Ser100-Phosphorylated RORα Orchestrates CAR and HNF4α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6.

We have previously shown that the RORα phosphorylation plays a pivotal role in Sult1e1 gene regulation within mouse liver. Here, we found serine 100 phosphorylated RORα orchestrates CAR and HNF4α to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPH). RORα knockdown using small interfering RNAs (siRNAs) suppressed CYP2B6 mRNAs in HPH, while transient expression of RORα in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (ChIP) and gel shift assays, we found that RORα in the form of phosphorylated S100 (p-Ser100 RORα) directly bound to a newly identified RORα response element (2B6-RORE, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB treated HPH, p-Ser100 RORα was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4α binding site. Chromatin conformation capture (3C) assay revealed direct contact between the PBREM and OARE only in PB treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORα at Ser100 residue in coimmunoprecipitation assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB treated mouse liver confirmed that HNF4α formed a complex with Ser 100 phosphorylated RORα as shown by super-shifted complexes with anti-p-Ser100 RORα and anti-HNF4α antibodies. Altogether, the results established that p-Ser100 RORα, bridging the PBREM and OARE, orchestrates CAR and HNF4α to form active chromatin complex during PB induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics and it is prone to induction by xenobiotics including phenobarbital via CAR and HNF 4α. Here, we show that RORα, through phosphorylated S100 residue, orchestrated CAR-HNF4α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role RORα in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation site within the DBD of the receptor.