Studies On The Role Of BMAL1O-GlcNAcylation In Circadian Rhythms

Objective:Various physiological, biochemical processes and behaviors show a circadian rhythm of about24hours, which exists in most light-sensitive organisms. From regulating the photosynthesis in cyanobacteria to affecting feeding of mammals, the circadian rhythm shows a close relationship with metabolism. The molecular mechanisms beneath how metabolism cycling regulates circadian rhythms leave us a lot of unknowns. Our research on identifying the regulatory functions of O-GlcNAcylation in circadian rhythms will improve the understanding of the crosstalk between metabolism and circadian rhythms.Methods:In current model of mammalian circadian timing system, transcription/translation feedback loops, where transcriptional activators BMAL1and CLOCK are in the core, are considered the prime mechanism sustaining internal oscillators. Using real time bioluminescence recording assay, we detected the clock gene expression in N1H3T3cells when the cells were treated with O-GlcNAcylation inhibitor DON.Succinylated wheat germ agglutinin enriches O-GlcNAcylated proteins specifically. We used various clock protein antibodies to identify which clock protein can be O-GlcNAcylated. In the meantime, we detected the O-GlcNAcylation of immunoprecipitated clock proteins using CTD110.6antibody. The interaction between clock proteins and OGT was testified by co-immunoprecipitation assay and GST pull down assay.We tested the stability of clock proteins when inhibiting the O-G1cNAcylation. The regulatory function of O-G1cNAcylation in the transcriptional activity of CLOCK/BAML1heterodimers was tested by the luciferase reporter assay.Results:1. The expression of clock gene Email decreased dramatically and the rhythms of NIH3T3cells attenuated quickly when the cells were treated with DON;2. sWGA enriched BMAL1protein specifically. The states of immunoprecipitated BMAL1O-GlcNAcylation increased with the overpression of OGT and decreased with the expression of OGA;3. The co-immunoprecipitation assay and GST pull down assay showed the interaction between BMAL1and OGT;4. The stability of BAML1decreased as DON treatment or OGA overexpression;5. The transcriptional activity of CLOCK/BMAL1heterodimers was inhibited by DON treatment or OGA overexpression.Conclusions:1. The core clock protein BMAL1can be O-GlcNAcylated;2. O-GlcNAcylation stabilizes BMAL1;3. The transcriptional activity of CLOCK/BMAL1heterodimers was inhibited by decreased O-GlcNAcylation;In this study, we proposed that the O-GlcNAcylation of BMAL1is a novel candidate for the mechanisms of metabolism regulating circadian rhythms. As O-GlcNAcylation is sensitive to the glucose level, such a modification may provide a new vision linking metabolism to circadian rhythms.