Fig. 4: EHF and CDX1 physically interact to regulate activity of differentiation markers in CRC cells.

A Representative ATAC-Seq tracks of the VIL1 gene in HCT116^EV, HCT116^EHF, HCT116^CDX1 and HCT116^{EHF + CDX1} cells. B Schematic of the structure of the VIL1 promoter. Shown are CDX1 and EHF binding sites. C, D Binding of C FLAG-EHF and D CDX1 to different regions of the VIL1 promoter in HCT116^EHF, HCT116^CDX1 and HCT116^{EHF + CDX1} cells normalised to IgG control and HCT116^EV by ChIP. E Re-ChIP analysis assessing binding of FLAG-EHF and CDX1 to various regions of the VIL1 promoter in HCT116^EHF, HCT116^CDX1 and HCT116^EHF+CDX1 cells normalised to IgG control and HCT116^EV. Values shown are mean ± SEM from a representative experiment in which the q-RT-PCR analysis was performed in triplicate. Similar results were obtained in a separate experiment. F, G Immunoprecipitation of F anti-FLAG-EHF probing for CDX1 and G anti-CDX1 probing for FLAG-EHF in HCT116 cells transiently transduced with either EHF, CDX1 or ΔPNT-EHF expression plasmids alone or in combination. H Schematic diagram of full-length EHF and ΔPNT-EHF expression plasmids. I, J Luciferase promoter reporter analysis of VIL1 co-transfected with EHF and CDX1 expression constructs in I HCT116 and J RKO cells for 72 h. Values shown are mean ± SEM from a representative experiment performed in quadruplicate, with results expressed as fold induction relative to pGL3 and normalised to Renilla luciferase activity. Similar results were obtained in a separate experiment. K HCT116 cells transiently transduced with EHF, CDX1 or ΔPNT-EHF expression plasmids alone or in combination and expression of EHF, CDX1 and differentiation markers determined by western blot.