Regulation of Expression of Citrate Synthase by the Retinoic Acid Receptor-Related Orphan Receptor a (RORα)

The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression

Regulation of Expression of Citrate Synthase by the Retinoic Acid Receptor-Related Orphan Receptor a (RORα)

The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression

Direct Regulation of CLOCK Expression by REV-ERB

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 1st 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

Regulation of Expression of Citrate Synthase by the Retinoic Acid Receptor-Related Orphan Receptor α (RORα)

The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression

Identification of a functional RevRE within the <i>CLOCK</i> gene by EMSA.

<p>(a) The sequences of the wild-type <i>CLOCK</i> RevRE and mutant <i>CLOCK</i> RevRE are shown. (b) Demonstration of direct binding of REV-ERBα to radiolabeled <i>CLOCK</i> RevRE DNA. Binding of REV-ERBα to labeled DNA decreased with addition of unlabeled (cold) <i>CLOCK</i> RevRE, but not mutant <i>CLOCK</i> 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.</p

Identification of a functional RevRE within the <i>CLOCK</i> gene using reporter-luciferase assays.

<p>(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 <i>BMAL1</i>/CLOCK heterodimers.</p

Identification of a REV-ERBα binding site within the <i>CLOCK</i> gene.

<p>(a) Significant REV-ERBα occupancy was observed within the <i>CLOCK</i> gene within the 1st intron. The genomic structure of <i>CLOCK</i> is shown with REV-ERBα occupancy illustrated as the gray line. The arrow indicates the direction of transcription. The raw ChIP/chip data is shown in a window beneath the gene as is a screen shot from the integrative genome browser. (b) The region of REV-ERBα occupancy was scanned for conserved RevRE using the Evolutionarily Conserved Region Browser and MatInspector. A RevRE was found to be conserved between mice and humans, the alignment of the RevRE is shown. (c) Alignment of the <i>CLOCK</i> RevRE to a characterized RevRE in the <i>Bmal1</i> promoter.</p

REV-ERBα is required for normal expression of <i>CLOCK</i>.

<p>HepG2 cells were transfected with siRNAs targeting RORα (78% reduction), RORγ (50% reduction) and REV-ERBα (57% reduction) to reduce their expression. <i>CLOCK</i> expression was then examined by RT-PCR. <i>CLOCK</i> expression was elevated when REV-ERBα expression was reduced (c), but unaffected by alteration of RORα (a) expression or RORγ (b). Data shown is mean ± SEM where n = 4. (d) ChIP assay illustrating REV-ERBα and NCoR occupancy of the 1^st intron of the <i>CLOCK</i> gene. (e) The synthetic REV-ERBα agonist GSK4112 suppresses <i>CLOCK</i> gene expression in HepG2 cells. *, p<0.05.</p

Regulation of Expression of Citrate Synthase by the Retinoic Acid Receptor-Related Orphan Receptor a (RORa)

The retinoic acid receptor-related orphan receptor a (RORa) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORa display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORa response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORa occupancy of the CS promoter and a putative RORE binds to RORa effectively in an electrophoretic mobility shift assay and confers RORa responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORa protein. Furthermore, we found that SR1001 a RORa inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORa target gene and one mechanism by which RORa regulates lipid metabolism is vi

Accosting the Golden Spire : a Financial Accounting Action Adventure

Accosting the Golden Spire, Fourth Edition mixes financial fraud, crime, ethics, and accounting practice together to provide a better way of learning the accounting process. Featuring a sleuth who handles balance sheets and income statements the way most detectives handle guns, Lenny Cramer and his humorous sidekick put accounting and business concepts into real-life context. Dr. Cramer, a professor at the Wharton School, operates a small forensic accounting firm. He teaches, testifies before Congress, and appears as an expert witness in a court battle. But the real action occurs when he investigates financial fraud in a friend’s jade shop. Using his forensic skills, he uncovers a plot to steal treasures from a remote Asian country and almost gets himself killed trying to stop the heist. Golden Spire is an educational novel designed as a supplement to financial accounting courses and professional ethics seminars at either the undergraduate or graduate level. It has been used successfully near the end of principles of accounting, at the beginning of intermediate accounting, and as basis for the discussion of professional codes of conduct in an accounting ethics course. The supplement would also be ideal for an MBA program which has a light coverage of accounting, or in CPA firms’ in-house training programs as an enrichment exercise. Classroom tests of early drafts of this third edition and the previous two published editions have demonstrated repeatedly that students enjoy reading the instructional thriller and learn the accounting concepts more readily than through traditional texts. -- publisherhttps://digitalcommons.bucknell.edu/books/1083/thumbnail.jp