Key Laboratory of Infection and Immunity of CAS, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China
Correspondence: Zusen Fan, Ph.D., The CAS key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China. Telephone: +86-10-64888457; Fax: +86-10-64871293; e-mail: email@example.com
Self-renewal and differentiation are the hallmarks of embryonic stem cells (ESCs). However, it is largely unknown about how the pluripotency is regulated. Here we demonstrate that Pcid2 is required for the maintenance of self-renewal both in mouse and human ESCs. Pcid2 plays a critical role in suppression of ESC differentiation. Pcid2 deficiency causes early embryonic lethality before the blastocyst stage. Pcid2 associates with EID1 and is present in the CBP/p300-EID1 complex in the ESCs. We show that MDM2 is an E3 ligase for K48-linked EID1 ubiquitination for its degradation. For the maintenance of self-renewal, Pcid2 binds to EID1 to impede the association with MDM2. Then EID1 is not degraded to sustain its stability to block the HAT activity of CBP/p300, leading to suppression of the developmental gene expression. Collectively, Pcid2 is present in the CBP/p300-EID1 complex to control the switch balance of mouse and human ESCs through modulation of EID1 degradation. Stem Cells2014;32:623–635
Embryonic stem cells (ESCs) have capability to self-renew in vivo and in vitro, which can maintain a certain number of themselves to differentiate into all cell types of a adult organism . The self-renewal of ESCs is achieved through expression of pluripotency genes and downregulation of commitment genes, while the differentiation of ESCs is obtained through activation of the specific transcriptional factors, upregulation of lineage-specific genes, and repression of the pluripotency genes [2, 3]. However, it has not been well-characterized about regulation of the fate switch between self-renewal and differentiation of ESCs.
Complicated epigenetic controlling mechanisms play important roles in regulating gene expression in embryonic development and in stem cell differentiation. The CBP (CREB-binding protein)/p300 family is well-known as a histone acetyltransferase (HAT) and a transcriptional coactivator that plays pivotal roles in embryogenesis, transcription regulation, and ESC differentiation [4–7]. Transcriptional activation of early fate-determining genes needs CBP/p300 activity, and many development-related genes have CBP/p300-bound regions in their promoters and enhancers [7, 8]. CBP/p300 can acetylate core histones in vitro and regulate transcription by linking transcriptional machinery with its acetylation-dependent regulation . p300 deficiency caused abnormal expression of germ layer markers in ESC differentiation . The HAT activities of CBP and p300 were reported to be involved in development of embryonic neural progenitors and myogenesis of ESCs [5, 10, 11]. EID1 (E1A-like inhibitor of differentiation one) was first identified to be an interactor of the retinoblastoma protein (pRb) . EID1 can inhibit the HAT activity of CBP/p300 to regulate MyoD transcription and fate determination of skeletal muscle.
Mammalian Pcid2 (PCI-domain containing protein two) is a homolog of the yeast Thp1 based on their structural similarity. Saccharomyces Thp1 is present in the ribonucleoprotein complex to participate in export of mRNAs from a nucleus to a cytoplasm . We recently reported that murine Pcid2 is selectively involved in the transport of MAD2 mRNA that regulates the mitotic checkpoint during B-cell proliferation . A genome-scale RNA interference (RNAi) screen identified Pcid2 as one of the genes that modulate self-renewal of mouse ESCs (mESCs) . However, it is still unknown how Pcid2 modulates this process as well as whether Pcid2 is involved in human ESC pluripotency. Here, we demonstrate that Pcid2 is present in the CBP/p300-EID1 complex to control the switch balance of mouse and human ESCs through modulation of EID1 degradation.
Materials and Methods
Mouse ESCs (MilliTrace Nanog GFP Reporter mouse embryonic stem cell, SCR089) and mouse ESC line 129/SVEV (CMTI-1) cells were purchased from Millipore (Billerica, MA, http://www.millipore.com). Mouse ESC D3 cells were obtained from the stem cell core facility of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). mESCs were cultured in DMEM supplemented with 15% FBS (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 1 mM nucleotide, 0.1 mM nonessential amino acids and 103 units per milliliter mouse LIF. Nanog GFP reporter mESCs were grown without feeder cells. D3 and 129/SVEV ESCs were grown on CF-1 mouse MEFs treated with mitomycin C. The hESC H1 cell line was obtained from Wicell Research Institute (Madison, WI, http://www.wicell.org) and was grown on CF-1 mouse MEFs treated with mitomycin C. The culture medium contained Dulbecco's modified Eagle's medium (DMEM)/F12 medium, 20% knockout serum replacement (Invitrogen) and 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, and 4 ng/mL human fibroblast growth factor (hFGF2) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Embryonic stem cell use was approved by the Institutional Medical Research Ethics Committee at Institute of Biophysics, Chinese Academy of Sciences.
