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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. [ABSTRACT FROM AUTHOR]

The deubiquitinating enzyme USP1 contains highly conserved motifs forming its catalytic center. Recently, the COSMIC mutation database identified a mutation in USP1 at Asp‐199 in endometrial cancer. Here, we investigated the role of Asp‐199 for USP1 function. The mutation of aspartic acid to alanine (D199A) resulted in failure of USP1 to undergo autocleavage and form a complex with ubiquitin, indicating D199A Usp1 is catalytically inactive. The D199A mutation did not affect the interaction with Uaf1. Moreover, D199A Usp1 had defects in deubiquitination of FANCD2 and PCNA and displayed reduced FANCD2 foci formation and DNA repair efficiency. Furthermore, mutation of Asp‐199 to glutamic acid resulted in phenotypes similar to the D199A mutation. Collectively, our findings demonstrate the importance of Asp‐199 for USP1 activity and suggest the implications of USP1 downregulation in cancer. [ABSTRACT FROM AUTHOR]

The histone demethylase lysine‐specific demethylase 4A (KDM4A) is reported to be overexpressed and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 or H3K36 methylation marks. However, the biological role and mechanism of KDM4A in prostate cancer (PC) remain unclear. Herein, we reported KDM4A expression was upregulation in phosphatase and tensin homolog knockout mouse prostate tissue. Depletion of KDM4A in PC cells inhibited their proliferation and survival in vivo and vitro. Further studies reveal that USP1 is a deubiquitinase that regulates KDM4A K48‐linked deubiquitin and stability. Interestingly, we found c‐Myc was a key downstream effector of the USP1‐KDM4A/androgen receptor axis in driving PC cell proliferation. Notably, upregulation of KDM4A expression with high USP1 expression was observed in most prostate tumors and inhibition of USP1 promotes PC cells response to therapeutic agent enzalutamide. Our studies propose USP1 could be an anticancer therapeutic target in PC. [ABSTRACT FROM AUTHOR]

PI3K‐Akt‐FoxO‐mTOR signaling is the central pathway controlling growth and metabolism in all cells. Ubiquitination of the protein kinase Akt prior to its phosphorylation is required for PI3K‐Akt activity. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63‐linked polyubiquitin chains on Akt to restrict PI3K‐Akt‐FoxO signaling in mouse muscle during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in mouse muscle increased Akt ubiquitination, PI3K‐Akt‐FoxO signaling, and glucose uptake during fasting. Co‐immunoprecipitation and mass spectrometry identified disabled homolog‐2 (Dab2), the tuberous sclerosis complex TSC1/TSC2, and PHLPP1 as USP1 bound proteins. During starvation, Dab2 is essential for Akt recruitment to USP1‐TSC1‐PHLPP1 complex, and for PI3K‐Akt‐FoxO inhibition. Surprisingly, USP1 limits TSC1 levels to sustain mTOR‐mediated basal protein synthesis rates and maintain its own protein levels. We propose that Dab2 recruits Akt to USP1‐TSC1‐PHLPP1 complex to efficiently terminate the transmission of growth signals when cellular energy level is low. Synopsis: The deubiquitinating enzyme USP1 restricts PI3K‐Akt‐FoxO signaling in muscle atrophy by removing K63‐linked polyubiquitin chains on Akt. During fasting, Dab2 promotes the recruitment of Akt to USP1, which together with the phosphatase PHLPP1 inhibits Akt, thereby suppressing PI3K‐Akt signaling when cellular energy level is low. USP1 directly deubiquitinates and inhibits Akt to sustain PI3K‐Akt‐FoxO signaling low in the muscle during prolonged starvation (low insulin condition).During fasting, Dab2 promotes the recruitment of Akt to USP1, which also binds the tuberous sclerosis complex TSC1/TSC2, and the phosphatase PHLPP1.USP1‐mediated deubiquitination of Akt reduces Akt phosphorylation at T308 in fasting, but loss of Akt phosphorylation at S473 requires PHLPP1.To maintain its own protein levels high, USP1 limits TSC1 levels to sustain mTOR‐mediated basal protein synthesis rates in fasting. [ABSTRACT FROM AUTHOR]

The Fanconi anemia ( FA) pathway regulates DNA interstrand crosslink ( ICL) repair. A critical step in this pathway is mono-ubiquitination of FANCD2 ( FANCD2-Ub). Deubiquitinase USP1 removes ubiquitin from FANCD2 resulting in inactivation of the FA pathway. USP1 is autocleaved and subsequently degraded for its down-regulation. Here, we investigated the functional consequences of Usp1-autocleavage defect. Usp1-autocleavage mutant (Usp1 GG/ AA) corrected the level of Fancd2-Ub similar to Usp1 WT in Usp1−/− MEFs. However, Usp1 GG/ AA only partially rescued MMC sensitivity with defective Fancd2 foci formation and homologous recombination defect. Contrary to this, Usp1 GG/ AA was capable of recovering UV resistance of Usp1−/− MEFs to a similar extent with Usp1 WT. Taken together, our findings suggest that Usp1 regulation by autocleavage is critical for Usp1 to exert its function in ICL repair. [ABSTRACT FROM AUTHOR]

Abstract: Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation plays a crucial role in maintaining genomic stability during DNA replication. DNA damage stalling the DNA replication fork induces PCNA ubiquitylation that activates DNA damage bypass to prevent the collapse of DNA replication forks that could potentially produce double-strand breaks and chromosomal rearrangements. PCNA ubiquitylation dictates the mode of bypass depending on the level of ubiquitylation; monoubiquitylation and polyubiquitylation activate error-prone translesion synthesis and error-free template switching, respectively. Due to the error-prone nature of DNA damage bypass, PCNA ubiquitylation needs to be tightly regulated. Here, we review the molecular mechanisms to remove ubiquitin from PCNA including the emerging role of USP1 and ELG1 in this fascinating process. [Copyright &y& Elsevier]