Fig. 4: The HDAC3/HNF4A complex regulates the transcriptional activity of TYMS.

A Information on H3K27Ac enrichment on the TYMS promoter region was acquired from the UCSC website. B GC cell lines, including MKN-45, HGC-27, were transfected with shNC or shHDAC3-2. H3K27Ac enrichment on the TYMS promoter was detected using ChIP. C Transcription factors (TFs) associated with the TYMS promoter were selected utilizing the UCSC website (threshold set at Minimum Score: 500), and TFs binding with HDAC3 were screened using the BioGRID4.4. An intersection was identified including HNF4A, MEF2A, GATA4, MAFK, KLF6, RARA, SNAI1. D The GEPIA database was used to screen the TFs, and the upregulated TFs (RARA and HNF4A) were presented. E The correction between HNF4A, RARA and TYMS were predicted based on the GEPIA database. F The binding of exogenous HDAC3 and HNF4A was tested using Co-IP in 293 T cells. G The binding of endogenous HDAC3 and HNF4A was detected in MKN-45 and HGC-27 cells using Co-IP. H The expression of HNF4A and TYMS in HNF4A depleted MKN-45 and HGC-27 cells was analyzed using Western blot. I Luciferase activity on the TYMS promoter was evaluated in HDAC3 and/or HNF4A overexpressed MKN-45 and HGC-27 cells using a dual-luciferase reporter assay. J The binding of HNF4A to the TYMS promoter was analyzed in MKN-45 and HGC-27 cells using ChIP-qPCR. K The binding of HDAC3 to the TYMS promoter was analyzed in MKN-45 and HGC-27 cells using ChIP-qPCR. L The binding of HDAC3 to the TYMS promoter in HNF4A knockdown MKN-45 and HGC-27 cells was tested using ChIP-qPCR. M The binding of HNF4A to the TYMS promoter in HDAC3-depleted MKN-45 and HGC-27 cells was detected using ChIP-qPCR. All experiments were repeated at least three times, *p < 0.05, **p < 0.01, ***p < 0.001.