FIG. 7.

Activation of cAMP/PKA pathway increases NRF2 enrichment at ARE like elements in human ABCC2 promoter. (A) 8-Br-cAMP increases NRF2 enrichment at ARE-like sites in ABCC2 promoter. Huh-7 cells were treated with 8-Br-cAMP (0.1 mM) for 45 min and were washed and cross-linked for 10 min with 1% formaldehyde and crosslinking terminated by 125 mM glycine. The chromatin were sheared and prepared and incubated overnight with Protein G magnetic beads with Anti-NRF2 antibody or preimmune IgG. The antibody-bound chromatins and input DNA (1/10 dilution) were analyzed by PCR for the presence of DNA-fragments containing NRF2 bound-ARE-like element (MARE_ARE) with primers as indicated in Table 1. (B) 8-Br-cAMP increases NRF2 enrichment at other known ABCC2 AREs. Huh-7 cells were treated in a manner similar to (A) and the NRF2-antibody bound chromatin and input DNA were analyzed by RT²-PCR for the presence of DNA fragments containing MARE_ARE as well as ARE-like elements described by Stockel et al. (47) in ABCC2 promoter as well as negative primers directed at a non ARE-containing promoter region (Table 1). (C) 8-Br-cAMP increases SIRT1 enrichment at ABCC2 ARE-like elements in Huh-7 cells. Huh-7 cells were treated in a manner similar to (A), and the sheared chromatin fragments were incubated with anti-SIRT1 antibody and preimmune IgG overnight and the antibody-bound chromatins were analyzed by RT²-PCR for DNA fragments containing MARE_ARE (Table 1). (D) 8-Br-cAMP increases SIRT1 enrichment at the NQO1 ARE. Huh-7 cells treated in a manner similar to (A) and incubated with anti-SIRT1 antibody and preimmune IgG overnight and the antibody-bound chromatins were analyzed by PCR for DNA fragments containing ARE-like elements as described by Dhakshinamoorthy et al. (13) using primers indicated in Table 1.