Antibodies and Reagents
A rabbit polyclonal antibody against human Pcid2 was raised from purified His-Pcid2 protein. The anti-Pcid2 antibody specifically recognized mouse and human Pcid2 both in recombinant and endogenous forms (data not shown). A mouse polyclonal antibody against mouse EID1 was raised from purified GST-EID1 protein. The specificity of anti-mouse EID1 antibody was verified (data not shown). The antibodies against Oct4, histone H3, acetyl-K9K14-histone H3, and K48-ubiquitination were purchased from Cell Signaling Technology (Danvers, http://www.cellsignal.com). The antibodies against Sox2, Fgf5, and Myc-tag were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com). The antibodies against Flag-tag and β-actin were from Sigma-Aldrich. The antibodies against Gata6, Brachury, and CBP were purchased from Abcam (Cambridge, MA, http://www.abcam.com). The antibodies against p300 and MDM2 were from Millipore. Anti-Ub antibody was from Enzo life sciences (Farmingdale, www.enzolifesciences.com). Anti-K63-ubiquitination antibody was from eBiosciences (San Diego, www.ebioscience.com). Secondary antibodies conjugated with Alexa-594, Alexa-488, Alexa-405, or Alexa-649 were purchased from Molecular Probes Inc (Eugene, OR, http://probes.invitrogen.com). All trans-RA, Curcumin, cycloheximide, and MG132 were from Sigma-Aldrich. DNaseI was purchased from Roche Molecular Biochemicals (Basel, Switzerland, http://www.roche-applied-science.com). MDM2 inhibitor (N-((3,3,3-trifluoro-2-trifluromethyl)propionyl) sulfanilamide) was from Millipore (Darmstadt, Germany). The recombinant LIF protein was from Millipore. His-bind resin was from Millipore. GST-sepharose was from GE Healthcare (Buckinghamshire, U.K.). The AP detection kit was purchased from Millipore. The SuperReal premix plus qPCR buffer was from TIANGEN Biotech (Beijing, China, www.tiangen.cn). The HAT activity colorimetric assay kit was from BioVision (Milpitas, www.biovision.com). The Nuclei EZ prep nuclei isolation kit was from Sigma-Aldrich.
The human Pcid2 cDNA (1–1,200 bp) was cloned into p3Xflag-CMV-9 expression vector. A swapped-Pcid2 mutant that cannot be targeted by Pcid2-shRNA was generated by introducing three synonymous mutations at cDNA sequence (753: T to A, 754: C to T, 756: G to C). The human full-length Pcid2 was also subcloned into pETDuet vector. The human EID1 cDNA (1–564 bp) was cloned into pCDNA4-MycHis expression vector. A K132R/K152R mutant which was reported as a stable form of EID1 was generated by the conventional mutagenesis method of DpnI digestion. Recombinant human EID1 fused with GST in the vector of pGEX-6P-1 (GE Healthcare, Fairfield, www.gelifesciences.com) was expression in Escherichia coli and purified using glutathione sepharose 4B beads. The human MDM2 cDNA (1–1,494 bp) was cloned into H-MBP-3C vector and purified using the Amylose Resin (New England BioLabs, Ipswich, www.neb.com) according to the manufacturer's instruction.
Generation of Pcid2−/− Embryos
Pcid2flox/flox mice were generated by homologous recombination as described . Pcid2+/− mice were produced by crossing Pcid2flox/flox mice with EIIa-Cre transgenic mice. Pcid2−/− embryos were generated by intercrossing of Pcid2+/− mice. Animal use and protocols were approved by the Institutional Animal Care and Use Committees at Institute of Biophysics, Chinese Academy of Sciences.
mESCs or hESCs with the feeder cells were grown on 0.01% poly-L-lysine-treated coverslips and fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 20 minutes at room temperature followed by 10% donkey serum blocking. Cells were then incubated with appropriate primary antibodies at 4°C overnight followed by incubation with corresponding fluorescence-conjugated secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained with Olympus FV1000 laser scanning confocal microscopy (Olympus, Tokyo Japan, www.olympus.co.jp).
In Vivo Assay of Teratomas
mESC D3 cells (1 × 106) was resuspended in 1 mL retrovirus supernatant supplied with 8 µg/mL Polybrene. Then, this mixture was added to six-well plate and centrifuged for 1 hour at 2,500 rpm at room temperature. Supernatant was removed and cells were resuspended in ESC medium and then split into two 60 mm dishes. Three days later, ESCs were trypsinized, washed twice with phosphate buffered saline (PBS), and then subcutaneously injected into the bilateral inguens of male Nonobese Diabetic-Severe Combined Immunodeficiency (NOD-SCID) mice (1 × 106 cells per injection). After 28 days, mice were sacrificed. Tumors were weighed, fixed in 4% PFA, sectioned and stained with H&E.
