Phenobarbital Induced Transcriptional Activation of CYP2B Genes by Chromatin Modification at the PBRU Region

Phenobarbital (PB) is xenobiotic compound that highly induces the transcription of CYP2B genes in the liver. PB-mediated CYP2B activation is regulated by the PB-dependent enhancer (PBRU) which contains binding sites for NF-1 and two nuclear receptor binding sites (NR-1,2). A third NR site is located at the 5' side of the PBRU. PB induces the nuclear translocation of a constitutively active nuclear receptor, constitutive androstane receptor (CAR) which binds to the NR sites as a heterodimer with RXR and activates CYP2B gene expression. To understand mechanisms by which the CYP2B genes is repressed in the untreated animal and by which CAR activates transcription, first, the repression of CAR transactivation by short heterodimer receptor (SHP) was examined and, second, the effect PB treatment on nucleosomal position and structure in native chromatin was examined. SHP did not affect the binding of CAR/RXR to the PBRU or the recruitment of the coactivator GRIP 1 by CAR/RXR in vitro. Direct interaction of SHP with corepressors and an enhanced inhibition of PBRU by mSin3A/HDAC1 in the presence of SHP were observed which suggested that SHP recruits corepressors to the PBRU to repress the Cyp2b10 gene, possibly in untreated animal. In the chromatin studies, the minimal PBRU (NR-1/NF-1/NR-2) was mapped in a nucleosome core while NR-3 was in the linker region. Nucleosomal structure at the proximal promoter of Cyp2b10 was not detected. Disruption, sliding, or remodeling of the PBRU nucleosome was not detected after PB treatment. By in vivo ChIP assays, the recruitment of GRIP1/CBP were observed at the PBRU, consistent with the observed increased acetylation of histones H3 and H4 and activation of Cyp2b10 after PB treatment. Brg-1, an ATPase of the Swi/Snf remodeling complex, was recruited at the PBRU. Brg-1 was shown to interact directly with CAR via the CAR DNA binding domain. Transcriptional activation by CAR in transient transfections was enhanced by both GRIP1/CBP and Brg-1, but not by the ATPase mutant of Brg-1. Brg-1 did not enhance activity of CAR DBD which is consistent with the binding studies. These studies demonstrated that the transcriptional activation of Cyp2b10 gene is mediated by PB-induced modifications of chromatin at the PBRU.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular and Integrative Physiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Bae, Yangjin

Contributors dc:contributor

• Kemper, Byron W.

• Biology, Molecular

Language dc:language

eng

Identifier

(MiAaPQ)AAI3223538

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/87240