Hey， Guys, I'm trying to identify a target gene for Foxo1 in Huh7 cell line. It's very strange that we have EMSA , luciferase promoter increase. However, when we over express Foxo1 in Huh7 cell line, the target gene is not change or seems to be decreased.
Therefore, have any one meet this situation?
Low accessibility of the promoter in the genome (epigenetics, acetylation/methylation markers or DNA methylation) could account for this. You would see efficient driving of the luciferase gene through an episomal promoter but not of the gene itself in its natural environment. Also, an unknown enhancer or repressor genomic region which is not part of the main promoter region could affect Foxo1 binding to its site in the genome.
For luciferase assay, it is much more straightforward. You just co-transfected Foxo1 and the promoter-containing luciferase plasmid. However, when you overexpressed Foxo1 in cells, the target gene can be regulated by multiple proteins rather than the solely Foxo1. The regulatory network is much more complicated.
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