ESCs were transfected with the indicated plasmids and harvested. Cells were lyzed with ice-cold RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% sodium desoxycholate, 0.1% SDS, 5 mM EDTA, 2 mM Phenylmethanesulfonyl fluoride (PMSF), 20 mg/mL aprotinin, 20 mg/mL leupeptin, 10 mg/mL pepstatin A, 150 mM benzamidine, and 1% Nonidet P-40) for 30 minutes. Lysates were incubated with the indicated antibodies followed by immunoprecipitation with protein A/G agarose.
mESCs were lyzed with low salt buffer (20 mM Hepes pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA). Nuclear components were extracted by high salt buffer (20 mM Hepes pH 7.9, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA) followed with micrococool MNase digestion. Gel filtration chromatography was performed by Superdex 200 10/300 GL (GE Healthcare) according to the manufacturer's instruction.
Total RNAs from mESCs or hESCs were extracted with the RNA miniprep Kit (LCsciences, Houston, TX) according to the manufacturer's protocol. For comparing the relative gene expression, 2 µg total RNA per aliquot was used for synthesizing cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, http://www.promega.com). qPCR analysis and data collection were performed on the Corbett 6200 qPCR System using the primer pairs shown in Supporting Information Table S1. All quantitation was normalized to an endogenous β-actin or GAPDH control. Primer sequences used for ChIP were shown in Supporting Information Table S2. DNA from each ChIP sample was normalized by the corresponding input sample.
RNA interference sequences were designed according to pSUPER system instructions (Oligoengine, Seattle). mESCs and hESCs were transfected with pSUPER vector encoding target sequence against mPcid2: 5′-AGAGGATGATTC TGATCTA-3′ , hPcid2: 5′-GATCATCACCTACAGGAAC, mEID1:5′-AGAGAG CAGT GACCTGCAGATGGAT-3′, mMDM2: 5′-GCTTCGGAACAAGAGACTC-3′, mCBP: 5′-AACAGTGGGAACCTTGTTC CA-3′, mp300: 5′-AATTGGGACT AACCAATG GTG-3′, or scramble sequences: 5′-AATTCTCCGAACGTGTCACGT-3′. Cells were transfected using lipofectin and silenced expression of the indicated genes was measured by immunoblotting.
mESCs were harvested and washed with ice-cold PBS. Cells were resuspended with TEB buffer (PBS containing 0.5% Triton X-100, 2 mM PMSF, and 0.02% NaN3) and lysed on ice for 10 minutes. Cells were centrifuged at 2,000 rpm for 10 minutes and washed with TBE buffer. Pellets were resuspended in 0.2N HCl and acid extracted overnight.
ESCs were dissociated and plated at 4.5 × 104 per well of the 24-well plates. For transfections, 3 µL of Lipofectamine 2000 (Invitrogen) was premixed with 100 µL of DMEM (Millipore), and then mixed with 0.6 µg shRNA plasmid plus 0.3 µg expression plasmid. Plasmid–lipid complexes were incubated at room temperature for 15–30 minutes and then added to the cells. After 24 hours, ESCs were replated at a ratio of 1:20 and cultured in ESC medium for another 5 days. After that, cells were fixed and stained for AP activity with the Alkaline Phosphatase ES Characterization Kit (Millipore) according to the manufacturer's instruction.
HAT Activity Assay
Nuclei were isolated in low salt buffer (20 mM Hepes pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA) followed by extraction of nuclear components in equal volume of low salt buffer and high salt buffer (20 mM Hepes pH 7.9, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA). CBP or p300 was immunoprecipitated by the indicated antibodies and protein A/G beads from the nucleus extracts and measured the HAT activity using the HAT activity kit (BioVision, Milpitas) according to the manufacturer's instruction.
Quantitative ChIP was performed according to a standard protocol (Upstate). Sheared chromatin (sonicated to 200–500 bp) from mESCs (2 × 106) fixed in 1% formaldehyde was incubated with 5 µg antibody overnight at 4°C followed by immunoprecipitation with salmon sperm DNA/protein agarose beads. After washing, elution and crosslink reversal, DNA from each ChIP sample and the corresponding input sample were purified and analyzed using qPCR.
Yeast Two Hybrid Screen
Yeast two-hybrid screening was performed using Matchmaker Gold Yeast Tow-Hybrid system (Clontech Laboratories, Mountain View, http://www.clontech.com) according to the manufacturer's protocol. Briefly, Pcid2 was cloned into pGBKT7 plasmid as a BD-Pcid2 bait. Yeast AH109 cells were transfected with BD-Pcid2 and plasmids containing human spleen cDNA library (Clontech). Clones selected with auxotroph were identified by DNA sequencing.
