Activation of antioxidant response and induction of HMOX1. (A) Representative immunoblots of total cellular proteins (20 μg) illustrating the effect of 25 μM arsenite on NRF2 and BACH1 protein levels. (B) Graphical representation of three separate experiments showing changes in expression of NRF2 and BACH1 proteins normalized to β-actin levels. (C) Representative immunoblots of nuclear (10 μg) and cytosolic proteins (20 μg) illustrating changes in subcellular localization of NRF2 and BACH1 following arsenite treatment (25 μM). (D) Graphical representation of three separate experiments showing changes in nuclear and cytosolic NRF2 and BACH1 proteins levels normalized to β-actin levels. The presence of NRF2 in nuclear extract at 0 h (N0) is provided as a positive immunoblot control for cytosolic NRF2. Quantification represents the mean ± SEM of three independent experiments. (E) Dose response of HMOX1 mRNA expression in HaCaT cells following a 6-h continuous treatment with the indicated concentration of arsenite. (F) Time course of HMOX1 mRNA expression in HaCaT cells following treatment with 25 μM arsenite. HMOX1 expression was determined using quantitative real-time PCR (qRT–PCR). HMOX1 mRNA concentrations were normalized to β-actin mRNA and expressed as fold change relative to untreated controls. Values represent at least three independent experiments quantified in triplicate.

Differential regulation of NRF2 and BACH1 activation. Immunoblots illustrating differential expression of NRF2 and BACH1 protein following treatment of HaCaT cells with 25 μM arsenite, 5 μM MG132, 25 μM hemin or MG132 + hemin combined. HaCaT cells were treated as indicated for 3 h or co-treated with 5 μM cycloheximide (CHX) following a 30-min CHX pretreatment. (A) Total cellular proteins (20 μg) from whole cell lysates. (B) Proteins extracts (10 μg) from nuclear lysates. (C) Proteins extracts (20 μg) from cytosolic lysates. Blots are representative of 2–3 separate experiments.

Time course of HMOX1 expression elicited by hemin and MG132 treatment. The time course of HMOX1 mRNA expression following treatment with 25 μM hemin or 5 μM MG132. HMOX1 mRNA was determined by quantitative real-time PCR (qRT–PCR), normalized to β-actin mRNA and expressed as percent maximum expression. Values represent at least three independent experiments quantified in triplicate ± SEM.

Induction of HMOX1 through the interplay of BACH1 and NRF2 with enhancer elements. (A) Binding of BACH1 (red ovals) at the E1 and E2 enhancer elements of HMOX1 in untreated control cells blocks NRF2 (blue ovals) binding and HMOX1 induction. Competition for ARE-binding elements between activated NRF2 and DNA-bound BACH1 is indicated by hypothetical rate constants representing stochastic binding (k_on) and dissociation (k_off) of NRF2. (B) Inactivation of BACH1 by hemin results in its removal from ARE motifs and elimination from the nucleus. Consequently, NRF2 can interact with exposed ARE enhancers to recruit RNA pol II (green oval) leading to high-level HMOX1 induction. (C) DNA-bound BACH1 blocks DNA binding of NRF2 despite its nuclear accumulation and prevents efficient HMOX1 induction. (D) Treatment with arsenite or co-treatment with hemin + MG132 results in BACH1 inactivation, nuclear accumulation and ARE binding of NRF2, and high-level HMOX1 induction.

Identification of NRF2 and BACH1 interactions with core ARE motifs of HMOX1. (A) The position of all 12 putative ARE motifs, relative to the HMOX1 transcription start site as annotated by the NCBI Homo sapiens Genome Map Viewer, Build 36.2. Open boxes = motif conforming to the consensus ARE sequence, shaded boxes = imperfect ARE motif. Boxed numerals indicate the number of motif repeats in that region. Arrows indicate the plus-strand orientation of each ARE motif relative to the consensus ARE sequence (RTGAYnnnGC). Relative DNA enrichment associated with NRF2 (B) and BACH1 (C) ChIP at each of the HMOX1 ARE-containing sites. Immunoprecipitated DNA was analyzed for enrichment by qRT–PCR using primers flanking ARE motifs at the positions indicated. Vertical bars and circle symbols graphically depict the relative magnitude of DNA enrichment at each position. Vertical bars indicate enrichment at positions −3928 bp (E1) and −9069 bp (E2) while circles correspond with regions where negligible DNA enrichment was observed. Values oriented vertically along the abscissa indicate the position of each DNA region amplified by qRT–PCR. Amplification was expressed in terms of ΔΔC_t as calculated by normalizing the C_t for each primer in chromatin immunoprecipitated samples to the C_t obtained from the respective input DNA and expressed as a percentage relative to the PCR primer having the maximum enrichment (–9069 bp). Values represent the mean ± SEM of at least three independent experiments performed in triplicate.

Association of NRF2 and BACH1 DNA binding with transcriptional activation. Time course of DNA binding by BACH1 (A) NRF2 (B) and RNA polymerase II (C) following treatment with 25 μM arsenite, 5 μM MG132 or 25 μM hemin. ChIP-enriched DNA was quantified using qRT–PCR with primers flanking the HMOX1 ARE motifs at positions −3992 (NRF2 and BACH1) and −148 (RNA Pol II) and is expressed as the value for each treatment normalized to its corresponding input and expressed as fold enrichment relative to untreated control. Figures represent the results of two independent experiments performed in triplicate ± SEM.

Differential regulation of HMOX1 and TXNRD1 by BACH1 and NRF2. (A) ChIP analysis of NRF2 and BACH1 interactions with ARE sites of HMOX1 and TXNRD1 before and after 25 μM arsenite treatment for 3 h. Binding of NRF2 (top panel) and BACH1 (bottom panel) to HMOX1 E1 and TXNRD1 is expressed relative to binding at the HMOX1 E2 enhancer element. Expression of HMOX1 (B) and TXNRD1 (C) mRNA was measured in HaCaT cells following treatment with 25 μM hemin, 5 μM MG132 or both in the presence (shaded bars) or absence (filled bars) of 5 μM CHX. Relative mRNA was determined by quantitative real-time PCR (qRT–PCR), normalized to β-actin mRNA and expressed as percent maximum expression. Values represent at least three independent experiments quantified in triplicate ± SEM.