Cell type and context-specific function of PLAG1 for IGF2 P3 promoter activity

The fetal transcription factor PLAG1 is found to be overexpressed in cancers,and has been suggested to bind the insulin like growth factor 2 (IGF2) P3 promoter,and to activate the IGF2 gene. The expression of IGF2 has partly been linked toloss of CTCF-dependent chromatin insulator function at the H19 imprinting controlregion (ICR). We investigated the role of PLAG1 for IGF2 regulation in Hep3B andJEG-3 cell lines. Chromatin immunoprecipitation revealed cell type-specific bindingof PLAG1 to the IGF2 P3 promoter, which was substantially insensitive to recombinantPLAG1 overexpression in the endogenous context. We hypothesized that the H19 chromatininsulator may be involved in the cell type-specific PLAG1 response. By using aGFP reporter gene/insulator assay plasmid construct with and without the H19 ICRand/or an SV40 enhancer, we confirm that the effect of the insulator is specificallyassociated with the activity of the IGF2 P3 promoter in the GFP reporter system,and furthermore, that the reporter insulator is functional in JEG-3 but not inHep3B cells. FACS analysis was used to assess the function of PLAG1 in low endogenouslyexpressing, but Zn-inducible stable PLAG1 expressing JEG-3 cell clones. Considerableincrease in IGF2 expression upon PLAG1 induction with a partial insulator overridingactivity was found using the reporter constructs. This is in contrast to the effectof the endogenous IGF2 gene which was insensitive to PLAG1 expression in JEG-3,while modestly induced the already highly expressed IGF2 gene in Hep3B cells.We suggest that the PLAG1 binding to the IGF2 P3 promoter and IGF2 expressionis cell type-specific, and that the PLAG1 transcription factor acts as a transcriptionalfacilitator that partially overrides the insulation by the H19 ICR.