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). Indeed, turnover of transcriptional factors such as octamer-binding factor 4 (Oct4), SRY box-containing factor 2 (Sox2), Krüppel-like factor 4 (Klf4), c-Myc, Nanog, Lin28, and Sall4 are mainly responsible for determination of the cell fate of ESCs [2
Maintenance of pluripotency in human ESCs (hESCs) is a balance between expression of a key set of proteins comprising Oct4, Sox2, and Nanog [7
]. Expression of Oct4 is high during the early embryonic stages and gradually declines during differentiation of hESCs, suggesting that Oct4 is a critical transcription factor in cell fate determination [9
]. Moreover, the expression level of Oct4 is a major determinant of maintenance of the pluripotent status of hESCs [9
Post-translational modifications (PTMs) of transcription factors, including Oct4, are fundamental regulators of cellular functions. Oct4 function is regulated by several PTMs including phosphorylation, ubiquitination, SUMOylation, and glycosylation [11
]. Among several PTMs of Oct4, ubiquitination is a major regulator of Oct4 protein turnover [2
]. The ubiquitination pathway is controlled by the ubiquitin proteasome system (UPS) that covalently ligates the small molecule ubiquitin (Ub) to specific sites on substrate proteins [17
]. Several E3 ligases that regulate protein turnover of Oct4 have been reported, including the WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) and Itch, which regulates Oct4 via K63-Ub linkages [12
]. DPF2 or ubi-d4/requiem (REQU) regulates Oct4 via K48-Ub linkage, and CHIP is a ubiquitin ligase (E3 ligase) that regulates Oct4 in breast cancer stem cells [20
]. Reversal of the ubiquitination process is performed by deubiquitinating enzymes (DUBs) that balance the action of E3 ligases to regulate key functions as well as the stability and localization of target proteins [22
]. Although extensive research has been performed to elucidate the role of Oct4 ubiquitination in the maintenance of pluripotency in stem cells, little is known about the DUBs that regulate Oct4 protein level in hESCs. However, a recent study by Wei et al. reported that the transcription factor Bach1 interacts with and facilitates Nanog, Sox2, and Oct4 deubiquitination by recruiting USP7. Bach1-mediated recruitment of USP7 helps maintain the identity and self-renewal of hESCs while the loss of Bach1 affects the differentiation fates of these cells [23
]. This report therefore highlights the importance of DUBs in regulating stem cell fate determination and differentiation by regulating the stability of key transcriptional factors in hESCs.
We recently demonstrated that USP3 interacts with and deubiquitinates the cell division cycle 25A (Cdc25A) protein, which regulates proper progression of the cell cycle [24
]. Cdc25 proteins are responsible for activating CDK complexes during the cell cycle, and Cdc25A plays a role in regulating G1/S cell cycle progression [24
]. Cdc25A is also known to regulate the cell cycle in stem cells [25
]. Interestingly, gene ontology (GO) analysis identified a positive correlation between Oct4 and various cell cycle genes, including Cdc25A
]. Previous studies have demonstrated that, in addition to Cdc25A, USP3 is a regulator of stemness associated-genes such as KLF5 and SUZ12 [29
Thus, we hypothesized that USP3 has an important role in regulating the protein level of key transcriptional factors in hESCs. In this study, we performed a loss-of-function study of USP3 in ESCs utilizing the CRISPR/Cas9 system. We demonstrate that USP3 interacts with and deubiquitinates endogenous Oct4 in hESCs. The loss of USP3 significantly destabilizes the protein level of Oct4 and affects normal morphology of hESCs.
Post-translational regulatory mechanisms such as ubiquitination and deubiquitination complement the translational regulation of proteins in a timely and selective manner during differentiation [6
]. Pluripotent stem cells have the properties of indefinite cell division while retaining their abilities to differentiate into various cell types and have been reported to exhibit high levels of proteasome activity [36
]. Various Ub linkages and conformations of Ub chains in cells are generally accepted to generate diversity in molecular signaling. However, the exact mechanisms by which components of the proteasomal pathway regulate the core factors involved in determining pluripotency have not been elucidated.
