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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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RORγ and RORα are recruited to the Npas2 promoter. (A) Circadian time-dependent recruitment of RORγ to the Npas2 promoter. ChIP analysis was performed using an anti-RORγ antibody and liver tissues (n = 4) isolated from WT and RORγ−/− mice at CT10 (low expression of RORγ) and CT22 (high expression of RORγ). Amplification of the Gapdh gene was used as a negative control. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) ChIP analysis was performed with anti-ROR antibodies using liver tissues (n = 4) isolated from WT, RORαsg/sg and RORγ−/− mice at CT22. QPCR amplification of a non RORE-containing distal site of the Npas2 gene was used as a negative control. Data present mean ± SEM, **P < 0.01, ***P < 0.001.
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Figure 4: RORγ and RORα are recruited to the Npas2 promoter. (A) Circadian time-dependent recruitment of RORγ to the Npas2 promoter. ChIP analysis was performed using an anti-RORγ antibody and liver tissues (n = 4) isolated from WT and RORγ−/− mice at CT10 (low expression of RORγ) and CT22 (high expression of RORγ). Amplification of the Gapdh gene was used as a negative control. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) ChIP analysis was performed with anti-ROR antibodies using liver tissues (n = 4) isolated from WT, RORαsg/sg and RORγ−/− mice at CT22. QPCR amplification of a non RORE-containing distal site of the Npas2 gene was used as a negative control. Data present mean ± SEM, **P < 0.01, ***P < 0.001.

Mentions: To determine whether RORγ was recruited to the ROREs in the Npas2(–1534/+81) promoter in vivo, ChIP-qPCR analysis was carried out using liver isolated from WT and ROR knockout mice. Tissues were collected at CT22, a time close to the peak expression of RORγ and Npas2 mRNAs, and at CT10, a time at which both RORγ and Npas2 mRNAs are expressed at their lowest level. On this basis, one would expect higher recruitment of RORγ to the Npas2 promoter at CT22 than at CT10. The data in Figure 4A confirmed that RORγ was more efficiently recruited to the −1380 region of the Npas2 promoter in WT liver collected at CT22 compared with the liver collected at CT10. Primers targeting a region within the Gapdh gene were used as negative control and did not show any specific recruitment of RORγ. As expected, at CT22, the level of RORγ associated with the Npas2 promoter in WT liver was significantly higher than in RORγ−/− liver, while RORγ was not recruited to a distal, non RORE-containing region of the Npas2 gene. Although the loss of RORα had only a small effect on Npas2 expression, ChIP analysis indicated that RORα was associated with the Npas2 promoter.Figure 4.


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)

RORγ and RORα are recruited to the Npas2 promoter. (A) Circadian time-dependent recruitment of RORγ to the Npas2 promoter. ChIP analysis was performed using an anti-RORγ antibody and liver tissues (n = 4) isolated from WT and RORγ−/− mice at CT10 (low expression of RORγ) and CT22 (high expression of RORγ). Amplification of the Gapdh gene was used as a negative control. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) ChIP analysis was performed with anti-ROR antibodies using liver tissues (n = 4) isolated from WT, RORαsg/sg and RORγ−/− mice at CT22. QPCR amplification of a non RORE-containing distal site of the Npas2 gene was used as a negative control. Data present mean ± SEM, **P < 0.01, ***P < 0.001.
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Related In: Results  -  Collection

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Figure 4: RORγ and RORα are recruited to the Npas2 promoter. (A) Circadian time-dependent recruitment of RORγ to the Npas2 promoter. ChIP analysis was performed using an anti-RORγ antibody and liver tissues (n = 4) isolated from WT and RORγ−/− mice at CT10 (low expression of RORγ) and CT22 (high expression of RORγ). Amplification of the Gapdh gene was used as a negative control. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) ChIP analysis was performed with anti-ROR antibodies using liver tissues (n = 4) isolated from WT, RORαsg/sg and RORγ−/− mice at CT22. QPCR amplification of a non RORE-containing distal site of the Npas2 gene was used as a negative control. Data present mean ± SEM, **P < 0.01, ***P < 0.001.
Mentions: To determine whether RORγ was recruited to the ROREs in the Npas2(–1534/+81) promoter in vivo, ChIP-qPCR analysis was carried out using liver isolated from WT and ROR knockout mice. Tissues were collected at CT22, a time close to the peak expression of RORγ and Npas2 mRNAs, and at CT10, a time at which both RORγ and Npas2 mRNAs are expressed at their lowest level. On this basis, one would expect higher recruitment of RORγ to the Npas2 promoter at CT22 than at CT10. The data in Figure 4A confirmed that RORγ was more efficiently recruited to the −1380 region of the Npas2 promoter in WT liver collected at CT22 compared with the liver collected at CT10. Primers targeting a region within the Gapdh gene were used as negative control and did not show any specific recruitment of RORγ. As expected, at CT22, the level of RORγ associated with the Npas2 promoter in WT liver was significantly higher than in RORγ−/− liver, while RORγ was not recruited to a distal, non RORE-containing region of the Npas2 gene. Although the loss of RORα had only a small effect on Npas2 expression, ChIP analysis indicated that RORα was associated with the Npas2 promoter.Figure 4.

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