Tissue-specific chromatin structure of the phenobarbital- responsive unit and proximal promoter of CYP2B1/2 and modulation by phenobarbital

摘要

Phenobarbital induction of transcription of CYP2B genes is mediated by an enhancer, termed a phenobarbital responsive unit (PBRU), ~2000 bp 5′ of the transcription start site. To further delineate the mechanism of phenobarbital induction, protein binding in native chromatin and the nucleosomal structure of the PBRU and proximal promoter were examined in liver and kidney, in which the CYP2B1/2 genes are expressed and not expressed, respectively. Protein binding to the PBRU in kidney chromatin was not detected even though in vitro DNase I footprints were not detectably different with nuclear extracts from liver and kidney. Likewise, protein binding to regulatory motifs was not detected in the proximal promoter region in kidney chromatin. In liver chromatin, however, DNase I hypersensitivity and partial protection of the regulatory motifs from DNase I digestion or reaction with dimethyl sulfate was observed and phenobarbital treatment increased the hypersensitivity but only modestly affected protection. Low resolution Southern analysis of micrococcal nuclease-digested chromatin from untreated rats revealed micrococcal nuclease hypersensitive regions in the proximal promoter and PBRU regions in liver, but not in kidney. Phenobarbital treatment increased hypersensitivity in liver in both regions. Micrococcal nuclease hypersensitivity in the PBRU was largely restricted to a linker region between phased nucleosomes while in the proximal promoter hypersensitivity extended over ~200 bp suggesting disruption of a nucleosome in this region. These data indicate that in liver phenobarbital treatment substantially alters protein binding to regulatory motifs in the PBRU, while not greatly affecting such binding in the proximal promoter, and substantially alters chromatin structure in both regions, presumably as a result of chromatin modifying factors recruited to the PBRU. In the kidney, chromatin is probably in a closed conformation that prevents binding of regulatory factors.