Project description:Circadian clocks coordinate time-of-day specific metabolic and physiological processes to maximize performance and fitness. In addition to light, which is considered the strongest time cue to entrain animal circadian clocks, metabolic input has emerged as an important signal for clock modulation and entrainment, especially in peripheral clocks. Circadian clock proteins have been to be substrates of O-GlcNAcylation, a nutrient sensitive post-translational modification (PTM), and the interplay between clock protein O-GlcNAcylation and other PTMs, like phosphorylation, is expected to facilitate the regulation of circadian physiology by metabolic signals. Here, we used mass spectrometry proteomics to identify PTMs on PERIOD, the key biochemical timer of the Drosophila clock, over the circadian cycle.
Project description:Circadian rhythms are daily oscillations in metabolism and physiology and are generated by the circadian clock. In fruit fly Drosophila, the circadian clock is generated by a transcription-translation feedback loop in which the positive arm components Clock and Cycle activate the expression of the Period and Timeless genes of negative arm, as well as other circadian clock-regulated genes. After being retained in the cytoplasm, the Period and Timeless proteins then migrate to the nucleus to inhibit the Clock/Cycle transactivity by protein-protein interactions (PPIs). The endogenous circadian clock is synchronized with the geological (solar) clock via photoreceptors. Drosophila Cryptochrome protein functions as a circadian photoreceptor. In the early morning, exposure of Cryptochrome to light causes a conformational change in it which results in the formation of new PPIs. Light-activated Cryptochrome interacts with the core clock protein Timeless and the E3 ubiquitin ligase-substrate adaptor protein Jetlag, which results in the ubiquitylation of Timeless by Jetlag-E3 ligase complex and then degradation of Timeless within minutes by proteasome system. Rapid degradation of Timeless and then its partner protein Period, because of its instability in the absence of Timeless, relieves the inhibition on the Clock/Cycle transcription factors suddenly. Therefore, Clock/Cycle-driven expression of circadian clock-regulated genes are induced again, which is the restart of the circadian oscillation or the resetting of the clock. Following Timeless degradation, Cryptochrome is also degraded so the photoreceptor mechanism does not start a new resetting signal until all the required factors are re-synthesized in a circadian manner. Light-dependent degradation of Drosophila Cryptochrome can be observed in Drosophila S2 cell line in culture. In this project, we aimed at finding the interactome of Cryptochrome protein in Drosophila S2 cell line under light and in the dark using proximity labeling method. Because of the fast kinetics of Cryptochrome degradation, we chose the enzymes that can saturate in less than one hour. TurboID (TID) and APEX2 enzymes label proteins with biotin in the proximity even though they work with different mechanisms. They were fused to Cryptochrome protein, and proximity labeling was performed in the dark or under light. We have identified novel light-dependent or -independent interactors of Drosophila Cryptochrome and confirmed some of them using classical coimmunoprecipitation technique.
Project description:CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation-tiling array assays (ChIP-chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4-6 hrs later by the transcriptional repressor PER, indicating that the majority of CLK targets are regulated similarly to core circadian genes (Menet et al. 2010). About 30% of target genes also show cycling Pol II binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation.
Project description:We report a large-scale transcriptomic analysis of several tissues of a reference Drosophila melanogaster strain as well as 141 Drosophila Genetic Reference Panel (DGRP) lines at high temporal resolution. Comprehensive data analysis has identified thousands of genes under clock- and tissue-specific control. By using a molecular time table approach, we uncovered that >20% of probed DGRP lines exhibit aberrant circadian expression, and the genetic dissection of one line (DGRP-796) revelled disrupted circadian gene expression in all analysed tissues, revealing a novel deletion in the cry gene. Together, this study revealed extensive variation in tissue-specific circadian expression, which acts upon tissue-specific regulatory networks to generate local oscillations in gene expression. Moreover, the many other lines identified here can be now used to better understand the mechanisms underlying the molecular clock, from tissue-specific to more central mechanisms.
Project description:Nearly half of the genes in the eye are regulated through the molecular circadian clock, a transcription-translation feedback loop that is governed by environmental stimuli. In aging, vision loss correlates with perturbations to the circadian clock. Rhythmic gene expression is substantially altered in the aging Drosophila melanogaster eye in that over 1/3 of genes have differential rhythmic expression. H3K4me3 is important for transcriptional activation of genes regulated by the circadian clock and is globally decreased in aging photoreceptors. It is unknown what mechanism underlies decreased H3K4me3. Glycine-N-methyltransferase (Gnmt) regulates the availability of the universal methyl donor S-adenosyl methionine (SAM) by methylating glycine to produce sarcosine and S-adenosylhomocysteine (SAH), a potent inhibitor of histone methyltransferase activity. Both Gnmt and SAH are increased in the aging eye, therefore we hypothesize that an age-dependent increase of Gnmt contributes to disruption of rhythmic gene expression. To test this hypothesis, nuclear RNA-seq was performed on photoreceptor nuclei that were collected every four hours from flies outcrossed to wild type or with overexpression of either active or partially active Gnmt