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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 by EMSA.(a) The sequences of the wild-type CLOCK RevRE and mutant CLOCK RevRE are shown. (b) Demonstration of direct binding of REV-ERBα to radiolabeled CLOCK RevRE DNA. Binding of REV-ERBα to labeled DNA decreased with addition of unlabeled (cold) CLOCK RevRE, but not mutant CLOCK RevRE. The arrow indicates increases amounts of cold DNA added (10×, 50×, and 100× molar excess). (c) EMSA illustrating that REV-ERBα binds to radiolabeled BMAL1 RevRE and that 100-fold molar excess of CLOCK RevRE DNA can compete for binding to REV-ERBα.
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pone-0017290-g003: Identification of a functional RevRE within the CLOCK gene by EMSA.(a) The sequences of the wild-type CLOCK RevRE and mutant CLOCK RevRE are shown. (b) Demonstration of direct binding of REV-ERBα to radiolabeled CLOCK RevRE DNA. Binding of REV-ERBα to labeled DNA decreased with addition of unlabeled (cold) CLOCK RevRE, but not mutant CLOCK RevRE. The arrow indicates increases amounts of cold DNA added (10×, 50×, and 100× molar excess). (c) EMSA illustrating that REV-ERBα binds to radiolabeled BMAL1 RevRE and that 100-fold molar excess of CLOCK RevRE DNA can compete for binding to REV-ERBα.

Mentions: The putative RevRE was examined for REV-ERBα binding using an EMSA. Synthetic oligonucleotides encoding the putative CLOCK RevRE were radiolabeled, and then incubated with REV-ERBα protein produced in vitro. The sequences of the oligonucleotides are shown in Figure 3a. Direct binding of REV-ERBα to the wild-type CLOCK RevRE is shown in Figure 3b. Addition of unlabeled wild-type CLOCK RevRE probe was able to displace the radiolabeled CLOCK RevRE from REV-ERBα, as shown in Figure 3b while an excess of unlabeled mutant CLOCK RevRE was unable to displace the radiolabeled CLOCK RevRE probe from the REV-ERBα protein demonstrating specificity. We also assessed the ability of the CLOCK RevRE probe to compete with the well-characterized BMAL1 RevRE. The BMAL1 RevRE was radiolabeled and then 100-fold molar excess of unlabeled CLOCK RevRE was included as a competitor. As illustrated in Figure 3c, the CLOCK RevRE was able to compete for binding of REV-ERBα to the BMAL1 RevRE.


Direct regulation of CLOCK expression by REV-ERB.

Crumbley C, Burris TP - PLoS ONE (2011)

Identification of a functional RevRE within the CLOCK gene by EMSA.(a) The sequences of the wild-type CLOCK RevRE and mutant CLOCK RevRE are shown. (b) Demonstration of direct binding of REV-ERBα to radiolabeled CLOCK RevRE DNA. Binding of REV-ERBα to labeled DNA decreased with addition of unlabeled (cold) CLOCK RevRE, but not mutant CLOCK RevRE. The arrow indicates increases amounts of cold DNA added (10×, 50×, and 100× molar excess). (c) EMSA illustrating that REV-ERBα binds to radiolabeled BMAL1 RevRE and that 100-fold molar excess of CLOCK RevRE DNA can compete for binding to REV-ERBα.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3066191&req=5

pone-0017290-g003: Identification of a functional RevRE within the CLOCK gene by EMSA.(a) The sequences of the wild-type CLOCK RevRE and mutant CLOCK RevRE are shown. (b) Demonstration of direct binding of REV-ERBα to radiolabeled CLOCK RevRE DNA. Binding of REV-ERBα to labeled DNA decreased with addition of unlabeled (cold) CLOCK RevRE, but not mutant CLOCK RevRE. The arrow indicates increases amounts of cold DNA added (10×, 50×, and 100× molar excess). (c) EMSA illustrating that REV-ERBα binds to radiolabeled BMAL1 RevRE and that 100-fold molar excess of CLOCK RevRE DNA can compete for binding to REV-ERBα.
Mentions: The putative RevRE was examined for REV-ERBα binding using an EMSA. Synthetic oligonucleotides encoding the putative CLOCK RevRE were radiolabeled, and then incubated with REV-ERBα protein produced in vitro. The sequences of the oligonucleotides are shown in Figure 3a. Direct binding of REV-ERBα to the wild-type CLOCK RevRE is shown in Figure 3b. Addition of unlabeled wild-type CLOCK RevRE probe was able to displace the radiolabeled CLOCK RevRE from REV-ERBα, as shown in Figure 3b while an excess of unlabeled mutant CLOCK RevRE was unable to displace the radiolabeled CLOCK RevRE probe from the REV-ERBα protein demonstrating specificity. We also assessed the ability of the CLOCK RevRE probe to compete with the well-characterized BMAL1 RevRE. The BMAL1 RevRE was radiolabeled and then 100-fold molar excess of unlabeled CLOCK RevRE was included as a competitor. As illustrated in Figure 3c, the CLOCK RevRE was able to compete for binding of REV-ERBα to the BMAL1 RevRE.

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