Exp Mol Med.  2012 Nov;44(11):642-652.

Circadian regulation of low density lipoprotein receptor promoter activity by CLOCK/BMAL1, Hes1 and Hes6

• Lee YJ
• Han DH
• Pak YK
• Cho S

Abstract

Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.

Keyword

ARNTL transcription factors; circadian rhythm; CLOCK proteins; HES1 protein, human; HES6 protein, human; receptors, LDL

MeSH Terms

ARNTL Transcription Factors/physiology
Animals
Base Sequence
Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism/physiology
CLOCK Proteins/physiology
Cholesterol/blood
*Circadian Rhythm
E-Box Elements
Exons
*Gene Expression Regulation
Hep G2 Cells
Homeodomain Proteins/*genetics/metabolism/physiology
Homeostasis
Humans
Liver/metabolism
Male
Mice
Mice, Inbred C57BL
*Promoter Regions, Genetic
Receptors, LDL/*genetics/metabolism
Repressor Proteins/*genetics/metabolism/physiology
Transcription, Genetic