Mechanism Study Of Circadian Phase Shift

During evolution,almost all creatures on the earth develop an internal circadian clock,which widely participates in the regulation of many physiological activities.Dysfunction of circadian clock leads to severe health problems and greatly decreases the quality of life.Although the modernization of lifestyle and the prevalence of circadian phase dysregulation related disease,the molecular mechanism of circadian phase regulation is unclear and there is no drug for circadian phase dysregulation treatment.In this study,we performed a chemical screen and identified multiple circadian phase shift compounds.CA01 dramatically shifts the circadian phase in both central and peripheral clock models.Using AID system(Auxin-inducible Degron System),we found CA01 target protein CDK9 directly regulates circadian phase.Using nascent RNA-seq,Real-time PCR,in vivo imaging and mathematical modeling experiments,we found CA01 shifts the clock phase by activating E-box gene transcription.Manipulating PER2(a classical E-box gene)protein level with AID system is enough to induce a dramatic phase shift.Besides,we found a nature product CA03 has a strong phase shift ability in both mammalian and plant circadian model,which indicates that the phase shift ability of CA03 is conserved among species.The molecular structure of CA03 is extremely similar to adenosine.CA03 enters cell via adenosine transporter.Inhibiting adenosine deaminase dramatically decreases the working concentration of CA03.Using wheelrunning behavioral assay and in vivo SCN rhythm recording,we found the natural product CA03 shifts the clock in both individual animal level and SCN nucleus,which makes it very likely to be a drug for circadian phase dysregulation treatment.Using Real-time PCR and in vivo imaging,we found CA03 shift the clock phase by activating E-box gene transcription.Using siRNA screen,biochemistry and cell biology approaches,we found CA03 shift mammalian clock phase via RUVBL2.CA03 directly binds to RUVBL2 and decrease its helicase activity.RUVBL2 rhythmically interacts with Bmall to specifically bind and regulates E-box gene transcription.CA03 decreases the interaction between RUVBL2 and Bmall to activate E-box gene transcription.In summary,this project discovered multiple circadian phase regulating compounds via chemical screen and preliminary studied the mechanism,which provides useful tools for circadian phase mechanism study and choices for treatment of circadian phase dysregulation disease.