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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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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.
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Figure 6: 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.

Mentions: Although NRF2 and BACH1 interact with ARE motifs, several lines of evidence support that these interactions with DNA are not identical, thereby suggesting that genes could be identified that are regulated by one factor but not by the other (12,29,30). TXNRD1 was identified as one such gene. ChIP analysis was used to test for NRF2 and BACH1 binding to the single ARE motif located 9 bp upstream of the TSS in the TXNRD1 5′-flanking region. As illustrated in the upper panel of Figure 6A, NRF2 binds the ARE motif of the TXNRD1 in ‘control cells’ with an affinity significantly greater than either at HMOX1 E1 or E2. After arsenite-induced oxidative stress, this site becomes more strongly bound by NRF2, reaching a level that is intermediate between NRF2 binding at the HMOX1 E1 and E2 sites after activation, and ∼6.5-fold greater than control levels. In contrast, the lower panel of Figure 6A shows negligible binding of BACH1 to the TXNRD1 ARE in either control or arsenite-treated cells. As a consequence of differential transcription factor binding, we anticipated that MG132, but not hemin, would elicit NRF2-dependent TXNRD1 induction. To test this prediction, we treated HaCaT cells with MG132 or hemin and quantified mRNA levels of HMOX1 and TXNRD1 by qRT–PCR. In untreated control cells, basal TXNRD1 mRNA levels are 35-fold greater than the levels of HMOX1 mRNA. Inhibition of NRF2 activation with CHX significantly inhibited basal TXNRD1 expression but left HMOX1 expression unchanged (Figure 6B and C). This pattern of expression is consistent with functionally active NRF2 residing basally in the nucleus and rules out the possibility that this resident nuclear NRF2 contributes to basal HMOX1 expression. The role of activated NRF2 is demonstrated by the MG132 treatments that trigger maximal TXNRD1 induction but mediate only weak HMOX1 expression (Figure 6B and C). Pretreatment with CHX prior to MG132 blocks NRF2 synthesis, returning HMOX1 mRNA to control levels and reducing TXNRD1 mRNA to the levels of CHX-treated control cells. These expression data reflect the primary role of NRF2 in basal and inducible expression of TXNRD1 but not HMOX1. Hemin treatment, which inactivates BACH1, has no effect on TXNRD1 expression (Figure 6C), reflecting the fact that TXNRD1 is regulated independently of BACH1. In contrast, hemin elicits a significant and nearly maximal increase in HMOX1 expression (Figure 6B). CHX pretreatment predominantly blocks this induction, demonstrating a requisite role for resident nuclear NRF2 in HMOX1 induction without necessitating nuclear accumulation of activated NRF2. Combined treatment with hemin plus MG132 results in induction of TXNRD1 to levels similar to those in cells treated with MG132 alone while the level of HMOX1 induction is similar to that elicited by hemin (Figure 6B and C). These results show that TXNRD1 induction is attributable solely to activation of NRF2 by proteasome inhibition but that HMOX1 induction requires inactivation of BACH1. Thus, BACH1 inactivation is predominantly responsible for HMOX1 induction and that NRF2 activation contributes nominally to this process. Interestingly, the combination of MG132 plus hemin also shows that, when preceded by CHX treatment, HMOX1 expression remains significantly induced despite the lack of NRF2 and hints at the possible role for BACH1 in repressing the activity of transcriptional activators in addition to NRF2. These data establish that inactivation of BACH1 is more important than activation of NRF2 for induction of HMOX1 expression and that not all NRF2-regulated genes respond in this manner, as is the case of TXNRD1.Figure 6.


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

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

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.
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Figure 6: 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.
Mentions: Although NRF2 and BACH1 interact with ARE motifs, several lines of evidence support that these interactions with DNA are not identical, thereby suggesting that genes could be identified that are regulated by one factor but not by the other (12,29,30). TXNRD1 was identified as one such gene. ChIP analysis was used to test for NRF2 and BACH1 binding to the single ARE motif located 9 bp upstream of the TSS in the TXNRD1 5′-flanking region. As illustrated in the upper panel of Figure 6A, NRF2 binds the ARE motif of the TXNRD1 in ‘control cells’ with an affinity significantly greater than either at HMOX1 E1 or E2. After arsenite-induced oxidative stress, this site becomes more strongly bound by NRF2, reaching a level that is intermediate between NRF2 binding at the HMOX1 E1 and E2 sites after activation, and ∼6.5-fold greater than control levels. In contrast, the lower panel of Figure 6A shows negligible binding of BACH1 to the TXNRD1 ARE in either control or arsenite-treated cells. As a consequence of differential transcription factor binding, we anticipated that MG132, but not hemin, would elicit NRF2-dependent TXNRD1 induction. To test this prediction, we treated HaCaT cells with MG132 or hemin and quantified mRNA levels of HMOX1 and TXNRD1 by qRT–PCR. In untreated control cells, basal TXNRD1 mRNA levels are 35-fold greater than the levels of HMOX1 mRNA. Inhibition of NRF2 activation with CHX significantly inhibited basal TXNRD1 expression but left HMOX1 expression unchanged (Figure 6B and C). This pattern of expression is consistent with functionally active NRF2 residing basally in the nucleus and rules out the possibility that this resident nuclear NRF2 contributes to basal HMOX1 expression. The role of activated NRF2 is demonstrated by the MG132 treatments that trigger maximal TXNRD1 induction but mediate only weak HMOX1 expression (Figure 6B and C). Pretreatment with CHX prior to MG132 blocks NRF2 synthesis, returning HMOX1 mRNA to control levels and reducing TXNRD1 mRNA to the levels of CHX-treated control cells. These expression data reflect the primary role of NRF2 in basal and inducible expression of TXNRD1 but not HMOX1. Hemin treatment, which inactivates BACH1, has no effect on TXNRD1 expression (Figure 6C), reflecting the fact that TXNRD1 is regulated independently of BACH1. In contrast, hemin elicits a significant and nearly maximal increase in HMOX1 expression (Figure 6B). CHX pretreatment predominantly blocks this induction, demonstrating a requisite role for resident nuclear NRF2 in HMOX1 induction without necessitating nuclear accumulation of activated NRF2. Combined treatment with hemin plus MG132 results in induction of TXNRD1 to levels similar to those in cells treated with MG132 alone while the level of HMOX1 induction is similar to that elicited by hemin (Figure 6B and C). These results show that TXNRD1 induction is attributable solely to activation of NRF2 by proteasome inhibition but that HMOX1 induction requires inactivation of BACH1. Thus, BACH1 inactivation is predominantly responsible for HMOX1 induction and that NRF2 activation contributes nominally to this process. Interestingly, the combination of MG132 plus hemin also shows that, when preceded by CHX treatment, HMOX1 expression remains significantly induced despite the lack of NRF2 and hints at the possible role for BACH1 in repressing the activity of transcriptional activators in addition to NRF2. These data establish that inactivation of BACH1 is more important than activation of NRF2 for induction of HMOX1 expression and that not all NRF2-regulated genes respond in this manner, as is the case of TXNRD1.Figure 6.

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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Related in: MedlinePlus