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Circadian clock function by quantitative proteomics and phosphoproteomics

Final Report Summary - CLOCKPROTEOMICS (Circadian clock function by quantitative proteomics and phosphoproteomics)

The main objective of this project was to identify, using SILAC-based quantitative mass spectrometry, global circadian oscillations in total proteome and phosphoproteome from mouse liver. During the time of this grant I have successfully completed the proteome aspect of the project by applying state of the art mass spectrometry in combination with quantitative proteomics. The proteome dataset was compared with published microarray data [1] to elucidate the significance of circadian post-transcriptional mechanisms in the regulation of liver metabolism. I found that approximately 6% of the liver proteins are cycling daily and interestingly, the majority of these protein oscillations diverge from the behavior of their transcripts. The data indicates that post-transcriptional mechanisms play an essential role in shaping the phase of rhythmic proteins downstream of transcription to ultimately drive cycles of metabolism. Moreover, the contribution of post-transcriptional regulation seems to differ among distinct metabolic pathways. Overall I found circadian oscillations of abundance in proteins not only involved in liver specific metabolic pathways but also in essential cellular processes. This work has been published in a peer review journal [2] and it is a landmark in the circadian field since it described the first circadian proteome in a mouse tissue obtained using state of the art mass spectrometry. Moreover, I am currently working assessing the circadian phosphoproteome in this same tissue. The data indicates that in the mouse liver a broad range of proteins show also circadian oscillations of their phosphorylation levels. Since up to day no reports have described daily changes of the global phosphoproteome in a mammalian tissue, we foresee that this study will be another breakthrough in the field and will ultimately contribute to the understanding of how metabolism is controlled by the circadian clock.
Another objective of this project was to isolate and analyse clock protein complexes bound to conserved DNA motifs in the per2 gene promoter. The complex isolation protocol consisted in incubating biotynilated DNA sequences (consensus and mutant as control) with nuclear extracts from mouse fibroblast (NIH3T3) differentially labeled with SILAC, as described [3]. Purified complexes were measured by MS. Analysis of the data allowed to identify several proteins in addition to BMAL and CLOCK that bind specifically consensus DNA sequences. Besides the cell culture experiments, I also performed in collaboration with the laboratory of Charles J. Weitz in Harvard Medical School similar interaction proteomics analyses with an in vivo set up, relevant for a more physiological outcome. In this case we used nuclear protein extracts from mouse livers sacrificed at specific circadian times to define a more time dependent complex. In addition we applied for quantification a label free method which has been successfully used in our department in the last years[4]. Control experiments were done using not only the mutant DNA sequence but also nuclear protein extracts obtained from BMAL1 knock out livers. This additional control allowed us to specifically find CLOCK-BMAL1 dependent DNA interactors. We have thus successfully identified a new potential component of the CLOCK-BMAL1 complex bound to DNA that has not been previously described. In particular, this protein has been described as an ubiquitin ligase that may play a role in chromatin modification, which could then mediate in clock-controlled transcription regulation. Further interaction and functional validation analyses are under investigation in order to define the role of this potential component in the molecular machinery of the clock.
1. Hughes ME, DiTacchio L, Hayes KR, Vollmers C, Pulivarthy S, et al. (2009) Harmonics of circadian gene transcription in mammals. PLoS Genet 5: e1000442.
2. Robles MS, Cox J, Mann M (2014) In-vivo quantitative proteomics reveals a key contribution of post-transcriptional mechanisms to the circadian regulation of liver metabolism. PLoS Genet 10: e1004047.
3. Butter F, Davison L, Viturawong T, Scheibe M, Vermeulen M, et al. (2012) Proteome-wide analysis of disease-associated SNPs that show allele-specific transcription factor binding. PLoS Genet 8: e1002982.
4. Cox J, Hein MY, Luber CA, Paron I, N. N, et al. (2014) MaxLFQ allows accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction. Molecular and Cellular Proteomics.