Abstract
Endogenous clocks generate rhythms in gene expression, which facilitates the organisms to cope through periodic environmental variations in accordance with 24-h light/dark time. A core question that needs to be elucidated is how such rhythms proliferate throughout the cells and regulate the dynamic physiology. In this study, we demonstrate the role of REGγ as a new regulator of circadian clock in mice, primary MEF, and SY5Y cells. Assessment of circadian conduct reveals a difference in circadian period, wheel mode, and the ability to acclimate the external light stimulus between WT and KO littermates. Compared to WT mice, REGγ KO mice attain the phase delay behavior upon light shock at early night. During the variation of 12/12 h light/dark (LD) exposure, levels of Per1, Per2, Cry1, Clock, Bmal1, and Rorα circadian genes in suprachiasmatic nucleus are significantly higher in REGγ KO than in WT mice, concomitant with remarkable changes in BMAL1 and PER2 proteins. In cultured cells depleted of REGγ, serum shock induces early response of the circadian genes Per1 and Per2 with the cyclic rhythm maintained. Mechanistic study indicates that REGγ directly degrades BMAL1 by the non-canonical proteasome pathway independent of ATP and ubiquitin. Silencing BMAL1 abrogates the changes in circadian genes in REGγ-deficient cells. However, inhibition of GSK-3β, a known promoter for degradation of BMAL1, exacerbates the action of REGγ depletion. In conclusion, our findings define REGγ as a new factor, which functions as a rheostat of circadian rhythms to mitigate the levels of Per1 and Per2 via proteasome-dependent degradation of BMAL1.
Highlights
Earth’s atmosphere has the evolutionarily fundamental characteristic of 24-h light/dark (LD) cycle that effectuate the predominant influence on sleep–wake and activity of organisms
Mice with REGγ deficiency exhibit circadian phase change circadian oscillations are self-sustained in individual cell lines, so to upon light stimulus validate the impact of REGγ on cyclic rhythm of circadian genes, Circadian rhythms of physiology, behavior, and biochemical reactions we treated MEF and SY5Y cells with 50% horse serum for 2 h are strongly synchronized by the endogenous circadian clocks. followed by serum-free medium and harvested the cells at
Alteration of circadian physiology induced by REGγ abrogation is GSK-3β-BMAL1 dependent Given that GSK-3β regulates BMAL1 protein stability and circadian function, we explored the regulation of circadian rhythms by REGγ following manipulation of BMAL1 protein levels in primary MEF and SY5Y cells
Summary
Earth’s atmosphere has the evolutionarily fundamental characteristic of 24-h light/dark (LD) cycle that effectuate the predominant influence on sleep–wake and activity of organisms. REGγ KO mice achieved the phase reaction (qPCR) and gel-based PCR analysis revealed that the basal delay behavior of running wheel upon light stimulus in constant mRNA expressions of circadian clock genes were significantly dark/dark (D/D); the REGγ WT mice did not attain the higher in SCN of REGγ KO mice than in WT control (Fig. S2A, B). We established a potential role of REGγ in the regulation of To determine the influence of REGγ on rhythmic expression of the circadian clocks by promoting direct proteasomal circadian genes in vitro, we conducted real-time qPCR and geldegradation of BMAL1, thereby, altering the circadian gene expres- based PCR analysis in REGγ WT/KO primary MEF and neuronal sion. Consistent with in vivo expression of circadian genes in SCN, Per1/2, Cry, Clock, Bmal, and Ror α were expressed higher in MEF KO (Fig. S3A, B) and SY5Y ShR cells (REGγ knockdown cells)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
