RESUMEN
We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light-dark and constant darkness conditions. Type II collagen and aggrecan genes--along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2--showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light-dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.
Asunto(s)
Cartílago/metabolismo , Relojes Circadianos , Proteínas Matrilinas/metabolismo , Fotoperiodo , Animales , Expresión Génica , Humanos , Masculino , Proteínas Matrilinas/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Several cis-acting elements play critical roles in maintaining circadian expression of clock and clock-controlled genes. Using in silico analysis, we identified 10 sequence motifs that are correlated with the circadian phases of gene expression in the cartilage. One of these motifs, an E-box-like clock-related element (EL-box; GGCACGAGGC), can mediate BMAL1/CLOCK-induced transcription, which is typically regulated through an E-box or E'-box. Expression of EL-box-containing genes, including Ank, Dbp, and Nr1d1 (Rev-erbα), was induced by BMAL1/CLOCK or BMAL1/NPAS2. Compared with the E-box, the EL-box elements had distinct responsiveness to DEC1, DEC2, and HES1: suppressive actions of DEC1 and DEC2 on the EL-box were less potent than those on the E-box. HES1, which is known to bind to the N-box (CACNAG), suppressed enhancer activity of the EL-box, but not the E-box. In the Dbp promoter, an EL-box worked cooperatively with a noncanonical (NC) E-box to mediate BMAL1/CLOCK actions. These findings suggest that in addition to known clock elements, the EL-box element may contribute to circadian regulation of clock and clock-controlled genes.
Asunto(s)
Factores de Transcripción ARNTL/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas CLOCK/metabolismo , Cartílago/metabolismo , Relojes Circadianos , Proteínas de Homeodominio/metabolismo , Animales , Secuencia de Bases , Secuencia de Consenso , Placa de Crecimiento/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Elementos Reguladores de la Transcripción , Factor de Transcripción HES-1 , TranscriptomaRESUMEN
DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E' boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E'-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E' box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1-/- mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiología , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas CLOCK , Ritmo Circadiano , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Células 3T3 NIH , Células Madre/citologíaRESUMEN
To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Relojes Biológicos/fisiología , Proteínas de Ciclo Celular/metabolismo , Ritmo Circadiano/fisiología , Ayuno/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Animales , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Proteínas Circadianas Period , Factores de TiempoRESUMEN
Circadian rhythms in cartilage have been reported repeatedly. However, previous studies used histological analysis or radioisotope-labeled precursors for DNA, collagen and proteoglycan synthesis, and thus it is difficult precisely to evaluate such studies on circadian rhythms in chondrocytes. On the other hand, circadian rhythms in plasma levels of several hormones, which play crucial roles in cartilage metabolism, proved to be significant both in human and animal models. In addition, clock genes--such as Clock, Bmal, Per, Cry and Dec--were identified in suprachiasmatic nucleus (SCN) and some peripheral tissues. These clock genes may be involved in circadian rhythms in cartilage.
Asunto(s)
Cartílago/metabolismo , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Animales , Proteínas CLOCK , División Celular/genética , Condrocitos/citología , Colágeno/biosíntesis , ADN/biosíntesis , Humanos , Proteoglicanos/biosíntesis , Transactivadores/fisiologíaRESUMEN
The basic helix-loop-helix transcription factor DEC1 is expressed in a circadian manner in the suprachiasmatic nucleus where it seems to play a role in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoter. The interaction of DEC1 with BMAL1 has been suggested as one of the molecular mechanisms of the suppression [Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. & Honma, K. (2002) Nature 419, 841-844]. Deletion analysis of DEC1 demonstrated that its N-terminal region, which includes the basic helix-loop-helix domain, was essential for both the suppressive activity and the interaction with BMAL1, as DEC1 lacking the basic region did not show any suppression or interaction. Furthermore, we found that Arg65 in the basic region, which is conserved among group B basic helix-loop-helix proteins, was responsible for the suppression, for the interaction with BMAL1 and for its binding to CACGTG E-boxes. However, substitution of His57 for Ala significantly reduced the E-box binding activity of DEC1, although it did not affect the interaction with BMAL1 or suppression of CLOCK/BMAL1-induced transcription. On the other hand, the basic region-deleted DEC1 acted in a dominant-negative manner for DEC1 activity, indicating that the basic region was not required for homodimer formation of DEC1. Moreover, mutant DEC1 also counteracted DEC2-mediated suppressive activity in a dominant-negative manner. The heterodimer formation of DEC1 and DEC2 was confirmed by pull-down assay. These findings suggest that the basic region of DEC1 participates in the transcriptional regulation through a protein-protein interaction with BMAL1 and DNA binding to the E-box.