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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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Comparison of the circadian expression of Npas2 in several peripheral tissues from WT, RORγ-/– and RORαsg/sg mice. (A–H) Various tissues from WT, RORγ−/− and RORαsg/sg mice (n = 4) were isolated every 4 h over a period of 24 h and expression of Npas2 was analyzed by QRT-PCR. The 24-h expression pattern was double plotted. (I) Tissues were isolated from RORγ−/−, RORαsg/sgRORγ−/− DKO mice (n = 4) at CT20. Npas2 expression in DKO and RORγ−/− mice was normalized to the expression in littermate WT mice. (J) Circadian pattern of RORγ1, RORα1 and RORα4 mRNA expression in liver from WT, RORγ−/− and RORαsg/sg mice and Rev-Erbα expression in WT mice (n = 4). Data present mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA.
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Figure 1: Comparison of the circadian expression of Npas2 in several peripheral tissues from WT, RORγ-/– and RORαsg/sg mice. (A–H) Various tissues from WT, RORγ−/− and RORαsg/sg mice (n = 4) were isolated every 4 h over a period of 24 h and expression of Npas2 was analyzed by QRT-PCR. The 24-h expression pattern was double plotted. (I) Tissues were isolated from RORγ−/−, RORαsg/sgRORγ−/− DKO mice (n = 4) at CT20. Npas2 expression in DKO and RORγ−/− mice was normalized to the expression in littermate WT mice. (J) Circadian pattern of RORγ1, RORα1 and RORα4 mRNA expression in liver from WT, RORγ−/− and RORαsg/sg mice and Rev-Erbα expression in WT mice (n = 4). Data present mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA.

Mentions: Several studies have provided evidence for a role of RORs in the regulation of several circadian clock genes (1,11,20,23,24,26,27,38). In this study, we focused on the regulation of Npas2 by RORα and RORγ. First, we analyzed the effect of the loss of RORα or RORγ expression on the circadian expression of Npas2 in the liver, brown adipose tissue (BAT), kidney and small intestines using RORαsg/sg and RORγ−/−mice. Consistent with a previous report, Npas2 mRNA showed a strong oscillatory pattern of expression in the liver of WT mice with peak expression between CT20 and CT0 and a low point at CT12 (Figure 1A and B) (51). However, the oscillatory expression of Npas2 was significantly altered in RORγ−/− liver. At CT0, when Npas2 is highly expressed in WT liver, expression of Npas2 mRNA was reduced by about 60% in the liver of RORγ−/− mice (Figure 1A). Npas2 expression exhibited a similar robust oscillatory pattern in BAT, kidney, small intestines and white adipose tissue (WAT) as in WT liver (Figure 1C–H). However, between CT20 and CT4 Npas2 expression was greatly decreased in BAT of RORγ−/− mice (Figure 1C), reduced modestly in RORγ−/− kidney (Figure 1E) and was slightly enhanced in the small intestines (Figure 1G). Little change was observed in the pattern of Npas2 expression in the liver and WAT from RORαsg/sg mice (Figure 1B and H), while a small shift in peak Npas2 expression was seen in RORαsg/sg kidney (Figure 1F) and an increase in RORαsg/sg BAT. RORα is not expressed in small intestines.Figure 1.


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)

Comparison of the circadian expression of Npas2 in several peripheral tissues from WT, RORγ-/– and RORαsg/sg mice. (A–H) Various tissues from WT, RORγ−/− and RORαsg/sg mice (n = 4) were isolated every 4 h over a period of 24 h and expression of Npas2 was analyzed by QRT-PCR. The 24-h expression pattern was double plotted. (I) Tissues were isolated from RORγ−/−, RORαsg/sgRORγ−/− DKO mice (n = 4) at CT20. Npas2 expression in DKO and RORγ−/− mice was normalized to the expression in littermate WT mice. (J) Circadian pattern of RORγ1, RORα1 and RORα4 mRNA expression in liver from WT, RORγ−/− and RORαsg/sg mice and Rev-Erbα expression in WT mice (n = 4). Data present mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA.
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Figure 1: Comparison of the circadian expression of Npas2 in several peripheral tissues from WT, RORγ-/– and RORαsg/sg mice. (A–H) Various tissues from WT, RORγ−/− and RORαsg/sg mice (n = 4) were isolated every 4 h over a period of 24 h and expression of Npas2 was analyzed by QRT-PCR. The 24-h expression pattern was double plotted. (I) Tissues were isolated from RORγ−/−, RORαsg/sgRORγ−/− DKO mice (n = 4) at CT20. Npas2 expression in DKO and RORγ−/− mice was normalized to the expression in littermate WT mice. (J) Circadian pattern of RORγ1, RORα1 and RORα4 mRNA expression in liver from WT, RORγ−/− and RORαsg/sg mice and Rev-Erbα expression in WT mice (n = 4). Data present mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA.
Mentions: Several studies have provided evidence for a role of RORs in the regulation of several circadian clock genes (1,11,20,23,24,26,27,38). In this study, we focused on the regulation of Npas2 by RORα and RORγ. First, we analyzed the effect of the loss of RORα or RORγ expression on the circadian expression of Npas2 in the liver, brown adipose tissue (BAT), kidney and small intestines using RORαsg/sg and RORγ−/−mice. Consistent with a previous report, Npas2 mRNA showed a strong oscillatory pattern of expression in the liver of WT mice with peak expression between CT20 and CT0 and a low point at CT12 (Figure 1A and B) (51). However, the oscillatory expression of Npas2 was significantly altered in RORγ−/− liver. At CT0, when Npas2 is highly expressed in WT liver, expression of Npas2 mRNA was reduced by about 60% in the liver of RORγ−/− mice (Figure 1A). Npas2 expression exhibited a similar robust oscillatory pattern in BAT, kidney, small intestines and white adipose tissue (WAT) as in WT liver (Figure 1C–H). However, between CT20 and CT4 Npas2 expression was greatly decreased in BAT of RORγ−/− mice (Figure 1C), reduced modestly in RORγ−/− kidney (Figure 1E) and was slightly enhanced in the small intestines (Figure 1G). Little change was observed in the pattern of Npas2 expression in the liver and WAT from RORαsg/sg mice (Figure 1B and H), while a small shift in peak Npas2 expression was seen in RORαsg/sg kidney (Figure 1F) and an increase in RORαsg/sg BAT. RORα is not expressed in small intestines.Figure 1.

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