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A molecular clock regulates angiopoietin-like protein 2 expression.

Kadomatsu T, Uragami S, Akashi M, Tsuchiya Y, Nakajima H, Nakashima Y, Endo M, Miyata K, Terada K, Todo T, Node K, Oike Y - PLoS ONE (2013)

Bottom Line: Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression.We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells.Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. tkado@gpo.kumamoto-u.ac.jp

ABSTRACT
Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

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Endogenous CLOCK binds to E-boxes of the human ANGPTL2 promoter in U2OS cells.A, Upper panel: Schematic diagram of the location of primers flanking E-box sites of the human ANGPTL2 promoter. Arrowheads indicate specific primers. Lower panel: Representative image of ChIP assays showing CLOCK binding to E-box sites of ANGPTL2 and PER2 promoters in unsynchronized U2OS cells. Purified DNA fragments derived from immunoprecipitated chromatin complexes were analyzed by PCR using primers specific for the E-box sites of ANGPTL2 and PER2 promoters or for the GAPDH promoter (negative control). B, Representative image of ChIP assays showing oscillatory CLOCK binding to E-boxes of the human ANGPTL2 promoter in U2OS cells after serum shock. Chromatin was collected at 16 h and 28 h after serum shock and subjected to ChIP assays of the human ANGPTL2 promoter or of the GAPDH promoter (negative control). Each experiment was performed at least three times.
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pone-0057921-g005: Endogenous CLOCK binds to E-boxes of the human ANGPTL2 promoter in U2OS cells.A, Upper panel: Schematic diagram of the location of primers flanking E-box sites of the human ANGPTL2 promoter. Arrowheads indicate specific primers. Lower panel: Representative image of ChIP assays showing CLOCK binding to E-box sites of ANGPTL2 and PER2 promoters in unsynchronized U2OS cells. Purified DNA fragments derived from immunoprecipitated chromatin complexes were analyzed by PCR using primers specific for the E-box sites of ANGPTL2 and PER2 promoters or for the GAPDH promoter (negative control). B, Representative image of ChIP assays showing oscillatory CLOCK binding to E-boxes of the human ANGPTL2 promoter in U2OS cells after serum shock. Chromatin was collected at 16 h and 28 h after serum shock and subjected to ChIP assays of the human ANGPTL2 promoter or of the GAPDH promoter (negative control). Each experiment was performed at least three times.

Mentions: Next, we performed chromatin immunoprecipitation (ChIP) assays in U2OS cells using a CLOCK antibody to evaluate binding of endogenous CLOCK to promoter E-boxes (Figure 5A). ChIP assays with primers flanking E-box sites revealed that endogenous CLOCK binds to the E2 and E4 sites. As a positive control we confirmed occupancy of an E-box of the human PER2 promoter by CLOCK. We also observed no binding of CLOCK to the human GAPDH promoter, which served as a negative control. Moreover, ChIP analysis of the human ANGPTL2 promoter using chromatin collected from U2OS cells at 16 h or 28 h after serum shock showed oscillatory binding of endogenous CLOCK to E-boxes (Figure 5B). These results suggest that the CLOCK/BMAL1 complex regulates rhythmic ANGPTL2 expression through the E2 and E4 sites.


A molecular clock regulates angiopoietin-like protein 2 expression.

Kadomatsu T, Uragami S, Akashi M, Tsuchiya Y, Nakajima H, Nakashima Y, Endo M, Miyata K, Terada K, Todo T, Node K, Oike Y - PLoS ONE (2013)

Endogenous CLOCK binds to E-boxes of the human ANGPTL2 promoter in U2OS cells.A, Upper panel: Schematic diagram of the location of primers flanking E-box sites of the human ANGPTL2 promoter. Arrowheads indicate specific primers. Lower panel: Representative image of ChIP assays showing CLOCK binding to E-box sites of ANGPTL2 and PER2 promoters in unsynchronized U2OS cells. Purified DNA fragments derived from immunoprecipitated chromatin complexes were analyzed by PCR using primers specific for the E-box sites of ANGPTL2 and PER2 promoters or for the GAPDH promoter (negative control). B, Representative image of ChIP assays showing oscillatory CLOCK binding to E-boxes of the human ANGPTL2 promoter in U2OS cells after serum shock. Chromatin was collected at 16 h and 28 h after serum shock and subjected to ChIP assays of the human ANGPTL2 promoter or of the GAPDH promoter (negative control). Each experiment was performed at least three times.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585275&req=5

pone-0057921-g005: Endogenous CLOCK binds to E-boxes of the human ANGPTL2 promoter in U2OS cells.A, Upper panel: Schematic diagram of the location of primers flanking E-box sites of the human ANGPTL2 promoter. Arrowheads indicate specific primers. Lower panel: Representative image of ChIP assays showing CLOCK binding to E-box sites of ANGPTL2 and PER2 promoters in unsynchronized U2OS cells. Purified DNA fragments derived from immunoprecipitated chromatin complexes were analyzed by PCR using primers specific for the E-box sites of ANGPTL2 and PER2 promoters or for the GAPDH promoter (negative control). B, Representative image of ChIP assays showing oscillatory CLOCK binding to E-boxes of the human ANGPTL2 promoter in U2OS cells after serum shock. Chromatin was collected at 16 h and 28 h after serum shock and subjected to ChIP assays of the human ANGPTL2 promoter or of the GAPDH promoter (negative control). Each experiment was performed at least three times.
Mentions: Next, we performed chromatin immunoprecipitation (ChIP) assays in U2OS cells using a CLOCK antibody to evaluate binding of endogenous CLOCK to promoter E-boxes (Figure 5A). ChIP assays with primers flanking E-box sites revealed that endogenous CLOCK binds to the E2 and E4 sites. As a positive control we confirmed occupancy of an E-box of the human PER2 promoter by CLOCK. We also observed no binding of CLOCK to the human GAPDH promoter, which served as a negative control. Moreover, ChIP analysis of the human ANGPTL2 promoter using chromatin collected from U2OS cells at 16 h or 28 h after serum shock showed oscillatory binding of endogenous CLOCK to E-boxes (Figure 5B). These results suggest that the CLOCK/BMAL1 complex regulates rhythmic ANGPTL2 expression through the E2 and E4 sites.

Bottom Line: Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression.We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells.Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. tkado@gpo.kumamoto-u.ac.jp

ABSTRACT
Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

Show MeSH
Related in: MedlinePlus