Characterization of glucocorticoid receptor and hepatocyte nuclear factor 4α (HNF4α) binding to the hnf4α gene in the liver

Hepatocyte nuclear factor 4α (HNF4α) plays a crucial role in hepatocyte differentiation, liver organogenesis and regulation of liver functions. In mouse liver, HNF4α is expressed from two promoters, P1 and P2, the latter being very weakly active and only in the embryo. Previously, using transfection assays we identified an enhancer upstream of P1 that mediates both HNF4α transactivation and glucocorticoid induction and showed that HNF4α1, originated from P1, represses activity of the P2 promoter, possibly through its indirect recruitment to the promoter. However, glucocorticoid receptor (GR) binding to the enhancer was not shown and HNF4α binding to P2, first reported in isolated human hepatocytes, was not confirmed in mouse liver. Here, to analyse glucocorticoid inducibility and auto-regulation of the hnf4α gene in the liver, we accurately mapped and quantitatively assessed GR and HNF4α binding to enhancer and HNF4α recruitment to the P2 promoter using chromatin immunoprecipitation (ChIP) and real-time PCR. We proved that GR binds to enhancer from embryonic day (E) 17.5 onward and HNF4α even earlier. We showed that HNF4α binds to P2 independently of the activation function (AF) 1 domain in adult liver. We mapped the binding region between −400 and −200 bp upstream of the transcription start site. Although Sp1 binds within this region in vitro, we did not find evidence of a role of this factor in HNF4α recruitment. Our results suggest that, in the liver, HNF4α expression may be induced by glucocorticoids around birth and positive auto-regulation of the gene may take place early in development. They support a model of P2 repression involving HNF4α recruitment to promoter, possibly through interaction with several promoter-bound factors.