Cell-cycle regulation by cyclins and CDKs is considered key towards maintenance of the self-renewal ability of stem cells [37
]. hESCs proliferate rapidly, displaying an elongated S phase and a short G1 phase, and express high levels of cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) [37
]. Oct4 plays an important role in maintaining cell-cycle-related genes in hESCs, and this regulation of the cell cycle has been postulated to have a major role in maintaining the pluripotent identity of stem cells [38
]. Oct4 acts as a cell cycle promoter by removing G1 phase blocks and promoting S phase entry, governing pluripotency in stem cells [39
]. There is a regulatory link between Oct4 and cell-cycle-regulating genes; Oct4 expression pattern was positively correlated with those of numerous cell cycle genes including Gspt1, Ccna1, Ccnd2, Ccne1, Ccnb1, Ccnf, Ppp1r8
, and Cdc25A
, indicating that Oct4 is a key cell cycle regulator [28
]. Thus, perturbation in Oct4 protein level could significantly impact cell-cycle progression and pluripotency in stem cells.
In a previous study, we identified USP3 as an important regulator of the cell cycle via its deubiquitinating effect on Cdc25A [24
]. The importance of Cdc25A in regulating the cell cycle in stem cells [25
] led us to initiate this study to elucidate the role of USP3 in stem cells [24
]. We first generated USP3
knockout NCCIT cells using the CRISPR/Cas9 system and investigated the protein levels of important stem cell transcription factors. Loss of USP3 led to a decrease in the stability of Oct4; conversely, overexpression of USP3 resulted in a dose-dependent increase in the stability of Oct4 (Figure 2
). Furthermore, we demonstrated that USP3 and Oct4 interact regardless of whether Oct4 is endogenously or exogenously expressed and co-localize in the nuclei of hESCs (Figure 3
Importantly, USP3 did not show a regulator effect on mRNA level of Oct4
), indicating that USP3 regulates Oct4 only at the post-translational level. We demonstrated a significant reduction in polyubiquitination of Oct4 in the presence of USP3 (Figure 4
). As a functional consequence of deubiquitination of Oct4 by USP3, loss of USP3 resulted in loss of the pluripotent morphology of hESCs (Figure 5
). Our findings highlight the importance of USP3 in regulating Oct4 protein level in human embryonic carcinoma cells and human embryonic stem cells. Thus, the interaction between USP3 and Oct4 is critical for maintenance of the pluripotency of hESCs.
4. Materials and Methods
Mammalian expression vectors encoding Flag-USP3 (cat. no. #22582, Watertown, MA, USA), HA-ubiquitin (cat. no. #18712, Watertown, MA, USA), and Cas9-2A-GFP (cat. no. #44719, Watertown, MA, USA) were purchased from Addgene (Watertown, MA, USA). To generate the catalytic mutant of USP3, we replaced the active cysteine residue at position 168 with serine by site-directed mutagenesis to produce USP3C168S (USP3CS). Cas9-2A-mRFP-2A-PAC was purchased from Toolgen (Seoul, Korea).
4.2. Antibodies and Reagents
The following antibodies were used: anti-USP3 (ab101473, 1:1000, abcam, Cambridge, MA, USA; GTX128238, 1:1000, Genetex, Irvine, CA, USA; and sc-135597, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA); anti-Oct4 (ab18976, 1:1000, Abcam, Cambridge, MA, USA; and sc-5279, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA); anti-Nanog (cat. no. #3580, 1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-Flag (anti-DDDDK-tag) (M185-3L, 1:1000, MBL International, Woburn, MA, USA).
Anti-Lin-28 (sc-374460, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GAPDH (sc-32233, 1:5000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Myc (sc-40, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-ubiquitin (sc-8017, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HA (sc-7392, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), normal mouse IgG (sc-2025, Santa Cruz Biotechnology, Dallas, TX, USA), and Protein A/G Plus Agarose beads (sc-2003, Santa Cruz Biotechnology, Dallas, TX, USA) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). TUBE 2 (cat. no. #UM402, Life Sensors, Malvern, PA, USA), MG132 (cat. no. #S2619, Selleckchem, Houston, TX, USA), and cycloheximide (CHX, cat. no. #C4859, Sigma-Aldrich, St. Louis, MO, USA) were also used. Normal rabbit IgG (cat. no. #12-370, Merck Millipore, Burlington, MA, USA), anti-Tra-1-60 (MAB4360, Merck Millipore, Burlington, MA, USA), and anti-SSEA4 (cat. no. #90231, Merck Millipore, Burlington, MA, USA) were purchased from Merck Millipore (Burlington, MA, USA).
4.3. Cell Culture and Treatments
CHA15 human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSC-NT4-S1), established by CHA University, Seoul, South Korea, were cultured under feeder-free conditions. CHA-15 were cultured on Matrigel (cat. no. #356231, Corning Life Sciences, Tewksbury, MA, USA)-coated 35 mm dishes in mTeSR1 (cat. no. #85850, STEMCELL Technologies, Vancouver, BC, Canada), while hiPSCs were cultured on Vitronectin (cat. no. #07180, STEMCELL Technologies, Vancouver, BC, Canada)-coated 35 mm dishes in TeSR-E8 (cat. no. #05990, STEMCELL Technologies, Vancouver, BC, Canada), and subcultured every 4–5 days using Gentle Cell Dissociation Reagent (cat. no. #07174, STEMCELL Technologies, Vancouver, BC, Canada). Cells were seeded onto 35 mm dishes in mTeSR1 or TeSR-E8 supplemented with 10 μM Y-27632 (ROCK inhibitor, cat. no. #1254, Tocris Bioscience, BS, United Kingdom) to enhance cell survival and attachment.
Human pluripotent embryonal carcinoma (NCCIT) cells were cultured in RPMI media (RPMI 1640, GIBCO BRL, Rockville, MD, USA). Human embryonic kidney (HEK293) cells were cultured in DMEM media (GIBCO BRL, Rockville, MD, USA). Both RPMI and DMEM media were supplemented with 10% fetal bovine serum (GIBCO BRL, Rockville, MD, USA) and 1% penicillin and streptomycin (GIBCO BRL, Rockville, MD, USA), and cells were grown at 37 °C in a humidified atmosphere with 5% CO2.
4.4. Embryoid Body (EB) Differentiation
hESCs were differentiated in vitro to EBs. Briefly, undifferentiated colonies were detached by treatment with collagenase IV (GIBCO BRL, Rockville, MD, USA) and incubated as floating aggregates for 5 days in ultra-low attachment 6-well plates with Essential 6TM Media (GIBCO BRL, Rockville, MD, USA).
4.5. Cas9 and sgRNA Constructs
We used plasmids encoding Cas9-2A-mRFP-2A-PAC (puromycin N-acetyl-transferase, puromycin resistance gene) and single-guide RNAs (sgRNAs) purchased from Toolgen (Seoul, South Korea). The sgRNA target sequences were designed on the basis of bioinformatics tools (www.broadinstitute.org
) and cloned into the vectors as described previously [40
]. Briefly, oligonucleotides containing each target sequence were synthesized (Bioneer, Seoul, South Korea), and T4 polynucleotide kinase was used to add terminal phosphates to the annealed oligonucleotides (Biorad, CA, USA). Vector was digested with BsaI and ligated the annealed oligonucleotides into the vector. Oligonucleotide sequences are listed in Table 1
4.6. T7E1 Assay
Protocols for T7E1 assay were described previously [41
]. Genomic DNA was isolated using DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. The region of DNA containing the nuclease target site was amplified by PCR using hemi-nested primers. PCR amplicons were denatured by heating and annealed to form DNA heteroduplex, which was then treated with 5 units of T7 E1 enzyme (New England Biolabs, MA, USA) for 20 min at 37 °C, followed by the separation of DNA fragments on 1% agarose gel by electrophoresis. Mutation frequencies were calculated using ImageJ software on the basis of band intensity. The following equation was used to calculate mutation frequency (Indel %): Indel % = 100 × (1 − [1 − fraction cleaved] 1/2), where the fraction cleaved was the total relative density of the cleavage bands divided by the sum of the relative density of cleavage and uncut bands.
