Project description:The circadian clock controls the expression of nearly 50% of protein coding genes in mice, and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.
Project description:The circadian clock is comprised of proteins that form negative feedback loops, which regulate the timing of global gene expression in a coordinated 24 hour cycle. As a result, the plant circadian clock is responsible for regulating numerous physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have combined high-throughput RNA-sequencing and mass spectrometry to comparatively characterize the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette transcriptome and proteome at the end-of-day and end-of-night.
Project description:The timing of many molecular and physiological processes in plants occurs at a specific time of day. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT INDUCIBLE AND CLOCK REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover new connections within the clock and between other pathways, here we use affinity purification coupled with mass spectrometry to discover time-of-day-specific protein interactors of the RVE8-LNK1/2 complex. Our clock proteins are tagged with a 6X-His-3X-FLAG C-terminal (HFC) affinity tag and purified through anti-FLAG immunoprecipitation and His-tag isolation affinity purification. We also performed affinity purification-mass spectrometry of 35S::YFP-COR27 and 35S::GFP-COR28, which were immunoprecipitated with anti-GFP TRAP beads. Experiment was performed on 10-day-old seedlings grown under 12 hours light: 12 hours dark 22 degrees Celsius.
Project description:Circadian rhythms are a series of endogenous autonomous 24-hour oscillations generated by the circadian clock. At the molecular level, the circadian clock is generated by a transcription-translation feedback loop, where BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. In this project, we aimed at finding the interactome of PER2 protein in human U2OS osteosarcoma cell line using proximity-dependent biotin identification (BioID) technique. U2OS clones overexpressing PER2-BioID2 or BioID2 were treated with dexamethasone in order to reset the circadian rhythm, and cells were then incubated in biotin-containing media for 12 hours to label the proteins in close proximity of PER2-BioID2. Samples were collected after 36 and 48 hours of the resetting to identify the labeled proteins by mass spectrometry. In addition to known interactors such as CRY1 and CRY2, many novel interactors were identified. In summary, we obtained a network of PER2 interactome and confirmed some of the novel interactions using classical the co-immunoprecipitation method.
Project description:Circadian clocks drive ~24 hr rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes.To gain insights into the role of the mammary clock, circadian time-series microarrays were performed to identify rhythmic genes in vivo. Breast tissues were isolated at 4 hr intervals for two circadian (24 hourly) cycles, from mice kept under constant darkness to avoid any light- or dark-driven genes.
Project description:Influence of diet and neuronal clk (clock) activity on hemolymph proteomics. We have shown that as photoreceptors die (in the fly) they necrose, which results in their intercellular contents leaking into the hemolymph. We hypothesize that this process is regulated by diet and circadian clock control.
Analysis of differential protein expression in the hemolymph from flies reared on a high protein diet. Comparison of flies with and without a functional circadian clock within their photoreceptors.
Species/Strain: Drosophila, Elav-GeneSwitch-GAL4>UAS-Clk-DN1 (+/- RU486), female
Project description:Plants are sessile organisms that have acquired highly plastic developmental strategies to adapt to the environment. Among these processes, the floral transition is essential to ensure reproductive success and is finely regulated by several internal and external genetic networks. The photoperiodic pathway, which controls the plant response to day length, is one of the most important pathways controlling flowering. In Arabidopsis photoperiodic flowering, CONSTANS (CO) is the central gene activating the expression of the florigen FLOWERING LOCUS T (FT) in the leaves at the end of a long day. CO expression is strongly regulated by the circadian clock. However, to date, no evidence has been reported regarding a feedback loop from the photoperiod pathway back to the circadian clock. Using transcriptional networks, we have identified relevant network motifs regulating the interplay between the circadian clock and the photoperiod pathway. Gene expression, chromatin immunoprecipitation experiments and phenotypic analysis allowed us to elucidate the role of CO over the circadian clock. Plants with altered CO expression showed a different internal clock period, measured by daily rhythmic movements in the leaves. We show that CO is able to activate key genes related to the circadian clock, such as CCA1, LHY, PRR5 and GI, at the end of a long day by binding to specific sites on their promoters. Moreover, a significant number of PRR5 repressed target genes are upregulated by CO, and this could explain the phase transition promoted by CO. The CO-PRR5 complex interacts with the bZIP transcription factor HY5 and helps to localize the complex in the promoters of clock genes. Our results indicate that there may be a feedback loop in which CO communicates back to the circadian clock, feeding seasonal information to the circadian system.
Project description:In mammals, circadian clocks are strictly suppressed during early embryonic stages as well as pluripotent stem cells, by the lack of CLOCK/BMAL1 mediated circadian feedback loops. During ontogenesis, the innate circadian clocks emerge gradually at a late developmental stage, then, with which the circadian temporal order is invested in each cell level throughout a body. Meanwhile, in the early developmental stage, a segmented body plan is essential for an intact developmental process and somitogenesis is controlled by another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM). In the present study, focusing upon the interaction between circadian key components and the segmentation clock, we investigated the effect of the CLOCK/BMAL1 on the segmentation clock Hes7 oscillation, revealing that the expression of functional CLOCK/BMAL1 severely interferes with the ultradian rhythm of segmentation clock in induced PSM and gastruloids. RNA sequencing analysis showed that the premature expression of CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These results suggest that the suppression of CLOCK/BMAL1-mediated transcriptional regulation during the somitogenesis may be inevitable for intact mammalian development.