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. 2024 Aug 27;102:skae251. doi: 10.1093/jas/skae251

Ubiquitination plays an important role during the formation of chicken primordial germ cells

Wei Gong 1,2, Xin Liu 3,4, Xiaoqian Lv 5,6, Yani Zhang 7,8, Yingjie Niu 9,10, Kai Jin 11,12, Bichun Li 13,14, Qisheng Zuo 15,16,✉
PMCID: PMC11452721  PMID: 39187982

Abstract

As an important posttranslational modification, ubiquitination plays an important role in regulating protein homeostasis in eukaryotic cells. In our previous studies, both the transcriptome and proteome suggested that ubiquitination is involved in the formation of chicken primordial germ cells (PGCs). Here, affinity enrichment combined with liquid chromatography–tandem mass spectrometry (MS/MS) was used to analyze the ubiquitome during the differentiation from embryonic stem cells to PGCs, and we identify that 724 lysine ubiquitinated sites were up-regulated in 558 proteins and 138 lysine ubiquitinated sites were down-regulated in 109 proteins. Furthermore, GO and KEGG enrichment analysis showed that ubiquitination regulates key proteins to participate in the progression of key events related to PGC formation and the transduction of key signals such as Wnt, MAPK, and insulin signals, followed by the detailed explanation of the specific regulatory mechanism of ubiquitination through the combined proteome and ubiquitome analysis. Moreover, both the activation and inhibition of neddylation were detrimental to the maintenance of the biological characteristics of PGCs, which also verified the importance of ubiquitination. In conclusion, this study provides a global view of the ubiquitome during the formation of PGCs by label‐free quantitative ubiquitomics, which lays a theoretical foundation for the formation mechanism and specific application of chicken PGCs.

Keywords: chicken, embryonic stem cell, primordial germ cell, ubiquitome

This study lays a theoretical foundation for the formation mechanism and specific application of chicken PGCs.

Introduction

It is well known that primordial germ cells (PGCs) are derived from embryonic stem cells (ESCs) and are the precursors of male and female germ cells (Magnúsdóttir and Surani, 2014). Poultry PGCs have been widely used in fields such as transgenic animal production and germplasm resource conservation because of their ability to migrate to the genital ridges through the blood circulation system (Nakamura, 2016). However, the instability of the culture system and low induction efficiency lead to insufficient amount of PGCs obtained in vitro, which limits the specific application of PGCs to a large extent. Therefore, a task of top priority for us is to construct the developmental regulatory network of chicken PGCs by focusing on the developmental regulatory mechanism of chicken PGCs, so as to optimize the culture and induction conditions.

As one of the most important protein posttranslational modifications, ubiquitination can regulate cell proliferation, differentiation and cell fate determination by participating in biological processes like gene expression regulation and signal transduction. Ubiquitin (Ub) is a small molecule protein consisting of 76 amino acids that is highly conserved in eukaryotes. Ubiquitin molecules mediate ubiquitination of the substrates under the combined action of ubiquitin-activating enzymes E1s, ubiquitin-conjugating enzymes E2s and ubiquitin ligases E3s, and the process is reversible (Van Wijk et al., 2019; Snyder and Silva, 2021). Ubiquitination can be divided into monoubiquitination and polyubiquitination. The former mainly regulates protein endocytosis and histone modification while the latter can mediate protein hydrolysis, signal transduction, and other biological processes (Baarends et al., 1999; Komander, 2009).

The formation of PGCs is dependent on the regulation of ubiquitination. No matter what species, the specification process of PGCs seems to depend on the induction of BMP4 signal (Zuo et al., 2020). It is generally accepted that BMP4 signal mainly induced some cells to exhibit the fate characteristics of germline such as the expression of transcription factors Blimp1, Dppa3/stella, and Tfap2c/AP2γ, the relative stability of which relies on ubiquitination during further specification of PGCs (Saitou and Yamaji, 2012; Yang et al., 2014; Shin et al., 2017; Cui et al., 2022). In addition, the cooperative regulation of many signaling pathways takes part in PGC formation (Zuo et al., 2023), and ubiquitination can affect the activation and inhibition of signaling pathways by regulating key signaling proteins. What has been universally acknowledged is that TGF-β/BMP4 is a critical signal during the specification and development of PGCs (Zuo et al., 2017, 2020; Yu et al., 2021). Studies have reported that E3 Smurf1/2 can change the ubiquitination level of MKK2 and TβRI (Yamashita et al., 2005; Zhang et al., 2013), and SUMO as well as USP15 is capable of regulating the ubiquitination of Smad4 and ALK3 (Yukita et al., 2012; Herhaus et al., 2014), thereby controlling the activation or inhibition of TGF-β/BMP4 signal. Importantly, PGCs undergo reprogramming of epigenetic modifications such as de novo DNA methylation, the erasure and reconstruction of histone methylation (Zhang et al., 2021; Zuo et al., 2023), which are fundamental to gene expression and signal transduction. DNA methyltransferase 1/3A (DNMT1/3A) can be modified by USP7 and CRL4BDCAF8 (Dong et al., 2022; Huang et al., 2022), and DNA demethylase TET can be modified by CRL4VPRBP (Yu et al., 2013).

Ubiquitomics based on liquid chromatography (LC)–tandem mass spectrometry (MS/MS) was developed to identify ubiquitinated proteins after affinity enrichment, and it has been widely utilized in recent studies to investigate the regulatory mechanisms of ubiquitination in diverse animal biological processes. Huang et al. (2020) provided a basis for exploring the regulatory mechanism of ubiquitination during spermatogenesis in mammals by the creation of the buffalo testis ubiquitome profile. In the research of Liu et al. (2023), the direct targets of TRIM59 were identified by conducting the ubiquitomics of human pulmonary artery smooth muscle cells overexpressing TRIM59, which could help explain how TRIM59 is involved in the pathogenic mechanisms of pulmonary hypertension. Cai et al. (2020) performed the ubiquitome analysis of rabies virus (RABV)-infected mouse brain and offered new ideas for studying the pathogenesis of RABV.

In order to explore the regulatory role of ubiquitination during the formation of chicken PGCs, we used a label-free quantitative strategy to investigate the ubiquitome of chicken ESCs and PGCs, and preliminarily revealed the regulatory mechanism of ubiquitination during PGC formation by GO, KEGG, and other analysis methods, providing a theoretical basis for further optimizing the culture and induction conditions of chicken PGCs.

Materials and Methods

Ethics statement

All of the procedures involving the care and use of animals were approved by the Institutional Animal Care and Use Committee of the Yangzhou University (approval number: SYXK [Su] 2021-0027). The fertilized eggs of Rugao yellow chicken were purchased from the Poultry Research Institute of Chinese Academy of Agricultural Sciences and hatched under the environment of 37 °C with 70% humidity.

Sample preparation

The freshly fertilized eggs from Rugao yellow chickens were provided by the Poultry Research Institute of CAAS, Yangzhou, Jiangsu Province, China. ESCs were isolated from the blastoderm cells of the X-stage fertilized eggs and cultured in KO-DMEM (Gibco, Carlsbad, CA, USA, 10829018), containing 10% KSR (Gibco, 10828028), 1% GlutaMax (Gibco, 35050061), 1% non-essential amino acids (Gibco, 11140050), 0.1 mM β-mercaptoethanol (Gibco, 21985023), 1% chicken serum (Gibco, 16110082), 10 ng/mL LIF (Sigma-Aldrich, St. Louis, MO, USA, ESG1106), 10 ng/mL bFGF (Sigma-Aldrich, GF446), 10 ng/mL hSCF (Sigma-Aldrich, GF021), 3 µM PD0325901 (MCE, Monmouth Junction, NJ, USA, HY-10254), 10 µM SB431542 (MCE, HY-10431) and 1% penicillin–streptomycin (Gibco, 15140148). PGCs were isolated from the genital ridge of the chicken embryos incubated for 5.5 days and cultured in DMEM (Gibco, 21068028) containing 24% H2O (Sigma-Aldrich, W3500), 0.15 mM CaCl2 (Sigma-Aldrich, C7902), 1% GlutaMax, 1% non-essential amino acids, 1.2 mM sodium pyruvate (Gibco, 11360070), 0.1 mM β-mercaptoethanol, 0.2% ovalbumin (Sigma-Aldrich, A5503), 2% B-27 (Gibco, 17504044), 1% EmbryoMax Nucleosides (Sigma-Aldrich, ES-008), 0.2% chicken serum, 0.01% heparin sodium (MCE, HY-17567A), 25 ng/mL activin A (MCE, HY-P70311), 4 ng/mL FGF-2 (MCE, HY-P70600) and 1% penicillin–streptomycin. ESCs and PGCs were maintained in a 5% CO2 humidified atmosphere at 37 °C. The isolation and culture methods for chicken ESCs and PGCs have been described previously (Zuo et al., 2020). After purification culture, ESCs and PGCs were collected and washed once with PBS, and the supernatant was discarded and the precipitation was frozen at −80 °C.

Protein extraction and trypsin digestion

5 × 107 ESCs and PGCs were ultrasonically treated in a 4-volume of lysis buffer respectively (8 M urea, 1% protease inhibitor, 50 μM PR-619). After centrifugation at 12,000 × g at 4 °C for 10 min, the supernatant was obtained and the protein concentration was determined by BCA kit. The protein solution was reduced with dithithreitol at a final concentration of 5 mM at 56 °C for 30 min. After that, iodoacetamide was added to a final concentration of 11 mM and incubation lasted for 15 min at room temperature away from light. The protein samples were then diluted by adding TEAB to dilute the concentration of urea to less than 2 M. Finally, the protein samples were digested by trypsin at a trypsin-to-protein mass ratio of 1:50 overnight and at a trypsin-to-protein mass ratio of 1:100 for 4 h.

Affinity enrichment of ubiquitinated peptides

The tryptic peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and incubated with pre-washed anti-K-ε-GG antibody-conjugated agarose beads at 4 °C overnight with gentle shaking. After incubation, the beads were washed 4 times with IP buffer and twice with deionized water. The bead-bound peptides were eluted 3 times with 0.1% trifluoroacetic acid and the collected eluent was dried by vacuum freezing, followed by desalted with C18 ZipTips.

LC–MS/MS analysis

The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and loaded onto a reversed-phase analytical column. The gradient comprised of an increase from 6% to 22% solvent B (0.1% formic acid in 98% acetonitrile) for 44 min, 22% to 30% for 12 min, climbing to 80% for 2 min, and then holding at 80% for the last 2 min, all at a constant flow rate of 400 nL/min on an EASY‐nLC 1000 UPLC system. The peptides were then subjected to NSI source followed by MS/MS in tims-TOF Pro (Bruker) coupled online to the UPLC. The electrospray voltage applied was 1.55 kV. The m/z scan range was 100 to 1,700 for all scans. A data-dependent procedure that alternated between one MS scan and 10 MS/MS scans was applied with 24 s dynamic exclusion.

Database search

The MS/MS data were processed using Maxquant search engine (v.1.6.6.0). Tandem mass spectra were searched against chicken (Gallus gallus) genome database concatenated with a reverse decoy database. Trypsin/P was specified as the cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 40 ppm in First search and 40 ppm in Main search, and the mass tolerance for fragment ions was set as 0.04 Da. Alkylation on Cys was set as the fixed modification, and oxidation on Met, acetylation on the N-terminus of proteins, ubiquitination on Lys were specified as variable modifications. False discovery rate thresholds were specified at 1%.

Bioinformatics analysis

The process and method of GO (The Gene Ontology Consortium, 2019) and KEGG (Kanehisa et al., 2008) enrichment analysis for the proteins with differentially ubiquitinated sites (DUSs) were referred to relevant studies. WoLF PSORT was used to predict the subcellular location. Motif-X was used to analyze the motif characteristics of the ubiquitinated site by the peptide sequences consisting of 10 amino acids upstream and downstream of the identified modification site. Occurrence and P-value were set to 20 and 0.000001.

VII-31 and MLN4924 treatments

VII-31 (MCE, HY-133558) or MLN4924(MCE, HY-70062) was dissolved by DMSO to prepare a concentrated stock solution, which was diluted with PGC culture medium to final concentrations of 0.05, 0.5, and 1 μM (the final concentration for DMSO was less than 0.1%). PGCs in good condition were collected by centrifugation at 1,000 rpm for 5 min and then re-suspended with a medium containing VII-31 or MLN4924 at different concentrations. PGCs were then inoculated in 24-well plates with 1 × 105 per well and maintained in a 5% CO2 humidified atmosphere at 37 °C for 24 h.

CCK-8 assay

CCK-8 assay was conducted to evaluate the proliferation ability of PGCs after different treatments. 1 × 105 PGCs in good condition inoculated in the 96-well plate were treated with VII-31 or MLN4924 at different concentrations for 24 h, and then 10 μL CCK-8 (Vazyme, Nanjing, China, A311-01) was added to each well. After incubation at 37 °C and 5% CO2 away from light for 2 h, the absorbance at 450 nm was read by microplate reader (TECAN, Shanghai, China).

Cell apoptosis assay

5 × 105 PGCs after different treatments were collected and washed with pre-cooled PBS. Then PGCs were respectively incubated with Annexin V-FITC and PI (Yeasen, Shanghai, China, 40302ES50) at room temperature for 15min without light. PGCs were then detected by flow cytometry (BD LSRFortessa, BD Biosciences, San Jose, CA, USA).

qRT-PCR

Total RNA was extracted from 2 × 106 PGCs after different treatments using TRNzol (Tiangen, Beijing, China, DP424) and reverse transcribed into cDNA with HiScript III RT SuperMix (Vazyme, R323-01). Gapdh was used as an internal control gene to estimate the expression of Blimp1, Ddx4, Cxcr4, and Oct4 (Lee et al., 2017; Zuo et al., 2020; Meng et al., 2022). The reaction mixture for qRT-PCR included 10 μL 2 × ChamQ Universal SYBR qPCR Master Mix, 0.6 μL each of upstream and downstream primer, 2 μL cDNA and 6.8 μL ddH2O. The reaction procedure was performed according to the instructions provided by ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme). The relative gene expression was calculated with the 2-ΔΔCt method (Rao et al., 2013). The primers are supplied in Supplementary Table 11.