Oligonucleotide primers used to amplify the PCR target for the T7E1 assay are listed in Table 1
. Amplicon size of the USP3 gene and expected cleavage sizes after the T7E1 assay are summarized in Table 2
4.7. Generation of DUB Knockout Single-Cell-Derived Clones
To generate USP3 knockout single-cell-derived clones, we co-transfected NCCIT or hESCs with the plasmids encoding Cas9 and sgRNA targeting USP3 or non-targeted sgRNAs (scrambled sgRNAs) at a 1:2 ratio using polyethyleneimine in NCCIT (PEI; Polysciences, Warrington, PA, USA) or Lipofectamine STEM reagent in hESCs (STEM00003, Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Transfected cells were sorted by FACS and reseeded into 24-well plates for recovery. Cells were dissociated and seeded again into 96-well plates to establish single-cell-derived colonies. After 15 days, single-round colonies were marked and expanded for screening by the T7E1 assay. USP3 KO-positive clones were confirmed by T7E1 and Sanger sequencing. Scrambled sgRNA-targeted cells that are T7E1-negative and showing no gene disruption of USP3 gene were used as controls for all the experiments.
T7E1-positive USP3 KO hESC clones #2, #5, and #9 generated by single-cell dilution were used for further characterization experiments described in Figure 5
For immunoprecipitation experiments, cells were lysed with RIPA buffer (cat. no. RC2002-050-00, Biosesang, Gyeonggi-do, South Korea) containing 50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and 1 mM PMSF, followed by preclearing with Protein A/G Plus Agarose beads. Lysates were immunoprecipitated with the indicated antibodies at 4 °C overnight, followed by incubation with 20μL of Protein A/G Plus Agarose beads at 4 °C for 4 h. Immunoprecipitates were washed with lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate, and eluted with 2X SDS sample loading buffer (cat. no. S3401, Sigma-Aldrich, St. Louis, Missouri, USA) containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris-HCl (pH 6.8). Co-immunoprecipitated proteins were denatured for 5 min at 95 °C, resolved by SDS-PAGE electrophoresis, and analyzed by immunoblotting.
4.9. Deubiquitination Assay
Cells transfected with various combinations of plasmids were lysed with RIPA buffer, followed by preclearing with Protein A/G Plus agarose beads as described above. Cell lysates were immunoprecipitated with anti-Oct4, anti-Myc antibodies, or TUBE agarose beads, as indicated. The immunoprecipitated proteins were eluted with 2X SDS buffer, denatured, and analyzed by Western blot with indicated antibodies.
For immunofluorescence assays, NCCIT or hESCs were cultured in 4-well cell culture dishes in their respective media for 36 h. Cells were fixed with 4% paraformaldehyde (cat. no. #163-20145, Wako, Richmond, VA, USA) for 15 min and washed twice with PBS, then permeabilized with 0.25% Triton X-100 (cat. no. #0694, Amresco, Solon, OH, USA) for 10 min and blocked with 1% BSA (cat. no. #A9418, Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h. Cells were stained with anti-USP3 and anti-OCT3/4 antibodies and incubated overnight at 4 °C. After washing with PBS, cells were incubated with Alexa Fluor-488- or Alexa Fluor-594-conjugated secondary antibodies for 1 h in the dark. DAPI was used to stain the nucleus of cells. Laser scanning confocal microscopy (TCS SP5, Leica, Wetzlar, Germany) was used to visualize fluorescence localization in cells.
Cells were harvested, and total RNA was isolated using TRIzol reagent (cat. no. #FATRR001, Favorgen, Ping-Tung, Taiwan). Then, 300ng of total RNA was reverse-transcribed into cDNA using Oligo dT primers (cat. no. #SO132, Thermo Scientific, Waltham, MA, USA) and Superscript III Reverse Transcriptase (cat. no. #18080-044, Invitrogen, Carlsbad, CA, USA). qRT-PCR amplification was performed using the SensiFAST SYBR No-ROX kit (cat. no. #BIO-98005, Bioline, GB, London) on a real-time PCR system (C1000 Thermal Cycler, Bio-rad, Hercules, CA, USA). GAPDH was used as the endogenous control gene. Primer information is provided in Table 3
All results are expressed as mean ± standard deviation. The significance of differences between groups was assessed using Student’s t-test. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc. San Diego, CA, USA). Differences were considered statistically significant at p < 0.05.