AH receptor complex coactivator recruitment and function Free

2890

The Aryl Hydrocarbon Receptor (AHR) mediates the toxicity of several environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR and its dimerization partner, the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), form a DNA-binding transcription factor complex termed the Aryl Hydrocarbon Receptor Complex (AHRC). Active gene transcription by the AHRC is enhanced by the recruitment of coactivator proteins, which facilitate a more open chromatin configuration. The aim of this study was to investigate the dioxin-induced recruitment and function of several coactivator proteins on the CYP1A1 and CYP1B1 genes in the human breast carcinoma cell line, MCF-7. By use of the Chromatin Immunoprecipitation assay, we have observed a dioxin-dependent recruitment of several coactivator proteins and histone modifications over the CYP1A1 and CYP1B1 genes. Specifically, we demonstrate a dioxin-dependent recruitment of the p300, Sp1, Brg-1, Brahma (Brm) coactivator proteins, as well as a dioxin- and time-dependent acetylation of lysine residues 5,8,12, and 16 on histone H4. We show that the dioxin-induced acetylation of lysines histone H4 is very robust at the promoter regions when compared to the enhancer regions of both genes. We are also able to inhibit the dioxin-dependent acetylation of histone H4 by knocking down the endogenous levels of the p300 coactivator protein using RNA interference. siRNA has also allowed us to identify the chromatin remodeling factors, Brg-1 and Brm, as gene-specific coactivators of dioxin induction of CYP1A1. The Loss of Brg-1 and Brm has a negative effect on CYP1A1 dioxin-induction, while having no significant effect on CYP1B1 induction, indicating that these genes may exhibit different modes of chromatin modification during TCDD induction in MCF-7 cells. In this study, we implicate p300 as a regulator of dioxin-induced acetylation of lysine residues on histone H4 on both CYP1A1 and CYP1B1 and demonstrate gene specific coactivation by the Brg-1 and Brm chromatin remodeling factors to CYP1A1. Together, these data reveal insight into the complex nature of AHRC coactivator recruitment and function.