EurekaMag.com logo
+ Site Statistics
References:
53,869,633
Abstracts:
29,686,251
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on LinkedInFollow on LinkedIn

+ Translate

Analysis of multiple nuclear receptor binding sites for CAR/RXR in the phenobarbital responsive unit of CYP2B2



Analysis of multiple nuclear receptor binding sites for CAR/RXR in the phenobarbital responsive unit of CYP2B2



Archives of Biochemistry and Biophysics 451(2): 119-127



The phenobarbital (PB) responsive enhancers in CYP2B genes contain a core of two direct repeat-4 nuclear receptor binding sites, NR-1 and NR-2, which flank an NF-1 site and appear to be most important for PB responsiveness. Additional sequences outside the core are required for maximal PB responsiveness, including a third direct repeat-4 site, NR-3. The PB response is mediated by constitutive androstane receptor (CAR) which binds as a CAR/RXR heterodimer to the NR sites. To determine the relative importance of the third NR site, each of the NR sites was mutated individually and in all combinations in the rat PB responsive unit (PBRU). Mutation of NR-3 resulted in similar effects on transactivation of the PBRU by CAR in HepG2 cells as did mutations of NR-1 and NR-2. The recruitment of GRIP1/SRC-2 by CAR/RXR to the PBRU assessed by gel shift assays was cooperatively enhanced if more than one NR site in the PBRU was occupied by CAR/RXR. NR-3 in combination with NR-1 or NR-2 was equal to NR-1 and NR-2 in mediating this cooperative recruitment. Recruitment of SRC-1 and GRIP1/SRC-2 was similar for all NR sites, while some selectivity of NR-1 for SRC-3 was observed. SRC-3 also exhibited CAR-independent activation of the PBRU in HepG2 cells. Micrococcal nuclease mapping of nucleosomes revealed that the NR-1/NR-2 core of the PBRU is present in a nucleosome while NR-3 is present in the linker adjacent to the nucleosome. In the linear sequence NR-3 is further from NR-1 than NR-2 is, but in a nucleosomal structure, NR-3 is well positioned for cooperative recruitment of GRIP1/SRC-2 by CAR/RXR that is bound to NR-3 and either NR-1 or NR-2, while NR-1 and NR-2 are on opposite sides of the nucleosome separated by the histone core. These results demonstrate that NR-3 is functionally similar to NR-1 and NR-2 in CAR transactivation of the PBRU in vitro and suggest that NR-3 may have a greater role in a chromatin context in vivo than is apparent from transient transfection studies. (c) 2006 Elsevier Inc. All rights reserved.

(PDF emailed within 0-6 h: $19.90)

Accession: 011757133

Download citation: RISBibTeXText

PMID: 16725103

DOI: 10.1016/j.abb.2006.04.016



Related references

The CYP2B2 phenobarbital response unit contains binding sites for hepatocyte nuclear factor 4, PBX-PREP1, the thyroid hormone receptor beta and the liver X receptor. Biochemical Journal 388(Pt 2): 407-418, 2005

Functional analysis of the phenobarbital-responsive unit in rat CYP2B2. Biochemical Pharmacology 62(1): 21-28, 1 July, 2001

Mutational analysis of the CYP2B2 phenobarbital response unit and inhibitory effect of the constitutive androstane receptor on phenobarbital responsiveness. Journal of Biological Chemistry 275(49): 38427-38436, 2000

Positive regulation of the rat CYP2B2 phenobarbital response unit by the nuclear receptor hexamer half-site.nuclear factor 1 complex. Biochemical Pharmacology 57(9): 1073-1076, 2000

Positive regulation of the rat CYP2B2 phenobarbital response unit by the nuclear receptor hexamer half-sitecntdotnuclear factor 1 complex. Biochemical Pharmacology 57(9): 1073-1076, May 1, 1999

Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit of nuclear factor-1, which binds simultaneously with constitutive androstane receptor /retinoid X receptor and enhances CAR/RXR-mediated activation of the PBRU. Journal of Biological Chemistry 276(10): 7559-7567, March 9, 2001

Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU. Journal of Biological Chemistry 276(10): 7559-7567, 2001

Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU. Journal of Biological Chemistry 276(10): 7559-7567, 2000

In vivo analysis of the phenobarbital-responsive rat CYP2B2 gene promoter and PBRU in transgenic mice. FASEB Journal 16(4): A180, March 20, 2002

Cell type specific activity of two glucocorticoid responsive unit of rat tyrosine aminotransferase gene is associated with multiple binding sites for c ebp and a novel liver specific nuclear factor. Nucleic Acids Research 19(1): 131-140, 1991

Phenobarbital alters protein binding to the CYP2B1/2 phenobarbital-responsive unit in native chromatin. Journal of Biological Chemistry 272(47): 29423-5, 1997

Localization of a phenobarbital-responsive element (PBRE) in the 5'-flanking region of the rat CYP2B2 gene. Gene 158(2): 263-268, 1995

A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer. Molecular and Cellular Biology 14(12): 8007-8017, 1994