| The circadian clock is a biological rhythm with a period of approximately 24 hours that controls the physiological and behavioral rhythms of an organism.This rhythm is established by a cell-autonomous oscillator and controlled by a negative transcription-translation feedback loop.Current studies have shown that posttranslational modification is an essential regulator of clock proteins,which regulates their transcriptional activity,subcellular localization and protein stability.CREB-binding protein(CREBBP)is a histone acetyltransferase that has been widely studied.Mutation of CBP can lead to a variety of diseases.Importantly,CBP plays an important role in the regulation of circadian rhythm,but its mechanism of action is not well understood.In addition,mouse Cbp homozygous mutants die around 1 week after birth,and deletion of the Drosophila CBP homolog nejire3(nej3)causes early embryonic lethality.These have limited the study of the role of CBP on the circadian clock in living organisms.Therefore,due to the lack of a homozygous mutant of Cbp,the specific mechanism of action involved in the circadian clock in vivo remains unclear.There are two copies of the cbp gene in zebrafish,cbpa and cbpb.Two hereditable cbp zebrafish mutants,cbpa-/-and cbpb-/-,were generated by CRISPR-Cas9 technology.Through behavioral analysis,we found that under normal light and dark conditions(LD),the average movement distance of cbpa-/-and cbpb-/mutants was shortened,and this difference mainly occurred when light was present during daytime.Under constant darkness(DD),both cbpa-/-and cbpb-/-mutants showed significant phase delay,period shortening and amplitude reduction.Quantitative real-time PCR(qRT-PCR)showed that the expression of clock genes per2,per1a and per1b was down-regulated and the rhythm was weakened under both LD and DD conditions in cbpa-/-and cbpb-/-zebrafish larvae mutants.The expression of clock genes in adult fish brain,eye,liver,muscle and intestines showed significant tissue specificity.At the same time,after the knockout of cbpa and cbpb genes,the rhythm,phase and amplitude of the three clock genes were significantly changed in liver tissue.These results indicate that Cbp also plays an important role in the regulation of peripheral circadian clock.In our study,we found that Cbpa and Cbpb could not only activate the expression of per2,per1a and per1b through cAMP/Ca2+response element binding protein(CREB)bind to CRE element,but also bind to the classical clock core protein Bmalb-Clockla to stimulate downstream gene expression through E-box.Further studies showed that Cbp activation of clock gene expression through CRE element requires the presence of E-box,while the CRE element is dispensable for Cbp activation of clock gene expression through E-box.These results suggest that the presence of E-box elements is a prerequisite for Cbp to regulate the circadian clock.Co1P assay also confirmed that Cbpa and Cbpb interacted with Clockla-Bmallb and Creb1a to regulate the circadian clock.We further found that Cbpa could form a complex with both Clock1a-Bmal1b and Creb1a.These results suggested that Cbpa formed a complex with Clockla-Bmallb and Crebla in the presence of E-box and CRE elements to regulate clock gene expression through the co-ordination of E-box and CRE elements.Since CBP plays an important role in metabolic regulation in mammals,our results demonstrated that zebrafish Cbp also plays an important role in regulating the circadian clock.So,does Cbp regulate metabolic rhythms?We found that in the liver tissue of cbpa-/-mutants,glycogen synthase 1(gys1),pyruvate dehydrogenase alpha(pdh1a),phosphofructokinase(pfkma)and lactate dehydrogenase(ldha),involved in glucose metabolism,were down-regulated and the rhythm and phase were significantly changed.In addition,key genes in the lipid metabolism pathway,acetyl-CoA carboxylase(acaca),Carnitine Palmitoyltransferase Ⅱ(cpt2),fatty acid binding protein(fabpla),fatty acid synthase(fasn),adipose triglyceride lipase(pnpla2)and stearoyl-CoA desaturase 1(scd1)were also down-regulated in cbpa-/liver tissue,the rhythm and phase were also significantly changed.These results suggest that Cbp plays an important role in regulating the rhythm of glucose and lipid metabolism in the liver.In summary,we have generated stable cbpa-/-and cbpb-/-zebrafish lines and found that mutations of cbp affected clock gene expression,motor behavior rhythm and peripheral circadian clock.We also revealed the mechanism of Cbp action on the circadian clock:E-box is an essential element of Cbp involved in the regulation of circadian clock.Cbp binds with CLOCK-BMAL to regulate the expression of E-box containing genes.When CRE element is present,Cbp binds with CLOCK-BMAL and Creb to form a complex to co-regulate the expression of genes both containing E-box and CRE element.Finally,we found that Cbp deficiency affected the rhythms of glucose and lipid metabolism. |
