Epigenetic program involved in the establishment of FOXA1-dependent enhancers during cellular differentiation. (A) Western blot assay showing induction of FOXA1 expression after stimulation of P19 cells with retinoic acid (RA) for 48 h. (B) FOXA1 ChIP-qPCR experiments were performed in P19 cells stimulated (+) or not (−) with RA for 48 h or 120 h. Fold enrichments relative to a negative control region are indicated. Results are from three independent experiments. (C) H3K4me1, 2, and 3 ChIP-qPCR experiments performed and analyzed as in B. (D) The percentage of cytosine methylation for CpG dinucleotides found within 200 bp of the center of FOXA1-binding sites was determined using bisulfite pyrosequencing. Shown is the average methylation levels of all CpGs analyzed for a given binding site. (E) RT-qPCR experiments performed in P19 cells stimulated (+) or not (−) with RA for 48 h or 120 h. Expression of analyzed genes was normalized using Rplp0 and is shown as fold induction relative to expression in undifferentiated P19 cells, which was set to 1. Results are from two independent experiments performed in duplicates. Note that, as expected, expression of the pluripotency transcription factor Nanog was strongly reduced upon induction of P19 cell differentiation with RA (data not shown). (ND) Not detectable.
