Ser100-Phosphorylated RORa Orchestrates CAR and HNF4a to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6 s

Abstract

We have previously shown that the retinoid-related orphan receptor alpha (RORa) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated RORa orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4a) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). RORa knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of RORa in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that RORa in the form of phosphorylated (p-) S100 directly bound to a newly identified RORa response element (RORa response element on CYP2B6 promoter, 2660/2649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 RORa was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4a binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORa at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4a formed a complex with Ser 100-phosphorylated RORa, as shown by supershifted complexes with anti-p-Ser100 RORa and anti-HNF4a antibodies. Altogether, the results established that p-Ser100 RORa bridging the PBREM and OARE orchestrates CAR and HNF4a to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes.