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Retinoic acid-related orphan receptor γ directly regulates neuronal PAS domain protein 2 transcription in vivo.

Takeda Y, Kang HS, Angers M, Jetten AM - Nucleic Acids Res. (2011)

Bottom Line: The activation of Npas2 by RORγ was repressed by co-expression with Rev-Erbα or addition of the ROR inverse agonist T0901317.Npas2 expression was also significantly enhanced during brown adipose differentiation and that this induction was greatly suppressed in adipose cells lacking RORγ.Our results indicate that RORγ and Rev-Erbα are part of a feed-back loop that regulates the circadian expression of Npas2 suggesting a regulatory role for these receptors in Npas2-dependent physiological processes.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

ABSTRACT
Retinoic acid-related orphan receptors (RORs) and the basic helix-loop-helix-PAS transcription factor Npas2 have been implicated in the control of circadian rhythm. In this study, we demonstrate that RORγ directly regulates Npas2 expression in vivo. Although the rhythmicity of Npas2 mRNA expression was maintained in RORγ(-/-) mice, the peak level of expression was significantly reduced in several tissues, while loss of RORα had little effect. Inversely, overexpression of RORγ in hepatoma Hepa1-6 cells greatly induced the expression of Npas2. RORγ-activated Npas2 transcription directly by binding two ROREs in its proximal promoter. ChIP analysis demonstrated that RORγ was recruited to this promoter in the liver of wild-type mice, but not RORγ-deficient mice. Activation of Npas2 correlated positively with chromatin accessibility and level of H3K9 acetylation. The activation of Npas2 by RORγ was repressed by co-expression with Rev-Erbα or addition of the ROR inverse agonist T0901317. Npas2 expression was also significantly enhanced during brown adipose differentiation and that this induction was greatly suppressed in adipose cells lacking RORγ. Our results indicate that RORγ and Rev-Erbα are part of a feed-back loop that regulates the circadian expression of Npas2 suggesting a regulatory role for these receptors in Npas2-dependent physiological processes.

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Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2(–1534/+81) promoter. (A) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10-Npas2(–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. (B) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2(–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. (C) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.
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Figure 3: Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2(–1534/+81) promoter. (A) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10-Npas2(–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. (B) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2(–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. (C) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.

Mentions: Earlier studies have shown that the nuclear receptor, Rev-Erbα, which also binds ROREs, can compete with RORs for RORE binding (22,23,37,54). Figure 3A shows that co-expression of Rev-Erbα repressed the activation of the Npas2 promoter by either RORα or RORγ in a dose-dependent manner. Moreover, expression of Rev-Erbα in Huh-7 cells repressed the endogenous activation of the Npas2(–1534/+81) promoter (Figure 3B). Mutation of RORE1/2 abolished this repression indicating that this repression was mediated through these ROREs. These observations are consistent with the hypothesis that Rev-Erbα functions as a repressor of Npas2 expression and competes with RORs for RORE binding. Recently, T0901317 was identified as an inverse agonist of RORα and RORγ (7). It was therefore of interest to examine the effect of T0901317 on the expression of Npas2 induction. As shown in Figure 3C, T0901317 was able to repress the activation of the Npas2 promoter by RORγ and RORα in Huh-7 cells.Figure 3.


Retinoic acid-related orphan receptor γ directly regulates neuronal PAS domain protein 2 transcription in vivo.

Takeda Y, Kang HS, Angers M, Jetten AM - Nucleic Acids Res. (2011)

Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2(–1534/+81) promoter. (A) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10-Npas2(–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. (B) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2(–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. (C) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.
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Figure 3: Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2(–1534/+81) promoter. (A) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10-Npas2(–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. (B) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2(–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. (C) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.
Mentions: Earlier studies have shown that the nuclear receptor, Rev-Erbα, which also binds ROREs, can compete with RORs for RORE binding (22,23,37,54). Figure 3A shows that co-expression of Rev-Erbα repressed the activation of the Npas2 promoter by either RORα or RORγ in a dose-dependent manner. Moreover, expression of Rev-Erbα in Huh-7 cells repressed the endogenous activation of the Npas2(–1534/+81) promoter (Figure 3B). Mutation of RORE1/2 abolished this repression indicating that this repression was mediated through these ROREs. These observations are consistent with the hypothesis that Rev-Erbα functions as a repressor of Npas2 expression and competes with RORs for RORE binding. Recently, T0901317 was identified as an inverse agonist of RORα and RORγ (7). It was therefore of interest to examine the effect of T0901317 on the expression of Npas2 induction. As shown in Figure 3C, T0901317 was able to repress the activation of the Npas2 promoter by RORγ and RORα in Huh-7 cells.Figure 3.

Bottom Line: The activation of Npas2 by RORγ was repressed by co-expression with Rev-Erbα or addition of the ROR inverse agonist T0901317.Npas2 expression was also significantly enhanced during brown adipose differentiation and that this induction was greatly suppressed in adipose cells lacking RORγ.Our results indicate that RORγ and Rev-Erbα are part of a feed-back loop that regulates the circadian expression of Npas2 suggesting a regulatory role for these receptors in Npas2-dependent physiological processes.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

ABSTRACT
Retinoic acid-related orphan receptors (RORs) and the basic helix-loop-helix-PAS transcription factor Npas2 have been implicated in the control of circadian rhythm. In this study, we demonstrate that RORγ directly regulates Npas2 expression in vivo. Although the rhythmicity of Npas2 mRNA expression was maintained in RORγ(-/-) mice, the peak level of expression was significantly reduced in several tissues, while loss of RORα had little effect. Inversely, overexpression of RORγ in hepatoma Hepa1-6 cells greatly induced the expression of Npas2. RORγ-activated Npas2 transcription directly by binding two ROREs in its proximal promoter. ChIP analysis demonstrated that RORγ was recruited to this promoter in the liver of wild-type mice, but not RORγ-deficient mice. Activation of Npas2 correlated positively with chromatin accessibility and level of H3K9 acetylation. The activation of Npas2 by RORγ was repressed by co-expression with Rev-Erbα or addition of the ROR inverse agonist T0901317. Npas2 expression was also significantly enhanced during brown adipose differentiation and that this induction was greatly suppressed in adipose cells lacking RORγ. Our results indicate that RORγ and Rev-Erbα are part of a feed-back loop that regulates the circadian expression of Npas2 suggesting a regulatory role for these receptors in Npas2-dependent physiological processes.

Show MeSH
Related in: MedlinePlus