In Vitro Ubiquitination Reconstitution Assay
Uba1, UbcH5c, p53, Pcid2, and ubiquitin were subcloned into pET-28a vectors. MDM2 was subcloned into pMAL-c2p vector. EID1 was subcloned into pGEX-6p-1 vector. Plasmids were then transformed into E. coli strain BL21 (DE3). Protein expression was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside at 16°C for 24 hours. Protein was purified by Ni-NTA resin column (Novagen) or GST-sepharose column. The in vitro ubiquitination reconstitution assay was performed by mixing E1 (Uba1), E2 (UbcH5c), E3 (MDM2), ubiquitin, Pcid2, and substrates in the ubiquitination buffer (50 mM Tris-HCl, 5 mM MgCl2, 2 mM dithiothreitol, and 2 mM ATP, pH 7.4) at 30°C for 2 hours.
Student's t test was used as statistical analysis by using Microsoft Excel.
Pcid2 Is Required for the Maintenance of Self-Renewal of mESC and hESC
To examine the in vivo role of Pcid2, we wanted to knock out Pcid2 during the early embryonic development of mice. We crossed Pcid2flox/flox mice with EIIa-Cre transgenic mice. However, Pcid2−/− blastocysts were rare to observe, and Pcid2 deficiency caused early embryonic lethality before the blastocyst stage (B. Ye, unpublished data). Pcid2 appeared in the inner cell mass (ICM) of blastocysts in wild-type (WT) mice (B.Ye, unpublished data), which suggests that Pcid2 may be of importance in pluripotency maintenance in early embryos. To determine the role of Pcid2 in regulation of pluripotency, we silenced Pcid2 expression in mESCs by using pSIN-shmPcid2 vector. Pcid2 was successfully knocked down over 80% 24 hours after transfection (data not shown). In Pcid2-silenced mESCs, Nanog promoter-driven green fluorescent protein (GFP) expression was remarkably reduced and Oct4 expression was significantly decreased (Fig. 1A). Pcid2 depletion resulted in failure of the three embryonic layer generation via an in vivo teratoma formation assay (Fig. 1B). To further confirm the in vivo role of Pcid2 in pluripotency, we designed a two-step targeting strategy to ablate Pcid2 expression in mESCs through homologous recombination and TALEN (transcription activator-like effector nucleases) technology as described . Pcid2-deficient mESCs were successfully established after infection with GFP-IRES-Cre retrovirus (Supporting Information Fig. S1). Pcid2 deficiency in mESCs led to morphological change, loss of Oct4 expression, and alkaline phosphatase (AP) activity (Fig. 1C). Taken together, Pcid2 plays a critical role in the maintenance of mESC pluripotency.
To further determine whether Pcid2 is involved in regulation of hESCs, we knocked down Pcid2 in human ES cell line hESC H1 cells. As expected, knockdown of Pcid2 in hESCs also significantly reduced Oct4 expression and AP activity (Fig. 1D). These data indicate that Pcid2 is involved in the regulation of pluripotency of both mESCs and hESCs.
Pcid2 Deletion Causes Differentiation of ESCs
To gain insight into the mechanism of Pcid2 to control pluripotency in ESCs, we wanted to test whether Pcid2 itself can promote stemness-related gene expression or repress differentiation-related gene expression. These two kinds of genes in Pcid2-silenced mESCs were analyzed by real-time quantitative polymerase chain reaction (qPCR). Pcid2 knockdown downregulated self-renewal-related genes, such as Oct4 and Nanog (Fig. 2A). In contrast, Pcid2 depletion upregulated differentiation-related genes, such as Gata6 (differentiated to endoderm), Brachyury (differentiated to mesoderm), and Pax6 (differentiated to ectoderm) in mESCs (Fig. 2A–2C). Similar results were obtained from Pcid2 knockout (KO) mESCs (Fig. 2D). Of note, retinoic acid (RA) induced differentiation of mESCs in vitro which served as a positive control. Overexpression of Pcid2 was able to suppress the RA-induced differentiation of mESCs (Fig. 2E). These observations imply that Pcid2 can rescue stemness in differentiated ESCs. Intriguingly, knockdown of Pcid2 in hESCs also reduced expression of the pluripotency genes (Pou5 and Nanog) (Fig. 2F) and induced expression of the ectoderm and mesoderm genes (VIM, TH, and Brachyury). However, the endoderm gene FoxA2 was not significantly changed in Pcid2-silenced hESCs (Fig. 2F). These results suggest that Pcid2 regulates the switch balance between self-renewal and differentiation in mESCs and hESCs, and it may modulate different gene loci of the developmental genes in these two kinds of ESCs.
