RESUMO
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.
Assuntos
Animais , Humanos , Masculino , Camundongos , Fatores de Transcrição ARNTL/fisiologia , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas CLOCK/fisiologia , Colesterol/sangue , Ritmo Circadiano , Elementos E-Box , Éxons , Regulação da Expressão Gênica , Células Hep G2 , Proteínas de Homeodomínio/genética , Homeostase , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Receptores de LDL/genética , Proteínas Repressoras/genética , Transcrição GênicaRESUMO
<p><b>OBJECTIVE</b>To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity.</p><p><b>METHODS</b>The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318. The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography. The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint. The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA.</p><p><b>RESULTS</b>COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter. Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity.</p><p><b>CONCLUSION</b>The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription. It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.</p>
Assuntos
Humanos , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Clonagem Molecular , DNA Complementar , Genética , Proteínas de Ligação a DNA , Genética , Farmacologia , Elementos E-Box , Genética , Células HeLa , Regiões Promotoras Genéticas , RNA Mensageiro , Genética , Receptores de Esteroides , Genética , Telomerase , Genética , Metabolismo , Fatores de Transcrição , Genética , Farmacologia , Transcrição Gênica , Leveduras , GenéticaRESUMO
Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.