Nrf2 activation is associated with Z-DNA formation in the human HO-1 promoter

Abstract

Using a luciferase reporter assay, we previously demonstrated that a Z-DNA-forming sequence of alternating thymine–guanine repeats in the human heme oxygenase-1 gene (HO-1) promoter is involved in nuclear factor erythroid-derived 2 (NF-E2)–related factor 2 (Nrf2)-mediated HO-1 promoter activation. However, the actual Z-DNA formation in this native genomic locus has not been experimentally demonstrated. To detect Z-DNA formation in vivo, we generated a construct containing the Z-DNA-binding domain of human adenosine deaminase acting on double-stranded RNA 1 fused with enhanced green fluorescence protein, designated as the Z-probe. A chromatin immunoprecipitation assay using an anti-GFP antibody showed that the Z-probe detects the well-characterized Z-DNA formation in the CSF1 promoter. Using this detection system, we demonstrated that the glutathione-depleting agent, diethyl maleate, induced Nrf2-dependent Z-DNA formation in the HO-1 promoter, but not in the thioredoxin reductase 1 gene promoter. Moreover, a time course analysis revealed that Z-DNA formation precedes HO-1 transcriptional activation. Concurrent with Z-DNA formation, nucleosome occupancy was reduced, and the recruitment of RNA polymerase II was enhanced in the HO-1 promoter region, suggesting that Z-DNA formation enhances HO-1 gene transcription. Furthermore, Nrf2-induced BRG1 recruitment to the HO-1 promoter temporarily occurred simultaneously with Z-DNA formation. Thus, these results implicate Nrf2-dependent Z-DNA formation in HO-1 transcriptional activation and suggest the involvement of BRG1 in Z-DNA formation.

Nrf2- and ATF4-Dependent Upregulation of xCT Modulates the Sensitivity of T24 Bladder Carcinoma Cells to Proteasome Inhibition

“…Chromatin immunoprecipitation (ChIP) analysis was performed as previously described with a few modifications (40). In brief, T24 cells were incubated with 100 nM bortezomib for 6 h and then fixed with 1% formaldehyde for 10 min at 37°C.…”

“…pSV-Luc-xCT int mt was constructed by site-directed mutagenesis using the following primer pair: forward, 5=-CAC ACT GAA TAG TGC TAA GCC CCT CTG AAT AGC AAA TTT CC-3=; reverse, 5=-GGA AAT TTG CTA TTC AGA GGG GCT TAG CAC TAT TCA GTG TG-3=. The expression plasmid pcDNA3-hNrf2 was prepared as previously described (40). pcDNA3-hATF4 was produced by subcloning PCR-amplified human ATF4 cDNA into the BamHI/EcoRI sites of the pcDNA3 vector as described previously.…”

The chromatin environment shapes DNA replication origin organization and defines origin classes

“…This is reminiscent of the nucleosome positioned downstream from the ORC binding site described in budding yeast origins (Eaton et al 2010) and in a panel of six mouse origins (Lombraña et al 2013). Moreover, TG-rich sequences can form Z-DNA structures (Wahls et al 1990(Wahls et al , 1991Majewski and Ott 2000) that have been detected in positioned nucleosomes, where they facilitate their remodeling (Liu et al 2006;Maruyama et al 2013). Remarkably, MCM helicase activity is weak on a nucleosome template, and the chromatin remodeling complex FACT is necessary to promote DNA unwinding (Tan et al 2006).…”

Gold nanoparticles induce heme oxygenase-1 expression through Nrf2 activation and Bach1 export in human vascular endothelial cells

“…These Abs were immobilized to magnetic protein G beads and used for the IP reactions. The precipitated DNA fragments were detected using RT-PCR and real-time PCR with the following previously described primers: 25 HO-1 E2 enhancer region: 5′-CCCTGCTGAGTAATCCTTTCC-3′ and 5′-GGCGGTGACTTAGCGAAAAT-3′; and HO-1 gene promoter region: 5′-GCCAGAAAGTGGGCATCAG-3′ and 5′-CTGAGGACGCTCGAGGGAG-3′. The real-time PCR was performed using a 7500 StepOne Real-Time PCR System (Thermo Fisher Scientific).…”