4α7(ΔDBD) protein was incubated with biotin-labeled oligonucleotides of S3. Coomassie blue staining of the purified recombinant GST-HNF4α7 and GST-HNF4α7(ΔDBD) proteins is indicated in (G). For ChIP, HGC-27 cells (with HNF4α7 overexpressed beforehand) or AGS cells were harvested, and the ChIP assay was carried out. All data were shown as the mean ± SEM. The difference significance was analyzed by one-way ANOVA (A) or unpaired two-tailed Student’s t-test (C, E).