Data analysis

All experiments were repeated at least 3 times, and the data are presented as mean ± standard error. After all data were sorted by EXCEL, SPSS 19.0 (SPSS, Chicago, IL, USA) and Graph Pad Prism 6 (GraphPad Software Inc., San Diego, CA, USA) were used to perform significance analysis and generate diagrams. Significant differences between the groups were determined by one-way ANOVA (*P < 0.05, significant difference. **P < 0.01, extremely significant difference).

Results

Proteome and transcriptome show inconsistency during PGC formation

During the combined transcriptome and proteome analysis of ESCs and PGCs, for some (73) genes inconsistent changes were found at the transcription level and protein level (Figure 1A and B; Supplementary Figure 1A). Ubiquitin-proteasome system (UPS) and autophagy are the main ways to degrade intracellular proteins (Pohl and Dikic, 2019). The results of GO enrichment analysis of differentially expressed proteins (DEPs) showed that 47 DEPs were enriched to 29 GO terms related to autophagy (Figure 1C; Supplementary Table 1) but more (69) DEPs were enriched to 58 GO terms related to UPS (Figure 1D; Supplementary Table 2), which indicated that UPS plays a more important role in the regulation of the overall protein level during the differentiation of ESCs to PGCs. Therefore, we conducted label‐free quantitative ubiquitomics of chicken ESCs and PGCs to explore the specific regulatory role of ubiquitination during PGC formation.

Figure 1.

Figure 1.

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Proteome and transcriptome show inconsistency during PGC formation. (A) Intersection of genes/proteins quantified in the transcriptome and the proteome. P, proteome. (B) Some of the intersection, as described in A, are inconsistent at the transcriptional and protein levels. (C and D) GO analyzed the enrichment of DEPs in terms related to autophage (C) and UPS (D).

Overview of the ubiquitomic analysis

In order to avoid the inconsistency of ubiquitination levels on proteins caused by individual differences, we collected cell samples from multiple individuals (n = 90) to mix into a pool. Two pools were prepared for each group to ensure the accuracy of the sequencing results. After digesting the proteins extracted from ESCs and PGCs, the ubiquitinated peptides were enriched by anti-K-ε-GG antibody for LC–MS/MS analysis.

A total of 147817 secondary spectra were obtained by mass spectrometry, of which 12.4% were effective. By spectrum analysis, 7,990 peptides were identified, including 5,037 ubiquitinated peptides, and the number of the amino acids composing the peptides was distributed in the range of 8 to 17 (Figure 2A; Supplementary Figure 2A). Comparison and database search were performed using the transcriptome data of ESCs and PGCs, and 5,105 sites on 2,192 proteins (100%, 5,105) can be identified and quantified (Figure 2A). Among the identified proteins, the number of proteins with only one ubiquitinated lysine residue was the highest (Figure 2B). With a threshold of FC > 1.5 or < 1/1.5, 724 lysine ubiquitinated sites were up-regulated in 558 proteins and 138 lysine ubiquitinated sites were down-regulated in 109 proteins (Figure 2C). PCA analysis showed that the 2 pools for sequencing in each group clustered obviously, and the relative standard deviation of the quantitative values between repeated samples in each group ranged between 0.1 and 0.2. In addition, Pearson’s correlation analysis revealed that the differences within the groups were small but the differences between the groups were large (Figure 2D to F). The results above suggested that the ubiquitome data are accurate and reliable for subsequent analysis.

Figure 2.

Figure 2.

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Overview of the ubiquitomic analysis. (A) The mass spectrometry results of the ubiquitome. (B) Distribution of the number of modification sites identified per protein in the ubiquitomics. (C) DUPs (left) and sites (right) in ESCs and PGCs were screened with the threshold criterion for FC (>1.5 or FC < 1/1.5) by the ubiquitomics. (D to F) The sample preparation was validated by PCA analysis (D), relative standard deviation values (E) and Pearson’s correlation analysis (F).

Ubiquitination modification shows diverse functions during the formation of chicken PGCs

To understand the structures of the ubiquitinated lysine sites during PGC formation, Motif-X was used to analyze and summarize the amino acid sequences around the ubiquitinated lysine residues. The results showed that 2,534 of the 5,105 Kub sites could be classified into 7 conserved motifs: RxxxxxxxKub, KubxxE, RxxxxxxKub, ExxxKub, KubxxD, KubE, KubQ (Figure 3A). Among them, KubxxE was the most abundant (Figure 3B). Meanwhile, it was found that alanine (A) at the ± 1 and ± 2 positions, glutamate (E) at the ± 1 positions and arginine (R) at the + 6 to + 10 positions were significantly enriched, while cysteine (C) at the − 1 to − 7 positions, lysine (K) at the + 1 to + 4 positions were significantly depleted.

Figure 3.

Figure 3.

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Ubiquitination modification shows diverse functions during the formation of chicken PGCs. (A) The conserved motifs of the ubiquitinated sites were analyzed by Motif-X. (B) The number of peptides identified with the conserved motifs, as described in A. (C) Amino acid sequence properties of the ubiquitinated sites. The heat map shows the occurrence frequency of the amino acids upstream and downstream of the ubiquitinated sites. (D) Intersection of proteins quantified in the proteome and the ubiquitome. Ub, ubiquitome. (E) The global pattern of the protein level associated with the ubiquitination level was indicated based on log2fold changes in the proteome and ubiquitome. (F) For UPSRPs (enriched in PU-UD and PD-UU modes), protein and ubiquitination levels changed in the different direction while for UPSURPs, protein and ubiquitination levels changed in the same direction. (G and H) The pattern of the protein level associated with the ubiquitination level was indicated for UPSRPs (G) and UPSURPs (H) based on log2fold changes in the proteome and ubiquitome.

The combined proteome and ubiquitome analysis of ESCs and PGCs showed that some proteins changed inconsistently at the protein level and ubiquitination level, which could be related to the function of ubiquitination. To further clarify the functions of ubiquitination during the formation of PGCs, the proteome and ubiquitome were compared and 148 proteins which changed at the protein level and ubiquitination level simultaneously were obtained (Figure 3D and E; Supplementary Figure 1B). Based on changes at the protein level and ubiquitination level, the proteins were enriched to 4 modes: protein level up-regulated, ubiquitination level up-regulated (PU-UU, n = 95); protein level up-regulated, ubiquitination level down-regulated (PU-UD, n = 4); protein level down-regulated, ubiquitination level up-regulated (PD-UU, n = 34); protein level down-regulated, ubiquitination level down-regulated (PD-UD, n = 17) (Figure 3F). Given that ubiquitination can mediate protein degradation by proteasome, for proteins enriched in PU-UD and PD-UU modes, ubiquitination could regulate through UPS, and these proteins were defined as UPS-related proteins (UPSRPs) (Figure 3G). Conversely, ubiquitination might regulate proteins enriched in PU-UU and PD-UD patterns (UPS-unrelated proteins, UPSURPs) through other ways, such as changing protein activity and subcellular localization (Figure 3H).

Ubiquitination plays an important role in the development of chicken PGCs

Differentially ubiquitinated proteins (DUPs) were mainly distributed in cytoplasm, nucleus, and plasma membrane (Supplementary Figure 2B). Moreover, there was little difference in the distribution of subcellular localization between ubiquitomics and proteomics (Supplementary Figure 2C). The results of GO enrichment analysis of DUPs showed that a total of 2,221 GO terms were enriched. Concretely, CDH2, NUDT21, GPM6B, etc. were enriched to GO terms related to cell differentiation (Figure 4A; Supplementary Table 3); CD47, RAC1, TTLL5, etc. were enriched to GO terms related to cell proliferation (Figure 4B; Supplementary Table 4), cell migration (Figure 4C; Supplementary Table 5) and germ cell formation (Figure 4D; Supplementary Table 6). At the same time, KEGG enrichment analysis revealed that MAPK, Wnt, HH, TGF-β, and other signaling pathways related to PGC formation were enriched (Kimura et al., 2014; Zuo et al., 2020; Lee et al., 2023; Shono et al., 2023) (Figure 4E; Supplementary Table 7). These results indicated that ubiquitination is involved in PGC formation.

Figure 4.

Figure 4.

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Ubiquitination plays an important role in the development of chicken PGCs. (A to D) GO analyzed the enrichment of DUPs in terms related to cell differentiation (A), cell proliferation (B), cell migration (C), and germ cell formation (D). (E) KEGG analysis indicated that the key signaling pathways related to PGC formation were enriched.

Ubiquitination modification can change the biological characteristics of PGCs

It has been reported that cullin-RING ubiquitin ligases (CRLs) mediate approximately 20% of protein degradation via UPS (Zhao and Sun, 2013; Song et al., 2021; Yu and Sun, 2021). This is consistent with the significant up-regulation of proteins related to CRLs in the proteome (Supplementary Figure 3A), which also indicated that CRLs are very active in catalyzing protein ubiquitination during PGC formation. In addition, GO terms related to proteasome formation were enriched (Supplementary Figure 3B and Supplementary Table 8). Specifically, proteins related to proteasome formation ADRM1, PSMF1, PSMC3, and PSMC6 were significantly up-regulated (Supplementary Figure 3C). Most importantly, we noted that key proteins regulating ubiquitination (involved in neddylation) exhibit dynamic changes during PGC formation (Supplementary Figure 3D).

In order to determine the effect of ubiquitination on the biological characteristics of PGCs, VII-31, and MLN4924 were used to activate and inhibit the ubiquitination process of PGCs, respectively. It was found that PGCs exposed to a low concentration (0.05 μM) of VII-31 exhibited normal morphology (large single round clones with smooth edges) (Figure 5A). Meanwhile, the proliferation of PGCs was significantly promoted (P < 0.05) (Figure 5B), but the apoptosis of PGCs was not obviously changed (Figure 5C, D). However, high concentrations (0.5 μM, 1 μM) of VII-31 extremely significantly inhibited PGC proliferation (P < 0.01) (Figure 5B) and extremely significantly increased PGC apoptosis (P < 0.01) (Figure 5C, D), accompanied by irregular cell shape (Figure 5A). After exposed to MLN4924, PGC proliferation was extremely significantly inhibited (P < 0.01) (Figure 5B) and PGC apoptosis was extremely significantly increased (P < 0.01) (Figure 5C, D), with the morphology of PGCs lost and cell differentiation observed (Figure 5A). Furthermore, qRT-PCR was used to detect the expression of PGC marker genes Blimp1, Ddx4, Cxcr4 and pluripotency marker gene Oct4 in PGCs. The results showed that different concentrations of VII-31 or MLN4924 significantly changed the expression of these genes to different degrees (P < 0.05) (Figure 5E), which indicated that both activation and inhibition of neddylation were detrimental to the maintenance of biological characteristics of PGCs. Taken together, the abnormal activation and inhibition of ubiquitination destroy the biological characteristics of PGCs.

Figure 5.

Figure 5.

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Ubiquitination modification can change the biological characteristics of PGCs. (A) Morphological observation of PGCs treated with VII-31 or MLN4924 for 24 h. Scale bar: 60 µm. (B) Cell proliferation of PGCs treated with VII-31 or MLN4924 for 24 h was detected by CCK-8 assay. (C) Cell apoptosis detection of PGCs treated with VII-31 or MLN4924 for 24 h. (D) Statistics of apoptosis rate (Q2), as described in C. (E) qRT-PCR was used to detect the expression of PGC marker genes Blimp1, Ddx4, Cxcr4, and pluripotency marker gene Oct4 in PGCs treated with VII-31 or MLN4924 for 24 h.

Regulatory targets of ubiquitination during PGC formation

To systematically analyze the molecular mechanism of ubiquitination in regulating the formation of chicken PGCs, KEGG enrichment analysis was conducted for UPSRPs and UPSURPs respectively. The results showed that 26 UPSRPs were enriched to MAPK, FoxO, Wnt, and other signaling pathways (Figures 3G and 6A; Supplementary Table 9), and 71 UPSURPs were enriched to signaling pathways such as Adipocytokine, Calcium (Figures 3H and 6B; Supplementary Table 10). In particular, NOD-like receptor, PPAR, Insulin and other signaling pathways were enriched by both UPSRPs and UPSURPs. The proteome and ubiquitome were combined to analyze the regulatory pattern of ubiquitination for the proteins in key signaling pathways (Figure 6C and D).

Figure 6.

Figure 6.

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Regulatory targets of ubiquitination in PGC formation. (A and B) KEGG analysis of UPSRPs (A) and UPSURPs (B). (C) Key ubiquitinated proteins was grouped by key signaling pathway and events associated with PGC formation. (D) Changes on the protein and ubiquitination levels of the key ubiquitinated proteins. Proteins with inconsistent protein and ubiquitination levels are defined to be degraded due to ubiquitination, and the remaining proteins are considered to experience other regulatory effects of ubiquitination.

Insulin signaling pathway was activated during PGC formation due to up-regulation of PPP1CB/PPP1CC (PU-UU), which is consistent with the reports (Whyte et al., 2015; Ye et al., 2023). On one hand, up-regulated PPP1CB/PPP1CC indirectly promoted glycogen synthesis by increasing the dephosphorylation of GYS, which facilitated the maintenance of glucose homeostasis. On the other hand, glycogen synthesis was enhanced through PHK (Kumar et al., 2018; Liu et al., 2019; Arends et al., 2022). Under the action of Insulin signaling pathway, lipogenesis was accelerated to maintain lipid homeostasis on account of up-regulation of FASN (PU-UU) (Liu et al., 2018; Xie et al., 2019; Zeng et al., 2022; Paulukinas and Penning, 2023). These are the basis for ensuring the biological characteristics of PGCs.

In MAPK signaling pathway, HSPA8 (PD-UU) was down-regulated owing to its increased degradation mediated by UPS. The declined inhibition of JNK by HSPA8 resulted in up-regulation of JNK, which then promoted the transcription factors AP1/JunD to regulate the transcription of downstream target genes (Hao et al., 2021; Al Moussawi et al., 2022). In Wnt signaling pathway, the up-regulated ubiquitination level was accompanied by the down-regulated protein level for CTNNB1 (PD-UU), the binding of which to the transcription factor TCF/LEF in the nucleus affected the transcription of downstream target genes (Doumpas et al., 2019; Koelman et al., 2022). In FoxO signaling pathway, down-regulation of PLK1 (PD-UU) affected the proliferation ability of PGCs (Li et al., 2019; Gao et al., 2020; Zhu et al., 2020).

In the early stage of this study, DEPs involved in key events in the formation of PGCs were systematically analyzed through the proteome (Zuo et al., 2023). Compared with the ubiquitome data, we found that DEPs in some key events were regulated by ubiquitination (Figure 6C and D). During glycolysis, ubiquitination down-regulated HK1 (PD-UU), GAPDH (PD-UU), and ENO1 (PD-UU) through UPS to inhibit the conversion of glucose to lactate (Liao et al., 2019; De Jesus et al., 2022; Huppertz et al., 2022). At the same time, up-regulated MDH1 (PU-UU) and UQCRFS1 (PU-UU) accelerated oxidative phosphorylation (Fernandez-Vizarra and Zeviani, 2021; Lin et al., 2023). What’s more, the level of histone acetylation was down-regulated because of up-regulation of histone deacetylation modification enzyme SFPQ (PU-UU) (Zuo et al., 2023). During autophagy, PIK3C2A (PU-UD) was up-regulated due to inhibition of UPS while SQSTM1 (PD-UD) was down-regulated, which ultimately triggered the enhancement of autophagy (Merrill et al., 2017; Sabbieti et al., 2022). These results suggested that ubiquitination is widely involved in key events during PGC formation.

Discussion

In this study, we performed a label-free quantitative ubiquitomics analysis and identified DUSs on DUPs. It was found that ubiquitination is widely involved in the progression of key events and the transduction of key signals by regulating the key proteins during the formation of chicken PGCs. Meanwhile, we preliminarily explained the specific regulatory mechanism of ubiquitination combined with the proteome.

The expression of the totipotency factor OCT4 is an important feature of PGCs (Saitou and Yamaji, 2012; Meng et al., 2022). It has been reported that the deletion of OCT4 hinders the normal formation of PGCs and even leads to reproductive defects. Interestingly, in vitro experiments revealed that the expression of OCT4 in PGCs cannot be as high as that in ESCs, otherwise PGCs would lose the characteristics of germline (Kimura et al., 2015; Li et al., 2018). However, too low expression of OCT4 is adverse to the proliferation and migration of PGCs (Kehler et al., 2004; Meng et al., 2022). These indicate that OCT4 depends on some mechanism to maintain its expression level in PGCs within a very small range. In our study, we found that abnormal activation or inhibition of ubiquitination changes the biological characteristics of PGCs, accompanied by the down-regulation of OCT4, which may demonstrate that ubiquitination is important for the homeostasis of the expression of OCT4 in chicken PGCs. Accordingly, OCT4 may be the focus of research on ubiquitination regulating PGC formation.

During our KEGG enrichment analysis of UPSRPs and UPSURPs, the other 2 signaling pathways drew our attention. In Adipocytokine signaling pathway, up-regulated ACSL3 (PU-UU) increased acyl-CoA synthesis (Yang et al., 2022). The increase of acyl-CoA facilitated ceramide synthesis and inhibited AKT, indirectly reducing insulin resistance (Ferreira et al., 2017; Chaurasia et al., 2019; Kim et al., 2022; Li et al., 2022). Additionally, the increased acyl-CoA promoted the synthesis of diglycerides and reduced the concentration of SOCS3, which would activate LEPR and eventually lead to the increase of glucose uptake and gluconeogenesis. The decrease of SOCS3 could also positively regulate cell growth and proliferation by activating JAK and strengthening the phosphorylation of SHP-2 (Singh et al., 2006; Luo et al., 2011; Xu et al., 2018; Borges et al., 2019; Zhu et al., 2021; Wee et al., 2022). Through our analysis, we found that Insulin signaling pathway interacts extensively with energy metabolic processes such as glycolysis to participate in PGC formation. In Calcium signaling pathway, down-regulation of ATP2B1 (PD-UD) brought about a slowdown in the rate of Ca2+ transport outside the cell (Görlach et al., 2015; Boczek et al., 2021), which may explain why PGCs are very sensitive to the concentration of Ca2+ during in vitro culture (Ge et al., 2009; Whyte et al., 2015; Niu et al., 2024).

Although the lysine residues ubiquitinated on the proteins can be identified and positioned, we cannot know whether monoubiquitination or polyubiquitination occurs at these sites, let alone the modification types of these sites. In fact, different types of ubiquitination correspond to different functions. K48-linked chain is the most linkage type and it mainly mediates proteasomal recognition and degradation of substrates. Following K48-linked chain is K63-linked chain which promotes autophagic degradation of substrates and associated substances such as damaged mitochondria and invading pathogens (Akutsu et al., 2016). In addition, K63-linked chains can also regulate non-degradative cellular processes such as protein transport, DNA damage repair and protein kinase activation (Yau and Rape, 2016; Waltho et al., 2024). In our analysis, we found that the abundance of CTNNB1 decreased, accompanied by the up-regulated ubiquitination levels, which were inconsistent with the activation of Wnt signaling pathway during PGC formation (Gong et al., 2024). This may be because ubiquitination on CTNNB1 mainly functions through inducing CTNNB1 translocation to the nucleus, rather than degradation. We expect that in the future improvements in sequencing technology can address the problem of identifying the modification types of the ubiquitinated sites, making it easier for us to focus on key modification sites for functional studies.

