Effects of overexpressing HNF4A on 3D cultured mouse and human colon organoid

To test the hypothesis that HNF4A should engage small intestine enhancers in colon and activate transcription, we over-expressed HNF4A in 2 independent mouse colonic orgnaoid lines and 5 independent human colonic organoid lines and performed RNA-seq studies. Both RNA-seq data revealed that small intestine functional pathways including fat, protein and carbohydrate digestion and absorption were activated after HNF4A OE. This gain-of-function study of HNF4A in mouse and human colon organoid supports HNF4A as a central mediator of small intestine gene activation in colonic plasticity, conserved across species. To investigate the effects of HNF4A in mouse and human colon, we established 2 independent mouse colon organoid lines and 5 independent human colon organoid lines and over-expressed HNF4A protein by lentivirus infection of the cultured organoids. We also included 2 control and 5 control groups for mouse and human respectively where the same colonic organoids were infected with control virus without HNF4A overexpression. We havested the cells, extracted RNA and used for RNA-profiling.