Pcid2 Associates with EID1 and Is Present in the CBP/p300-EID1 Complex of ESCs
To define the molecular mechanism of Pcid2 in regulation of self-renewal and differentiation in ESCs, we screened a human spleen cDNA library to identify Pcid2 interactors using a yeast two-hybrid approach. EID1 was identified to interact with Pcid2 (Fig. 3A). Recombinant glutatione S-transferase (GST)-EID1 protein could precipitate recombinant His-Pcid2 (Fig. 3B), which indicates a direct interaction between Pcid2 and EID1. Their association was also verified in cotransfected 293T cells by coimmunoprecipitation (Fig. 3C). EID1 was previously reported to be an inhibitor of CBP/p300 HAT activity to block histone acetylation . We found that anti-mEID1 antibody could precipitate endogenous Pcid2, CBP, and p300 in mESCs (Fig. 3D). Additionally, Pcid2 was eluted with the p300-EID1 complex around 600 kD from mESC lysates through gel filtration chromatography (Fig. 3E). Moreover, Pcid2 colocalized with EID1 and CBP/p300 in nuclei of mESCs (Fig. 3F). These data indicate that Pcid2 associates with EID1 and is present in the CBP/p300-EID1 complex of ESCs.
Pcid2 Blocks the HAT Activity of CBP/p300 to Suppress Expression of Developmental Genes
We assessed the effect of Pcid2 on regulation of CBP/p300 HAT activity by an in vitro HAT assay. We found that Pcid2 depletion remarkably increased CBP/p300 HAT activity (Fig. 4A). To further confirm this specificity of Pcid2, we carried out a rescue experiment by generating a swapped-Pcid2 mutant (Pcid2mt) that could not be targeted by Pcid2-short hairpin RNA (shRNA). Expectedly, Pcid2mt overexpression rescued CBP/p300 HAT activity in Pcid2-silenced mESCs (Fig. 4A).
K9/K14 lysine residues of histone H3 were identified as the substrates of CBP/p300 and their acetylation usually indicates transcriptionally active regions in chromatin [4, 17, 18]. We found that K9/K14 acetylation of histone H3 was increased in Pcid2-silenced mESCs (Fig. 4B). As expected, cotransfection of Pcid2mt with Pcid2-shRNA caused the rescue of K9K14 acetylation of histone H3 (Fig. 4Bb). Additionally, Pcid2−/− mESCs enhanced K9K14 acetylation of histone H3 (Fig. 4c). Chromatin immunoprecipitation (ChIP) analysis using anti-K9K14-H3 antibody showed that Pcid2 silencing resulted in enhanced acetylation in the promoters of developmental genes but not the pluripotency genes (Fig. 4D). These observations suggest that Pcid2 maintains ESC pluripotency through suppression of the expression of developmental genes.
In addition, DNase I digestion assays showed that Pcid2 knockdown increased chromatin accessibility to DNase I digestion at the promoters of developmental genes but not the promoters of pluripotency genes (Supporting Information Fig. S2 and data not shown), indicating open status of chromatin regions of these developmental genes. Indeed, Pcid2 knockdown dramatically augmented the expression of developmental genes (Fig. 4E), whereas Pcid2mt overexpression could counteract the effect of Pcid2 knockdown. Consistently, Pcid2 knockdown in hESCs caused elevated K9K14 acetylation of histone H3 (Fig. 4F) and enhanced the expression of developmental genes (Fig. 2F). Moreover, Pcid2 overexpression significantly decreased CBP/p300 HAT activity (Fig. 4G) and reduced the K9K14 acetylation of histone H3 (Fig. 4F). Together, these results indicate that Pcid2 blocks the HAT activity of CBP/p300 to repress histone acetylation in ESCs, leading to blocking of development-related gene expression.
We further tested the critical role of CBP/p300 HAT activity in Pcid2-mediated pluripotency of ESCs. Depletion of CBP or p300 in mESCs could counteract Pcid2 knockdown-induced transcriptional activation of developmental genes (Supporting Information Fig. S3a). Additionally, a specific CBP/p300 HAT inhibitor curcumin could block Pcid2 knockdown-induced transcriptional activation of developmental genes (Supporting Information Fig. S3b). Furthermore, curcumin could repress acetylation of histone H3 and rescue Oct4 expression in Pcid2-silenced mESCs (Supporting Information Fig. S3c). These results were verified by AP staining (Supporting Information Fig. S3d). Similar results were obtained in RA-induced mESCs (Supporting Information Fig. S3c, S3d). To further confirm the role of HAT activity in maintaining ESC phenotype, we used curcumin in an embryoid body (EB) induction system which has been widely used as a process of spontaneous differentiation of ES cells. We observed that curcumin significantly inhibited EB differentiation and promoted multiple-layer gene expression (Supporting Information Fig. S3e, S3f). We also observed similar results in hESCs (Supporting Information Fig. S3g and data not shown). These results suggest that the CBP/p300 HAT activity is essential for the activation of developmental genes in ESCs.