Unfortunately, current ubiquitomics is unable to reflect changes in the subcellular localization of a protein and can only annotate the subcellular localization of a protein in a non-tissue-specific way, which is unfavorable for our analyzing the ubiquitome since altering the subcellular localization is one of the most important functions of ubiquitination (Yang et al., 2021; Cruz Walma et al., 2022). In addition, we intended to construct the relationship between motif characteristics and ubiquitination functions by statistical analysis of amino acid preferences near lysine ubiquitinated sites, but our statistical results did not successfully reveal the specificity of the functions corresponding to the motifs. This may be due to the lack of depth of the existing sequencing technology, which resulted in the loss of some proteins during sequencing and indirectly affected the statistics of the data.

Supplementary Material

skae251_suppl_Supplementary_Tables_S1-S11_Figures_S1-S3
skae251_suppl_supplementary_tables_s1-s11_figures_s1-s3.docx (711.9KB, docx)

Acknowledgments

We thank the Poultry Institute of the Chinese Academy of Agricultural Sciences Experimental Poultry Farm for providing experimental materials. This work was supported by the STI 2030-Major Projects [2023ZD0407503], the Excellent Youth Foundation of Jiangsu [BK20220117], the National Natural Science Foundation of China [32372861], the International Science and Technology Cooperation Projects of Yangzhou [YZ2022206], the China Postdoctoral Science Foundation [2020M681746].

Glossary

Abbreviations

A

alanine

C

cysteine

CRL

cullin-RING ubiquitin ligase

DEP

differentially expressed protein

DNMT1/3A

DNA methyltransferase 1/3A

DUP

differentially ubiquitinated protein

DUS

differentially ubiquitinated site

E

glutamate

ESC

embryonic stem cell

FDR

false discovery rate

K

lysine

LC

liquid chromatography

MS/MS

tandem mass spectrometry

PD-UD

protein level down-regulated, ubiquitination level down-regulated

PD-UU

protein level down-regulated, ubiquitination level up-regulated

PGC

primordial germ cell

PU-UD

protein level up-regulated, ubiquitination level down-regulated

PU-UU

protein level up-regulated, ubiquitination level up-regulated

R

arginine

RABV

rabies virus

RSD

relative standard deviation;

Ub

ubiquitin

UPS

Ubiquitin-proteasome system

UPSRP

UPS-related protein

UPSURP

UPS-unrelated protein

Contributor Information

Wei Gong, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Xin Liu, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Xiaoqian Lv, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Yani Zhang, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Yingjie Niu, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Kai Jin, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Bichun Li, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Qisheng Zuo, Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, P.R. China; Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, P.R. China.

Conflict of interest statement

The authors declare no competing interests.

Author contributions

Z.Q.S. conceived and designed the experiments. G.W. performed the experiments. G.W. and L.X. analyzed the data. G.W. and L.X. wrote the manuscript. L.B.C., L.X.Q., Z.Y.N., N.Y.J., and J.K. edited the manuscript.

Data availability

The data used to support the findings of this study are included in the article. All details and materials of the experimental process can be obtained by contacting the corresponding author. Data and code related to this paper may be requested from the authors. The data of RNA-seq for ESCs and PGCs in this paper has been deposited in the GEO database with the Accession No. GSE159511. The data of proteome for ESCs and PGCs are available via ProteomeXchange with identifier PXD035490 (Username: reviewer_ pxd035490@ebi.ac.uk; Password: qXE68mgp For Reviewer). The data of ubiquitome for ESCs and PGCs are available via ProteomeXchange with identifier PXD052543 (Username: reviewer_pxd052543@ebi.ac.uk; Password: hJ5DibdJ4zDy For Reviewer).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

skae251_suppl_Supplementary_Tables_S1-S11_Figures_S1-S3
skae251_suppl_supplementary_tables_s1-s11_figures_s1-s3.docx (711.9KB, docx)

Data Availability Statement

The data used to support the findings of this study are included in the article. All details and materials of the experimental process can be obtained by contacting the corresponding author. Data and code related to this paper may be requested from the authors. The data of RNA-seq for ESCs and PGCs in this paper has been deposited in the GEO database with the Accession No. GSE159511. The data of proteome for ESCs and PGCs are available via ProteomeXchange with identifier PXD035490 (Username: reviewer_ pxd035490@ebi.ac.uk; Password: qXE68mgp For Reviewer). The data of ubiquitome for ESCs and PGCs are available via ProteomeXchange with identifier PXD052543 (Username: reviewer_pxd052543@ebi.ac.uk; Password: hJ5DibdJ4zDy For Reviewer).

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