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Heme oxygenase-1 induction by NRF2 requires inactivation of the transcriptional repressor BACH1.

Reichard JF, Motz GT, Puga A - Nucleic Acids Res. (2007)

Bottom Line: In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1.By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction.Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati, Cincinnati, OH 45267-0056, USA. john.reichard@childrens.harvard.edu

ABSTRACT
Oxidative stress activates the transcription factor NRF2, which in turn binds cis-acting antioxidant response element (ARE) enhancers and induces expression of protective antioxidant genes. In contrast, the transcriptional repressor BACH1 binds ARE-like enhancers in cells naïve to oxidative stress and antagonizes NRF2 binding until it becomes inactivated by pro-oxidants. Here, we describe the dynamic roles of BACH1 and NRF2 in the transcription of the heme oxygenase-1 (HMOX1) gene. HMOX1 induction, elicited by arsenite-mediated oxidative stress, follows inactivation of BACH1 and precedes activation of NRF2. BACH1 repression is dominant over NRF2-mediated HMOX1 transcription and inactivation of BACH1 is a prerequisite for HMOX1 induction. In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1. By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction. Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

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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 ΔΔCt as calculated by normalizing the Ct for each primer in chromatin immunoprecipitated samples to the Ct 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.
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Figure 2: 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 ΔΔCt as calculated by normalizing the Ct for each primer in chromatin immunoprecipitated samples to the Ct 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.

Mentions: To identify the maximum possible BACH1- and NRF2-binding sites, we searched 10 kb upstream of the HMOX1 TSS for core ARE motifs conforming to the sequence RTGAYNNNGC or its reverse complement (5). Twelve consensus elements were identified (Table 1) and each of these sites were investigated for NRF2 and BACH1 interactions by ChIP analysis (Figure 2A). Of the 12 ARE motifs, NRF2 and BACH1 interact with the same two sites containing multiple ARE motifs; one, a more proximal site located at −3928 bp upstream of the TSS (E1) and the other a more distal site at −8979 bp upstream (E2). NRF2 binds both of these sites after arsenite treatment (Figure 2B) while BACH1 binds both of them in arsenic naïve cells (Figure 2C). The proximal E1 element is composed of two slightly different ARE core motifs having the sequences GCtgcGTCAT and GCtgaGTCAC and separated by 54 nt. The more distal E2 site consists of four identical repeats of the core ARE motif having the sequence GCtraGTCAC, each separated by 19 nt. An additional single motif is located at −9491 bp, 415 bp further upstream from the end of this ARE tetrad for a total of five elements in this DNA region. In control cells, BACH1 binding at the E2 site is 5-fold greater than at E1. Following arsenite treatment, NRF2 binding at the distal E2 tetrad is greater than at the E1 site by ∼2.5-fold. None of the other five remaining ARE motifs were bound by either NRF2 or BACH1. These data show that BACH1 and NRF2 undergo reciprocal binding at the E1 and E2 enhancers following oxidative stress initiated by arsenite treatment.Figure 2.


Heme oxygenase-1 induction by NRF2 requires inactivation of the transcriptional repressor BACH1.

Reichard JF, Motz GT, Puga A - Nucleic Acids Res. (2007)

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 ΔΔCt as calculated by normalizing the Ct for each primer in chromatin immunoprecipitated samples to the Ct 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.
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Figure 2: 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 ΔΔCt as calculated by normalizing the Ct for each primer in chromatin immunoprecipitated samples to the Ct 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.
Mentions: To identify the maximum possible BACH1- and NRF2-binding sites, we searched 10 kb upstream of the HMOX1 TSS for core ARE motifs conforming to the sequence RTGAYNNNGC or its reverse complement (5). Twelve consensus elements were identified (Table 1) and each of these sites were investigated for NRF2 and BACH1 interactions by ChIP analysis (Figure 2A). Of the 12 ARE motifs, NRF2 and BACH1 interact with the same two sites containing multiple ARE motifs; one, a more proximal site located at −3928 bp upstream of the TSS (E1) and the other a more distal site at −8979 bp upstream (E2). NRF2 binds both of these sites after arsenite treatment (Figure 2B) while BACH1 binds both of them in arsenic naïve cells (Figure 2C). The proximal E1 element is composed of two slightly different ARE core motifs having the sequences GCtgcGTCAT and GCtgaGTCAC and separated by 54 nt. The more distal E2 site consists of four identical repeats of the core ARE motif having the sequence GCtraGTCAC, each separated by 19 nt. An additional single motif is located at −9491 bp, 415 bp further upstream from the end of this ARE tetrad for a total of five elements in this DNA region. In control cells, BACH1 binding at the E2 site is 5-fold greater than at E1. Following arsenite treatment, NRF2 binding at the distal E2 tetrad is greater than at the E1 site by ∼2.5-fold. None of the other five remaining ARE motifs were bound by either NRF2 or BACH1. These data show that BACH1 and NRF2 undergo reciprocal binding at the E1 and E2 enhancers following oxidative stress initiated by arsenite treatment.Figure 2.

Bottom Line: In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1.By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction.Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati, Cincinnati, OH 45267-0056, USA. john.reichard@childrens.harvard.edu

ABSTRACT
Oxidative stress activates the transcription factor NRF2, which in turn binds cis-acting antioxidant response element (ARE) enhancers and induces expression of protective antioxidant genes. In contrast, the transcriptional repressor BACH1 binds ARE-like enhancers in cells naïve to oxidative stress and antagonizes NRF2 binding until it becomes inactivated by pro-oxidants. Here, we describe the dynamic roles of BACH1 and NRF2 in the transcription of the heme oxygenase-1 (HMOX1) gene. HMOX1 induction, elicited by arsenite-mediated oxidative stress, follows inactivation of BACH1 and precedes activation of NRF2. BACH1 repression is dominant over NRF2-mediated HMOX1 transcription and inactivation of BACH1 is a prerequisite for HMOX1 induction. In contrast, thioredoxin reductase 1 (TXNRD1) is regulated by NRF2 but not by BACH1. By comparing the expression levels of HMOX1 with TXNRD1, we show that nuclear accumulation of NRF2 is not necessary for HMOX1 induction; rather, BACH1 inactivation permits NRF2 already present in the nucleus at low basal levels to bind the HMOX1 promoter and elicit HMOX1 induction. Thus, BACH1 confers an additional level of regulation to ARE-dependent genes that reveals a new dimension to the oxidative stress response.

Show MeSH
Related in: MedlinePlus