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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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Overexpression of RORα/γ or Rev-Erbα in Hepa1-6 cells, respectively, induced or repressed Npas2 activation. (A) Npas2 gene expression was examined by QPCR analysis in Hepa1-6 cells (n = 5) stably expressing empty vector, Flag-RORα, Flag-RORγ or Flag-Rev-Erbα. The expression of Npas2 in Hepa1-6(Empty) was normalized to 1. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) RORs and Rev-Erbα were recruited to the Npas2 promoter in Hepa1-6 cells. ChIP analysis was performed with the Hepa1-6 stable cell lines and anti-Flag M2 antibody. Hepa1-6(Empty) served as a negative control. Data present mean ± SEM, ***P < 0.001.
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Figure 5: Overexpression of RORα/γ or Rev-Erbα in Hepa1-6 cells, respectively, induced or repressed Npas2 activation. (A) Npas2 gene expression was examined by QPCR analysis in Hepa1-6 cells (n = 5) stably expressing empty vector, Flag-RORα, Flag-RORγ or Flag-Rev-Erbα. The expression of Npas2 in Hepa1-6(Empty) was normalized to 1. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) RORs and Rev-Erbα were recruited to the Npas2 promoter in Hepa1-6 cells. ChIP analysis was performed with the Hepa1-6 stable cell lines and anti-Flag M2 antibody. Hepa1-6(Empty) served as a negative control. Data present mean ± SEM, ***P < 0.001.

Mentions: To obtain further evidence for the involvement of RORα/γ and Rev-Erbα in the regulation of Npas2, we established murine hepatoma Hepa1-6 cell lines stably expressing Flag-tagged RORα, RORγ or Rev-Erbα. Immunocytochemistry using anti-Flag M2 antibody showed that these receptors were expressed in these cells and localized to the nucleus (data not shown). Figure 5A shows that Npas2 expression was dramatically induced in Hepa1-6 cells expressing either RORα or RORγ. In contrast, Rev-Erbα overexpression significantly downregulated the expression of Npas2.Figure 5.


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)

Overexpression of RORα/γ or Rev-Erbα in Hepa1-6 cells, respectively, induced or repressed Npas2 activation. (A) Npas2 gene expression was examined by QPCR analysis in Hepa1-6 cells (n = 5) stably expressing empty vector, Flag-RORα, Flag-RORγ or Flag-Rev-Erbα. The expression of Npas2 in Hepa1-6(Empty) was normalized to 1. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) RORs and Rev-Erbα were recruited to the Npas2 promoter in Hepa1-6 cells. ChIP analysis was performed with the Hepa1-6 stable cell lines and anti-Flag M2 antibody. Hepa1-6(Empty) served as a negative control. Data present mean ± SEM, ***P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3113563&req=5

Figure 5: Overexpression of RORα/γ or Rev-Erbα in Hepa1-6 cells, respectively, induced or repressed Npas2 activation. (A) Npas2 gene expression was examined by QPCR analysis in Hepa1-6 cells (n = 5) stably expressing empty vector, Flag-RORα, Flag-RORγ or Flag-Rev-Erbα. The expression of Npas2 in Hepa1-6(Empty) was normalized to 1. Data present mean ± SEM, ***P < 0.001 by ANOVA. (B) RORs and Rev-Erbα were recruited to the Npas2 promoter in Hepa1-6 cells. ChIP analysis was performed with the Hepa1-6 stable cell lines and anti-Flag M2 antibody. Hepa1-6(Empty) served as a negative control. Data present mean ± SEM, ***P < 0.001.
Mentions: To obtain further evidence for the involvement of RORα/γ and Rev-Erbα in the regulation of Npas2, we established murine hepatoma Hepa1-6 cell lines stably expressing Flag-tagged RORα, RORγ or Rev-Erbα. Immunocytochemistry using anti-Flag M2 antibody showed that these receptors were expressed in these cells and localized to the nucleus (data not shown). Figure 5A shows that Npas2 expression was dramatically induced in Hepa1-6 cells expressing either RORα or RORγ. In contrast, Rev-Erbα overexpression significantly downregulated the expression of Npas2.Figure 5.

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