Pcid2 Prevents EID1 Degradation to Maintain Self-Renewal of ESCs
We next wanted to assess protein levels of Pcid2 and EID1 undergoing ESC differentiation. Surprisingly, we observed that Pcid2 and EID1 simultaneously degraded during mESC differentiation induced by leukemia inhibitory factor (LIF) withdrawal or RA treatment (Fig. 5A, 5B). Notably, EID1 degradation was blocked by the proteasome inhibitor MG132 (Fig. 5C), indicating that its degradation is dependent on proteasomes. We found that endogenous EID1 was indeed ubiquitinated in mESCs during both RA-induced differentiation and EB spontaneous differentiation (Fig. 5D, 5E). Furthermore, Pcid2 knockdown also induced EID1 ubiquitination in mESCs (Fig. 5F), whose ubiquitination was verified to be K48-linked but not K63-linked (Fig. 5G). Pcid2mt overexpression attenuated the EID1 ubiquitination and rescued the protein level of EID1 in Pcid2-silenced mESCs (Fig. 5H–5J). In contrast, overexpression of Pcid2 impeded EID1 ubiquitination and degradation in mESCs (Fig. 5K, 5L). Similar observations were achieved in hESCs (data not shown).
The Sacchromyces homolog Thp1 was reported to participate in export of mRNAs in yeast and the similar function augmenting a selective mRNA export has been recently identified in mammals . However, Pcid2 depletion in mESCs had no effect on mRNA cytoplasm/nucleus distribution of either stemness-related or differentiation-related genes that we tested (Supporting Information Fig. S4a). Actually, Pcid2 silencing did not change EID1 mRNA cytoplasm/nucleus distribution either. Moreover, Pcid2 overexpression failed to alter the mRNA level of EID1 (Supporting Information Fig. S4b). These results suggest the alternative function of Pcid2 that regulates EID1 stability through its protein level.
MDM2 Is an E3 Ligase for K48-Linked Ubiquitination of EID1 for Its Degradation
Through immune-precipitation assays, we found that anti-mEID1 antibody could precipitate endogenous Pcid2 and MDM2 (Fig. 6A). Moreover, EID1 colocalized with either Pcid2 or MDM2 in nuclei of mESCs (Fig. 6B). We next wanted to test whether MDM2 is an E3 ligase for EID1 ubiquitination. We transfected Myc-EID1, pSIN-shMDM2, and His-Ub vectors into 293T cells followed by ubiquitination assays. Intriguingly, EID1 was indeed ubiquitinated in the cotransfected 293T cells, and MDM2 knockdown dramatically reduced the ubiquitination of EID1 (Fig. 6C). Actually, EID1 was rapidly degraded even with ectopic expression of EID1 (Supporting Information Fig. S5). Overexpression of MDM2 accelerated EID1 degradation, while Pcid2 overexpression significantly declined EID1 proteolysis (Supporting Information Fig. S5). These data suggest that EID1 is a substrate for MDM2, and Pcid2 impairs the ubiquitination of EID1. EID1 was verified to be ubiquitinated by MDM2 via an in vitro ubiquitination reconstitution assay (Fig. 6D). Addition of recombinant Pcid2 really repressed the ubiquitination of EID1. Furthermore, overexpression of Pcid2 remarkably attenuated the MDM2-mediated ubiquitination of EID1 (Fig. 6E). Of note, EID1 was degraded in Pcid2 knockdown-induced differentiated mESCs by confocal microscopy, whereas MDM2 depletion abolished EID1 degradation (Fig. 6F).
Pcid2 Abolishes the Activity of MDM2 Through Competitive Binding to EID1
The above results showed that Pcid2 can impair MDM2-mediated EID1 ubiquitination. To determine the inhibitory mechanism of Pcid2 against the enzymatic activity of MDM2, we conducted domain mapping between Pcid2 and EID1 interaction. We identified that the aa115–177 fragment of EID1 was sufficient and necessary for binding to Pcid2 (Supporting Information Fig. S6a). We further demonstrated that both Pcid2 and MDM2 bound to the same aa115–177 fragment of EID1 (Supporting Information Fig. S6b). These results indicate that both Pcid2 and MDM2 can directly bind to EID1. Notably, overexpression of Pcid2 could disrupt the interaction of MDM2 with EID1 in mESCs (Fig. 6G), indicating that Pcid2 and MDM2 competitively bind to EID1. Additionally, Pcid2 did not directly bind to MDM2 (Supporting Information Fig. S7a) and had no direct inhibitory effect on the enzymatic activity of MDM2 (Supporting Information Fig. S7b). Our results suggest that Pcid2 protects EID1 from degradation via competitive binding inhibition.
