Xenobiotic response element binding enriched in both nuclear and microsomal fractions of rat cerebellum Nobuyuki Kuramoto, Katsuhiro Baba, 1 Keiko Gion, Chie Sugiyama, Hideo Taniura and Yukio Yoneda Laboratory of Molecular Pharmacology, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa, Japan Abstract Xenobiotic response element (XRE) is a core nucleotide sequence at the upstream of inducible target genes for the transcription factor aryl hydrocarbon receptor (AhR) that is responsible for signal transduction of exogenous environ- mental pollutants in eukaryotic cells. Immunoblotting analysis revealed the constitutive expression of AhR-related proteins in rat liver and brain, while specific binding of a radiolabelled probe containing XRE was detected in nuclear preparations of both liver and brain on gel retardation electrophoresis. Among discrete rat brain structures examined, cerebellum exhibited the highest XRE binding with less potent binding in hypotha- lamus, midbrain, medulla-oblongata, hippocampus, cerebral cortex and striatum. In contrast to liver and hippocampus, cerebellum also contained unusually higher XRE binding in microsomal fractions than that in either nuclear or mitoch- ondrial fractions. Limited proteolysis by V8 protease did not markedly affect XRE binding in cerebellar nuclear extracts, with concomitant diminution of that in hepatic and hippocam- pal nuclear extracts. In primary cultured cerebellar neurons, indigo was effective in significantly increasing XRE binding only when determined immediately after sustained exposure for 120 min in the presence of high potassium chloride. These results suggest the abundance of as-yet unidentified proteins with high affinity for XRE and responsiveness to indigo in both nuclear and microsomal fractions of rat cerebellum. Keywords: cerebellum, DNA binding, microsomes, xeno- biotic response element. J. Neurochem. (2003) 85, 264–273. In eukaryotic cells, de novo synthesis of functional proteins is mainly controlled at the level of transcription of genomic genesbyRNApolymeraseIIresponsiblefortheformationof mRNA from DNA in the nucleus. Transcription factors are nuclear proteins that specifically recognize particular core nucleotidesequencesatpromoterand/orenhancerregionson target genes and thereby elicit quantitative control of formation of mRNA as a result of modulation of the activity of RNA polymerase II in the nucleus. Transcription factors often have one of the most major protein motifs, leucine- zipper/helix-loop-helix motif, through which homo and/or hetero dimeric protein complex is formed. Xenobiotic response element (XRE; TNGCGTG) is a core nucleotide sequence specifically recognized by the transcription factor of a nuclear receptor type, aryl hydrocarbon receptor (AhR) (Saatcioglu et al. 1990). AhR is shown as a receptor for particularlipophilicligandsthatarehighlytoxicenvironmen- tal pollutants including 3-methylcholanthrene, benzo[a]pyrene Received July 5, 2002; revised manuscript received October 21, 2002; accepted December 27, 2002. Address correspondence and reprint requests to Dr Yukio Yoneda, Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology, 13–1 Takara-machi, Kan- azawa, Ishikawa 920–0934, Japan. E-mail: yyoneda@anet.ne.jp 1 The present address of Katsuhiro Baba is Biochemical Pharmacology, Nagoya Laboratories, Pfizer Inc. Aichi 470–2393, Japan. Abbreviations used: AhR, aryl hydrocarbon receptor; Arnt, Ah receptor nuclear translocator; Cdk, cyclin dependent kinase; CYP, cytochrome P-450; DIV, days in vitro; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; FCS, fetal calf serum; GAP-43, growth-associated protein 43; GFAP, glial fibrillary acidic protein; HKRB, HEPES buffered Krebs–Ringer buffer; HSP90, heat shock protein 90; MAP-2, microtubule associated protein 2; mXRE, mutated xenobioticresponseelement;NaGP,sodium b-glycerophosphate; NeuN, neuronal nuclear antigen; SDS, sodium dodecylsulfate; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TRE, 12-O-tetradecanoylphoepbor 13-acetate response element; VD3, vitamin D3; XRE, xenobiotic response element. Journal of Neurochemistry , 2003, 85, 264–273 doi:10.1046/j.1471-4159.2003.01679.x 264 Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 264–273