Study On The Function Of Biological Clock Rev - Erbα In Zebrafish Autophagy

Autophagy is evolutionally conserved machinery for cellular homeostasis and cell fate decisions. A previous study indicated that the circadian clock plays an important role in mouse autophagy. However, the mechanisms underlying circadian regulation of autophagy is not clear. Here TEM analysis showed that the number of autophagosomes changes daily and peaks in early morning in zebrafish, and quantitative real-time PCR showed that autophagy genes atg4a, atg4d, lc3b, gabarapa, ulk1a and ulk1b, and autophagy-related genes beclin1, bnip3and atp6v1d are all rhythmically expressed in zebrafish under both LD (light/dark) and DD (constant dark) conditions but lose their rhythmic expression in both zebrafish per1b and rev-erba mutants. Luciferase reporter assays showed that while the promoter activities of ulk1a are activated by Roraa but repressed by Rev-erba. We found that there were two classic RORE elements in ulkla’s promoter, when RORE elements were mutated, the luciferase activities of ulk1a was down-regulated by co-transferase with Roraa and Rev-erba.ChIP (Chromatin Immunoprecipitation) assays showed that Rev-erba bind to the RRE boxes in the promoter region of ulkla in zberafish. Moreover, the JC-1staining results indicated that Rev-erba plays a critical role in balancing the cell energy through autophagy. Taken together, our findings indicate that circadian clock genes rev-erba and per1b are essential for autophagy rhythms, and circadian regulation of autophagy is fulfilled through direct transcriptional control of ulk1a in zebrafish.