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Direct regulation of CLOCK expression by REV-ERB.

Crumbley C, Burris TP - PLoS ONE (2011)

Bottom Line: A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice.Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene.Our data suggests that REV-ERBα plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Jupiter, Florida, United States of America.

ABSTRACT
Circadian rhythms are regulated at the cellular level by transcriptional feedback loops leading to oscillations in expression of key proteins including CLOCK, BMAL1, PERIOD (PER), and CRYPTOCHROME (CRY). The CLOCK and BMAL1 proteins are members of the bHLH class of transcription factors and form a heterodimer that regulates the expression of the PER and CRY genes. The nuclear receptor REV-ERBα plays a key role in regulation of oscillations in BMAL1 expression by directly binding to the BMAL1 promoter and suppressing its expression at certain times of day when REV-ERBα expression levels are elevated. We recently demonstrated that REV-ERBα also regulates the expression of NPAS2, a heterodimer partner of BMAL1. Here, we show that REV-ERBα also regulates the expression another heterodimer partner of BMAL1, CLOCK. We identified a REV-ERBα binding site within the 1(st) intron of the CLOCK gene using a chromatin immunoprecipitation - microarray screen. Suppression of REV-ERBα expression resulted in elevated CLOCK mRNA expression consistent with REV-ERBα's role as a transcriptional repressor. A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice. Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene. Our data suggests that REV-ERBα plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes.

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Identification of a functional RevRE within the CLOCK gene using reporter-luciferase assays.(a) The reporter constructs contain a three-times repeat of the putative CLOCK RevRE cloned upstream of the firefly luciferase gene. (b) REV-ERBα was co-transfected with a luciferase reporter containing the wild-type 3×RevRE leading to reduced expression of luciferase relative to the reporter alone. The expression of luciferase from the mutant 3×RORE was unaffected by REV-ERBα co-transfection. Data shown is mean ± SEM, n = 8. *, p<0.05. (c) Proposed model for coordinated regulation of BMAL1/CLOCK heterodimers.
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pone-0017290-g004: Identification of a functional RevRE within the CLOCK gene using reporter-luciferase assays.(a) The reporter constructs contain a three-times repeat of the putative CLOCK RevRE cloned upstream of the firefly luciferase gene. (b) REV-ERBα was co-transfected with a luciferase reporter containing the wild-type 3×RevRE leading to reduced expression of luciferase relative to the reporter alone. The expression of luciferase from the mutant 3×RORE was unaffected by REV-ERBα co-transfection. Data shown is mean ± SEM, n = 8. *, p<0.05. (c) Proposed model for coordinated regulation of BMAL1/CLOCK heterodimers.

Mentions: Due to the location of the putative RevRE within the 1st intron of the CLOCK gene, we generated a three-times repeat of the RevRE to clone upstream of the firefly luciferase gene (Figure 4a). When the wild-type 3×RevRE reporter construct was transfected into HepG2 cells, the co-transfection of REV-ERBα suppressed luciferase expression relative to reporter alone (Figure 4b). The expression of luciferase from the mutant 3×RevRE reporter construct was unaffected by co-transfection of REV-ERBα when transfected into HepG2 cells. Similar results were observed in HEK293 cells (data not shown).


Direct regulation of CLOCK expression by REV-ERB.

Crumbley C, Burris TP - PLoS ONE (2011)

Identification of a functional RevRE within the CLOCK gene using reporter-luciferase assays.(a) The reporter constructs contain a three-times repeat of the putative CLOCK RevRE cloned upstream of the firefly luciferase gene. (b) REV-ERBα was co-transfected with a luciferase reporter containing the wild-type 3×RevRE leading to reduced expression of luciferase relative to the reporter alone. The expression of luciferase from the mutant 3×RORE was unaffected by REV-ERBα co-transfection. Data shown is mean ± SEM, n = 8. *, p<0.05. (c) Proposed model for coordinated regulation of BMAL1/CLOCK heterodimers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066191&req=5

pone-0017290-g004: Identification of a functional RevRE within the CLOCK gene using reporter-luciferase assays.(a) The reporter constructs contain a three-times repeat of the putative CLOCK RevRE cloned upstream of the firefly luciferase gene. (b) REV-ERBα was co-transfected with a luciferase reporter containing the wild-type 3×RevRE leading to reduced expression of luciferase relative to the reporter alone. The expression of luciferase from the mutant 3×RORE was unaffected by REV-ERBα co-transfection. Data shown is mean ± SEM, n = 8. *, p<0.05. (c) Proposed model for coordinated regulation of BMAL1/CLOCK heterodimers.
Mentions: Due to the location of the putative RevRE within the 1st intron of the CLOCK gene, we generated a three-times repeat of the RevRE to clone upstream of the firefly luciferase gene (Figure 4a). When the wild-type 3×RevRE reporter construct was transfected into HepG2 cells, the co-transfection of REV-ERBα suppressed luciferase expression relative to reporter alone (Figure 4b). The expression of luciferase from the mutant 3×RevRE reporter construct was unaffected by co-transfection of REV-ERBα when transfected into HepG2 cells. Similar results were observed in HEK293 cells (data not shown).

Bottom Line: A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice.Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene.Our data suggests that REV-ERBα plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Jupiter, Florida, United States of America.

ABSTRACT
Circadian rhythms are regulated at the cellular level by transcriptional feedback loops leading to oscillations in expression of key proteins including CLOCK, BMAL1, PERIOD (PER), and CRYPTOCHROME (CRY). The CLOCK and BMAL1 proteins are members of the bHLH class of transcription factors and form a heterodimer that regulates the expression of the PER and CRY genes. The nuclear receptor REV-ERBα plays a key role in regulation of oscillations in BMAL1 expression by directly binding to the BMAL1 promoter and suppressing its expression at certain times of day when REV-ERBα expression levels are elevated. We recently demonstrated that REV-ERBα also regulates the expression of NPAS2, a heterodimer partner of BMAL1. Here, we show that REV-ERBα also regulates the expression another heterodimer partner of BMAL1, CLOCK. We identified a REV-ERBα binding site within the 1(st) intron of the CLOCK gene using a chromatin immunoprecipitation - microarray screen. Suppression of REV-ERBα expression resulted in elevated CLOCK mRNA expression consistent with REV-ERBα's role as a transcriptional repressor. A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice. Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene. Our data suggests that REV-ERBα plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes.

Show MeSH
Related in: MedlinePlus