The circadian clock is generated by a molecular timekeeping mechanism coordinating daily oscillations of physiology and behaviors in mammals. In the mammalian circadian clockwork, basic helix-loop-helix ARNT-like protein 1 (BMAL1) is a core circadian component whose defects lead to circadian disruption and elicit behavioral arrhythmicity. To identify previously unknown regulators for circadian clocks, we searched for genes influencing BMAL1 protein level by using a CRISPR/Cas9-based genome-wide knockout library. As a result, we found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1 (USP1) positively affects BMAL1 protein abundance. Overexpression of wild-type USP1, but not a deubiquitinase-inactive mutant USP1, upregulated BMAL1 protein level, whereas genetic ablation of USP1 downregulated BMAL1 protein level in U2OS cells. Furthermore, treatment with USP1 inhibitors led to significant downregulation of BMAL1 protein in U2OS cells as well as mouse tissues. Subsequently, genetic ablation or pharmacological inhibition of USP1 resulted in reduced mRNA levels of a panel of clock genes and disrupted circadian rhythms in U2OS cells. Mechanistically, USP1 was able to de-ubiquitinate BMAL1 and inhibit the proteasomal degradation of BMAL1. Interestingly, the expression of Usp1 was much higher than the other two deubiquitinases of BMAL1 (Usp2 and Usp9X) in the mouse heart, implying a tissue-specific function of USP1 in the regulation of BMAL1 stability. Our work thus identifies deubiquitinase USP1 as a previously unknown regulator of the mammalian circadian clock and highlights the potential of genome-wide CRISPR screens in the identification of regulators for the circadian clock.
