Thèse de doctorat en Aspects moléculaires et cellulaires de la biologie
Sous la direction de Franck Delaunay.
Soutenue en 2006
à Nice , dans le cadre de École doctorale des Sciences de la vie et de la santé (Sophia Antipolis, Alpes-Maritimes) .
Circadian clock are endogenous time-keeping systems that control a variety of physiological functions in all organisms ranging from cyanobacteria to mammals. Circadian clock generate a 24h oscillation by way of a molecular oscillator. The synchronization of this oscillator and its molecular mechanisms has been extensively characterized these last years, but we still don’t understand how this oscillator regulates rhythmically downstream pathways. The aim of this study is to understand how these regulations occur in the mouse liver, in which many biological processes are under circadian control. Recent work in Drosophila highlighted that most of the circadian control is indirect, suggesting that the oscillator regulate genes like transcription factor to convey the temporal signal. To understand how the oscillator regulates rhythmically downstream pathways, we performed a microarray analysis to characterize the clock-controlled gene expression in mouse liver over a 24h LD12:12 cycle. These microarrays allow the study of more than 12,000 genes. We found that 6500 genes were significantly expressed, which represent about 70% of the liver transcriptome. By using an optimized algorithm, we identified 237 genes with a robust circadian expression pattern. These genes control a wide variety of biological processes, and a phase analysis suggests that some processes are co-regulated to specific times of the day. Of these 237 genes, 20 encode transcription factors. For 15 of them, the circadian regulation was unknown before this work. One of these transcription factors, Stra13, encode a transcriptional repressor involved in apoptosis, cell proliferation, differentiation, and adaptation to hypoxia. A detailed study of its circadian regulation indicates that this gene is circadianly expressed in many peripheral organs of mice kept in constant darkness (DD). This circadian regulation of Stra13 must have an important role in the physiology since it also conserved in Zebrafish. Importantly, this circadian regulation disappeared in tumoral cells and hypoxic cultured cells. A functional study demonstrated that the Stra13 promoter is activated by the clock core transcription factors CLOCK and BMAL1, and that this activation was repressed by CRY1 and STRA13 itself. The circadian clock of mice lacking the Stra13 gene isn’t affected, indicating that Stra13 may not play a role in the circadian oscillator mechanism, although we observed an up-regulation of BHLHB3, a Stra13 paralog. On the other hand, 20 out of 42 Stra13 target genes are circadianly expressed in wild type mice, and we demonstrated that this circadian regulation disappeared in Stra13 deficient-mice, proving that Stra13 is a clock output regulating physiological processes in the mouse liver. Altogether our results demonstrate the functional diversity of the circadian transcriptome in the mouse liver; and that near 10% of this transcriptome encode transcription factors, which confirmed the initial hypothesis. Finally, the detailed study of Stra13 demonstrates a novel indirect regulatory mechanism of the circadian gene expression in the mouse liver.
The hepatic circadian transcriptome : the transcriptional regulator Stra13 regulates a subset of peripheral circadian output
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