| Circadian rhythm is a 24-hour periodic rhythm in the behavior and physiological processes of organisms.These rhythms are maintained by periodic oscillations generated by the circadian clock at the molecular level,and they are widely found in different species and are highly conserved.The post-translational modifications(such as acetylation,phosphorylation,etc.)of proteins play an important role in the maintenance of circadian oscillations in behavior and physiological processes.In recent years,many new lysine acylation modifications have been discovered,but how these modifications are involved in physiological processes is unknown.Previous results in our lab showed that protein lysine propionylations exhibited global circadian rhythms in fly heads,and this rhythm was regulated by the core circadian clock gene period.A time series propionylomic analysis of fly heads identified 341 propionylated sites of 171 proteins,79(23%)of which showed obvious rhythmic oscillations throughout the day.To explore the regulatory mechanism of rhythmic propionylation,one of the most obvious rhythmic propionylation site,propionylation at Lysine 17 of histone H2B(H2BK17pr)was selected.Since food can regulate circadian rhythm,and it is known that most propionyl groups in the body come from the metabolite of amino acids and fatty acids,we speculated that the rhythm of H2BK17 pr is regulated by rhythmic feeding.Therefore,we restricted the feeding time of wildtype flies to daytime or nighttime.We found that,compared with ad libitum feeding,daytime restricted feeding enhanced the amplitude of H2BK17 pr in the heads of male flies,while nighttime restricted feeding resulted in loss of rhythmic propionylation oscillation at this site,suggesting that rhythmic feeding is involved in regulating the rhythm of H2BK17 pr.Next,we further explored the regulatory mechanism of H2BK17 pr at the molecular level.Since some of the histone acetyltransferases(HAT)and deacetylases(HDAC)may also be involved in the regulation of propionylation,we screened these enzymes in S2 cell lines using inhibitors and si RNA.Type I HDAC(HDAC1and HDAC3)and CBP/NEJIRE of the HAT family were found to be involved in the regulation of H2BK17 pr.In order to investigate the physiological function of H2BK17 pr,we obtained fly line with H2BK17 mutated to alanine(H2BK17A),which rendered the site unable to undergo propionylation.The mutation had no obvious effect on locomotor rhythm or core clock gene expression.By time-series RNA sequencing of wildtype and mutant fly heads,we found that some of the transcripts cycling in wild type lost their oscillations(436 under light/dark cycles and 943 in constant darkness),while some of the non-cycling transcripts gained rhythm(459 under light/dark cycles and 1277 under constant darkness).This suggests that H2BK17 pr can systematically regulate the rhythmic expression of clock-controlled genes.Some genes involved in carbohydrate metabolism,glycolysis and fat storage lost their rhythms,while those involved in redox reaction and rhabdoid muscle development gained their rhythms in H2BK17 A mutants.Subsequently,some differentially expressed genes were further verified by real-time quantitative PCR.In summary,we have identified a preliminary mechanism regarding the regulation of H2BK17 pr in flies,and found that H2BK17 pr may be involved in regulating the rhythmic expression of genes related to many important physiological processes.This study provides a basis for further studies on propionylation in the regulation of circadian rhythm and physiological homeostasis. |
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