To further confirm that Pcid2 regulates ESC pluripotency through MDM2-mediated EID1 degradation, two EID1 mutants were constructed for rescue assays. One was K132R/K152R mutant (EID1-st) that was reported to be a stable form of EID1 , whose mutation significantly attenuated EID1 ubiquitination (Supporting Information Fig. S8a) but did not lose the inhibition to p300 HAT activity (Supporting Information Fig. S8b). The other was the mutant EID1(aa1–157Δ53–62Δ92–115) (ΔEID1) which was reported to disrupt the binding capability to CBP/p300 . We observed that EID1 knockdown promoted differentiation of mESCs analogous to Pcid2 depletion (Fig. 6H). Overexpression of the stable mutant EID1-st caused the rescue of stemness, and MDM2 knockdown also rescued the ESC pluripotency. Additionally, the MDM2 inhibitor (N-((3,3,3-trifluoro-2-trifluromethyl)propionyl) sulfanilamide)  rescued the stemness of mESCs. However, the mutant ΔEID1 failed to rescue the stemness of mESCs (Fig. 6H). Similar results were observed in hESCs (data not shown). Of note, Pcid2 deletion in mESCs dramatically augmented the ubiquitination of EID1 (Fig. 6I). Moreover, the MDM2 inhibitor was able to repress EID1 ubiquitination. Collectively, Pcid2 regulates the MDM2-mediated EID1 degradation to modulate the switch balance between self-renewal and differentiation of ESCs.
In this study, we demonstrate that Pcid2 plays a pivotal role in the regulation of switch balance between self-renewal and differentiation in mouse and human ESCs. Pcid2 is present in the CBP/p300-EID1 complex in the ESCs. We found that MDM2 is an E3 ligase for K48-linked EID1 ubiquitination for its degradation. For the maintenance of self-renewal, Pcid2 binds to EID1 to impede the association with MDM2 (Supporting Information Fig. S9). Then EID1 is not ubiquitinated by MDM2 for its degradation to sustain its stability. The stable EID1 binds to CBP/p300 to block their HAT activity, leading to blockade of the developmental gene expression. Undergoing ESC differentiation, Pcid2 is degraded to release EID1. EID1 is ubiquitinated by MDM2 for its degradation. CBP/p300 is activated to acetylate core histones, resulting in the expression of developmental genes. It needs to be further investigated for degradation of Pcid2 in the process of ESC differentiation.
We found that Pcid2−/− blastocysts were difficult to generate through intercrossing of Pcid2flox/+ with EIIA-Cre mice, and Pcid2 deficiency caused early embryonic lethality before the blastocyst stage. Pcid2 has positive staining signals in the ICM of blastocysts in WT mice, indicating that Pcid2 is likely to be expressed in pluripotent cells in embryos. Given that Pcid2 is critically required for the development of a zygote to the blastocyst, Pcid2 may be of great importance in pluripotency maintenance in early embryos. The expression pattern and function of Pcid2 in precise developmental stages of embryos need to be further explored. Overexpression of Pcid2 could block RA-induced ESC differentiation and rescue the expression of pluripotency genes. Additionally, Pcid2 dramatically enhanced Yamanaka four factors (KMOS)-induced pluripotent stem cell (iPSC) generation (data not shown). These suggest that Pcid2 may be involved in the stemness rescue of differentiated cells.
The balance of pluripotency and differentiation of ESCs is refinedly controlled by a series of mechanisms such as transcriptional factors, signaling molecules, and cytokines or extracellular ligands [20–24]. Additionally, a recent report showed that an intracellular signaling network is co-ordinated in hESCs . Phosphoinositide 3-kinase (PI3K)/Akt activity sustains self-renewal by limiting differentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling. Actually, ESCs maintain their stemness both through simultaneously activating pluripotency gene transcription network and inhibiting differentiation-related genes. Herein we found that Pcid2 suppresses developmental gene expression by inhibiting CBP/p300 HAT activity. Meanwhile, overexpression of Pcid2 could also upregulate the expression of the pluripotency genes (Fig. 2E and data not shown). We demonstrated that Pcid2 maintains ESC pluripotency mainly through the Pcid2-EID1-MDM2 axis. However, the overall effect of Pcid2 in pluripotency may be the consequence of activation of multiple mechanisms, including a feedback loop of the pluripotency transcriptional factors, signaling pathways, and epigenetic complexes. These issues still need to be further investigated.
We showed that Pcid2 is required for the pluripotency maintenance of mESCs and hESCs. Pcid2-silenced mESCs or hESCs caused the transcriptional activation of developmental genes. However, Pcid2 knockdown did not affect the expression of the endoderm genes such as FoxA2 and H19 (data not shown) in hESCs, Actually, hESCs and mESCs differ remarkably in morphology, proliferation rates , growth factor requirement, signaling dependence [27, 28], and transcriptional profiling in differentiation . Our observations suggest Pcid2 may act on different germ layer development in mice and humans by targeting different gene loci.
