Phenobarbital induction of transcription of CYP2B genes is mediated by an enhancer, termed a
phenobarbital responsive unit (PBRU), approximately 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 hyper-sensitivity 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 approximately 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.