Sumoylation controls CLOCK-BMAL1-mediated clock resetting via CBP recruitment in nuclear transcriptional foci.

CLOCK-BMAL1 is a key transcription factor complex of the molecular clock system that generates circadian gene expression and physiology in mammals. Here, we demonstrate that sumoylation of BMAL1 mediates the rapid activation of CLOCK-BMAL1 by CREB-binding protein (CBP) in nuclear foci and also the resetting of the circadian clock. Under physiological conditions, a bimolecular fluorescence complementation-based fluorescence resonance energy transfer (BiFC-FRET) assay revealed that CLOCK-BMAL1 rapidly dimerized and formed a ternary complex with CBP in discrete nuclear foci in response to serum stimuli. We found that the formation of this ternary complex requires sumoylation of BMAL1 by SUMO3. These processes were abolished by both the ectopic expression of the SUMP2/3-specific protease, SUSP1, and mutation of the major sumoylation site (Lys259) of BMAL1. Moreover, molecular inhibition of BMAL1 sumoylation abrogated acute Per1 transcription and severely dampened the circadian gene oscillation triggered by clock synchronization stimuli. Taken together, these findings suggest that sumoylation plays a critical role in the spatiotemporal co-activation of CLOCK-BMAL1 by CBP for immediate-early Per induction and the resetting of the circadian clock.

Mechanism of photosignaling by Drosophila cryptochrome: role of the redox status of the flavin chromophore.

Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. Upon light absorption, dCRY undergoes a conformational change that enables it to bind to Timeless (dTIM), as well as to two different E3 ligases that ubiquitylate dTIM and dCRY, respectively, resulting in their proteolysis and resetting the phase of the circadian rhythm. Purified dCRY contains oxidized flavin (FADox), which is readily photoreduced to the anionic semiquinone through a set of 3 highly conserved Trp residues (Trp triad). The crystal structure of dCRY has revealed a fourth Trp (Trp-536) as a potential electron donor. Previously, we reported that the Trp triad played no role in photoinduced proteolysis of dCRY in Drosophila cells. Here we investigated the role of the Trp triad and Trp-536, and the redox status of the flavin on light-induced proteolysis of both dCRY and dTIM and resetting of the clock. We found that both oxidized (FADox) and reduced (FAD) forms of dCRY undergo light-induced conformational change in vitro that enable dCRY to bind JET and that Trp triad and Trp-536 mutations that block known or presumed intraprotein electron transfer reactions do not affect dCRY phototransduction under bright or dim light in vivo as measured by light-induced proteolysis of dCRY and dTIM in Drosophila S2R+ cells. We conclude that both oxidized and reduced forms of dCRY are capable of photosignaling.

Core clock regulators in dexamethasone-treated HEK 293T cells at 4 h intervals.

OBJECTIVE: The study of the circadian clock and its mechanisms is easily facilitated through clock resetting in cell culture. Among the various established synchronizers of the circadian clock in cell culture (temperature, serum shock, glucocorticoids), the artificial glucocorticoid Dexamethasone (DEX) is the most widely used. DEX treatment as a protocol to reset the circadian clock in culture gives simple readout with minimal laboratory requirements. Even though there are many studies regarding clock resetting in culture using DEX, reference points or expression patterns of core clock genes and their protein products are scarce and sometimes contradict other works with similar methodology. We synchronise a cell line of human origin with DEX to be used for studies on circadian rhythms. RESULTS: We treat HEK 293T cells with DEX and describe the patterns of mRNA and proteins of core clock regulators, while making a clear point on how CLOCK is less than an ideal molecule to help monitor rhythms in this cell line.