Abstract
Oct4 is an important mammalian POU family transcription factor expressed by early human embryonic stem cells (hESCs). The precise level of Oct4 governs the pluripotency and fate determination of hESCs. Several post-translational modifications (PTMs) of Oct4 including phosphorylation, ubiquitination, and SUMOylation have been reported to regulate its critical functions in hESCs. Ubiquitination and deubiquitination of Oct4 should be well balanced to maintain the pluripotency of hESCs. The protein turnover of Oct4 is regulated by several E3 ligases through ubiquitin-mediated degradation. However, reversal of ubiquitination by deubiquitinating enzymes (DUBs) has not been reported for Oct4. In this study, we generated a ubiquitin-specific protease 3 (USP3) gene knockout using the CRISPR/Cas9 system and demonstrated that USP3 acts as a protein stabilizer of Oct4 by deubiquitinating Oct4. USP3 interacts with endogenous Oct4 and co-localizes in the nucleus of hESCs. The depletion of USP3 leads to a decrease in Oct4 protein level and loss of pluripotent morphology in hESCs. Thus, our results show that USP3 plays an important role in controlling optimum protein level of Oct4 to retain pluripotency of hESCs.
Highlights
Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of mammalian blastocysts and are characterized by their ability to differentiate into multiple cell types and undergo self-renewal [1]
We demonstrate that ubiquitin-specific protease 3 (USP3) interacts with and deubiquitinates endogenous octamer-binding factor 4 (Oct4) in human ESCs (hESCs)
Given that USP3 acts as a protein stabilizer of Oct4 and our observation of transient reduction in expression of USP3 at day 2 during stem cell differentiation, we investigated whether knockout of USP3 in hESCs affected the pluripotent nature of hESCs
Summary
Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of mammalian blastocysts and are characterized by their ability to differentiate into multiple cell types and undergo self-renewal [1]. ESC self-renewal and differentiation ability are mainly regulated by a network of transcriptional factors (1). 4 (Klf4), c-Myc, Nanog, Lin, and Sall are mainly responsible for determination of the cell fate of ESCs [2,3,4,5,6]. Maintenance of pluripotency in human ESCs (hESCs) is a balance between expression of a key set of proteins comprising Oct, Sox, and Nanog [7,8]. Expression of Oct is high during the early embryonic stages and gradually declines during differentiation of hESCs, suggesting that Oct is a critical transcription factor in cell fate determination [9]. The expression level of Oct is a major determinant of maintenance of the pluripotent status of hESCs [9,10]
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