Covalent histone modifications play vital roles in the regulation of pluripotency . Histone modifications, including acetylation, methylation, and phosphorylation, can be either repressive or permissive for transcriptional activation, which depends on its location and context. The mammalian Polycomb repressive complex two was reported to trimethylate Lys27 on histone H3 and occupy the differentiation-related genes to prevent full expression of these genes, which modulates the balance of self-renewal and differentiation [30, 31]. Histone deacetylases (HDACs) and HATs were both reported to regulate pluripotency [18, 32]. ESCs were reported to undergo a wave of global histone modifications including acetylation in the beginning of differentiation. Histone acetylation was also reported to occur on either developmental genes or pluripotency genes depended on dynamics of differentiation [17, 18, 32, 33]. It is still not clear regarding how histone acetylation modulates the pluripotency. EID1 harbors two acid patches and a C-terminal pRB-binding motif (LXCXE) , which was first identified to be an interactor of pRB. The intact C-terminal region of EID1 is needed to bind to pRB and p300, which links cell cycle control and epigenetic regulation. EID1 binding to CBP/p300 can repress their HAT activity to block histone acetylation leading to inactivation of differentiation in somatic cells. CBP/p300 can respond to a variety of intracellular and extracellular signaling pathways and act as molecular switches that control cell fate decisions . CBP/p300 was reported to be involved in the regulation of embryo development and differentiation [4, 10, 11]. In this study, we demonstrate that CBP/p300 participates in the regulation of pluripotency through promoting histone acetylation leading to the expression of developmental genes of ESCs.
Pcid2 is present in the CBP/p300-EID1 complex in the mESCs and hESCs. For the maintenance of ESC pluripotency, Pcid2 binds to EID1 to impede binding of EID1 to MDM2. However, undergoing ESC differentiation, Pcid2 is rapidly degraded to release EID1. We found that that mRNA level of Pcid2 was not changed and Pcid2 was downregulated in its protein level during the differentiation of ESCs (data not shown). Herein we show that MDM2 is an E3 ligase for K48-linked EID1 ubiquitination for its degradation. EID1 is ubiquitinated by MDM2 to mediate proteasome-dependent degradation in ESCs. The C-terminal segment aa115–177 of EID1 is the binding region for both Pcid2 and MDM2 in ESCs. Through competitive binding to EID1, Pcid2 impairs the enzymatic activity of MDM2 to block the ubiquitination of EID1. Our findings indicate that MDM2-mediated EID1 degradation is involved in the modulation of pluripotency of ESCs. Of note, Pcid2 can inhibit EID1 ubiquitination but not p53 ubiquitination. p300 was reported to act as an adaptor that facilitates MDM2-mediated p53 degradation . It was reported that p53 expression is downregulated during ESC differentiation . In RA-induced ESC differentiation, p53 is acetylated by p300/CBP and disassociated with MDM2 [36, 37]. Whether MDM2-mediated p53 degradation is involved in regulation of pluripotency needs to be further elucidated. Pcid2 is not a substrate for MDM2, whose degradation undergoing differentiation remains to be further investigated. Collectively, our study provides new insights into the molecular mechanism of Pcid2 in the regulation of switch balance between maintenance of pluripotency and differentiation in mouse and human embryonic stem cells.
We thank Dr. James Thomson at University of Wisconsin-Madison and Dr. Xiaoyan Ding at Core facility for stem cell research of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences for providing human ESC line H1; Dr. William G. Kaelin, Jr for providing pCDNA4-EID1 1–157Δ53–62Δ92–115 plasmid; Dr. Carol Prives for providing pcDNA3-MDM2 plasmid; Zhenwei Yang for real-time qPCR technique support; Yan Teng for imaging technique support; Chunchun Liu, Junying Jia, Xudong Zhao, and Su Liu for technical support; Zhaoxia Liu and Lu Hao for Yeast-two hybrid screening; Drs. Lan Huang and Geng Zhang for helpful advices and suggestions; Jun Chen, Shengwu Liu and Man Li for critical reading and suggestions. This work was supported by the National Natural Science Foundation of China (30830030, 30901302, 31100971), 973 Program of the MOST of China (2010CB911902), the Strategic Priority Research Programs of the Chinese Academy of Sciences (XDA01010407), and China Postdoctoral Science Foundation (20110490617, 2012T50145).
B.Y.: designed and performed experiments, analyzed data, and wrote the paper; Z.D.: performed experiments and analyzed data; B.L. and X.Y.: performed experiments; R.W.: constructed plasmids and screened yeast-two hybrid library; C.L.: analyzed data.; G.H.: performed ubiquitination and pulldown assays; S.W. and P.X.: performed ubiquitination and pulldown assays, analyzed data; K.K.: established Pcid2flox/flox mice and analyzed data; N.S.: established Pcid2flox/flox mice and analyzed data; Z.F.: initiated the study, organized, designed, and wrote the paper. B.Y. and Z.D. contributed equally to this article.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.