Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 gene by xenobiotics

Abstract

CAR (constitutive active/androstane receptor) regulates both the distal enhancer PBREM (phenobarbital-responsive enhancer module) and the proximal element OARE [OA (okadaic acid) response element] to synergistically up-regulate the endogenous CYP2B6 (where CYP is cytochrome P450) gene in HepG2 cells. In this up-regulation, CAR acts as both a transcription factor and a co-regulator, directly binding to and enhancing PBREM upon activation by xenobiotics such as TCPOBOP {1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene} and indirectly associating with the OARE in response to OA [Swales, Kakizaki, Yamamoto, Inoue, Kobayashi and Negishi (2005) J. Biol. Chem. 280, 3458–3466]. We have now identified the cohesin protein SMC1 (structural maintenance of chromosomes 1) as a CAR-binding protein and characterized it as a negative regulator of OARE activity, thus repressing synergy. Treatment with SMC1 small interfering RNA augmented the synergistic up-regulation of CYP2B6 expression 20-fold in HepG2 cells, while transient co-expression of spliced form of SMC1 abrogated the synergistic activation of a 1.8 kb CYP2B6 promoter. SMC1 indirectly binds to a 19 bp sequence (−236/−217) immediately downstream from the OARE in the CYP2B6 promoter. Both DNA affinity and chromatin immunoprecipitation assays showed that OA treatment dissociates SMC1 from the CYP2B6 promoter, reciprocating the indirect binding of CAR to OARE. These results are consistent with the conclusion that SMC1 binding represses OARE activity and its dissociation allows the recruitment of CAR to the OARE, synergizing PBREM activity and the expression of the CYP2B6 gene.