Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 gene by xenobiotics.
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.
Inoue
Kaoru
K
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Borchers
Christoph H
CH
Negishi
Masahiko
M
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency / Q000302:isolation & purification / Q000378:metabolism*; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression; D006801:Humans; D051379:Mice; D019319:Okadaic Acid / Q000494:pharmacology; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015262:Xenobiotics / Q000494:pharmacology*; D000098550:Structural Maintenance of Chromosome Protein 1
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D000090702:Constitutive Androstane Receptor; D011725:Pyridines; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D014157:Transcription Factors; D015262:Xenobiotics; D000098550:Structural Maintenance of Chromosome Protein 1; D019319:Okadaic Acid; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating