Kinetic Analysis of Transcriptional and Post-Transcriptional Processes During Circadian Cycles

Every morning the sun rises, flooding the earth with its light and heat and every night it disappears leaving the Earth in the darkness of the night. Organisms living on this planet have adapted to this solar rhythm and have spread their activities throughout the day. Early in the development of life, an internal molecular clock appeared in primitive organisms and the evolutionary advantage of being able to anticipate, rather than simply passively face, environmental changes led virtually all organisms to have an internal timing system called "circadian clock." In most species, this clock is constituted of a genetic network ticking in every cell of the body. This genetic network organized in interlocked feedback loops generates oscillations with a 24-hour period. A variable number of proteins and RNAs is thus accumulating in a rhythmic fashion. For example, in mouse liver, which was the main biological model I used during my thesis, it is estimated that thousands of genes are expressed periodically. The molecular processes leading to rhythmic gene expression and protein accumulation are highly regulated. Dynamical control is needed to sustain the oscillations and to maintain their timing. During my PhD, I was particularly interested in the regulation of rhythmic gene activation via transcription factor binding in their promoter region. I also focused on gene transcription itself and its dynamics as well as on transcription associated changes in epigenetic marks. Another interest was given to the regulation of transcripts (messenger RNA) after transcription, especially to understand whether their degradation in the cytoplasm was regulated rhythmically. Finally, I contributed to a project aiming at the detection of transcripts whose translation into protein was specifically regulated in a circadian manner. The four projects on which I worked were done in collaborations with experimental biology labs. Our partners performed the experiments and provided the biological material while I was in charge of the data processing and their statistical analysis. For some projects, I also developed different mathematical models for a more accurate and complete understanding of the phenomena studied and to take into account the inherent variability of biological data. Notably in the project on the post-transcriptional regulation of transcripts, the model-based approach we developed allowed estimating the role of this regulation in a way that is less prone to arbitrary choice of criteria or thresholds compared to previous studies addressing this question. To summarize, the results I obtained show that, in human cell lines, the major transcription factor of the clock (the heterodimer CLOCK/BMAL1) binds to DNA at specific binding sites and their number is comparable to the number of binding sites observed in the mouse liver. However the fraction of rhythmic transcript among the genes with CLOCK/BMAL1 binding sites is about 10 times lower than the corresponding fraction observed in mouse liver. The results of the second project showed that, in mouse liver, rhythmic transcription is followed by dynamical changes in epigenetic marks with delays specific to the marks (tri-methylation of lysines 4 and 36 of histone 3 of nucleosomes) and we highlighted the role of post-transcriptional regulation for the rhythmic expression of a subset of mRNAs. In the third project, we further investigated the regulation at the post-transcriptional level and combining measurements of pre-mRNA and mRNA with a mathematical model, we showed that the post-transcriptional regulation plays a limited role in the rhythmic accumulation of transcripts and is involved in the regulation of only 20 % of rhythmic transcripts. Laboratories of Michael Brunner (University of Heidelberg, Germany), Frederic Gachon, Nouria Hernandez (University of Lausanne, Switzerland) and Ueli Schibler (University of Geneva, Switzerland) 3 Finally, helped by the analysis I performed on micro array data from an experiment aiming at comparing the fraction of translated mRNA to the total accumulation of these mRNAs, the team of Frederic Gachon showed that several transcripts implied in the composition of ribosomes are translated specifically during the night, when rodents are active. All of these results and their links with other published studies on circadian rhythms, show that biological tissues have largely incorporated the clock for the regulation of many other functional pathways in order to optimally synchronize their physiological actions with environmental changes due to the alternation of days and nights. In mouse liver, links with the metabolism are particularly strong and disruptions of the clock induce phenotypes reflecting a disturbed metabolism.