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Interaction of the Primordial Germ Cell-Specific Protein C2EIP With Zuo et al. Cell Death and Disease (2018) 9:497 DOI 10.1038/s41419-018-0557-2 Cell Death & Disease ARTICLE Open Access Interaction of the primordial germ cell- specific protein C2EIP with PTCH2 directs differentiation of embryonic stem cells via HH signaling activation Qisheng Zuo1,KaiJin1, Jiuzhou Song2, Yani Zhang1, Guohong Chen1 and Bichun Li1 Abstract Although many marker genes for germ cell differentiation have been identified, genes that specifically regulate primordial germ cell (PGC) generation are more difficult to determine. In the current study, we confirmed that C2EIP is a PGC marker gene that regulates differentiation by influencing the expression of pluripotency-associated genes such as Oct4 and Sox2. Knockout of C2EIP during embryonic development reduced PGC generation efficiency 1.5-fold, whereas C2EIP overexpression nearly doubled the generation efficiency both in vitro and in vivo. C2EIP encodes a cytoplasmic protein that interacted with PTCH2 at the intracellular membrane, promoted PTCH2 ubiquitination, activated the Hedgehog (HH) signaling pathway via competitive inhibition of the GPCR-like protein SMO, and positively regulated PGC generation. Activation and expression of C2EIP are regulated by the transcription factor STAT1, histone acetylation, and promoter methylation. Our data suggest that C2EIP is a novel, specific indicator of PGC generation whose gene product regulates embryonic stem cell differentiation by activating the HH signaling pathway fi 1234567890():,; 1234567890():,; via PTCH2 modi cation. Introduction re-programming them to differentiate into spermatogo- – Primordial germ cells (PGC) can be used for therapeutic nial stem cells (SSC)7 10. Moreover, RNAi-mediated purposes and genetic modification as an alternative to knockout of LIN28 downregulates expression of multi- + embryonic stem cells1,2. Unfortunately, harvesting plen- ple germ cell surface markers during STELLA cell dif- tiful PGC in vitro is often problematic because of the ferentiation, including key PGC development genes such limited understanding of their ontogenetic mechanism, as Blimp1, Prdm14, and endogenous STELLA11,12. which greatly confines their clinical application3. Multiple Although these genes can mark PGC and be used to track gene products are specifically involved in PGC differ- their migration and colonization, not all of them are entiation4. For example, hypoxia induces PGC generation uniquely expressed in PGC and their regulatory by maintaining HIF1 expression and down-regulating mechanisms are not conspicuous. Oct4 expression5,6, whereas Blimp1 and Prdm14 regulate In the present study, we identified a novel PGC-specific PGC generation in mice by epigenetically modifying and marker gene, C2EIP13,14, and characterized its molecular function in detail. We previously reported that C2EIP was not noticeably expressed in embryonic stem cells (ESC), Correspondence: Bichun Li ([email protected]) SSC, or chick embryo fibroblasts but are specifically 1Key Laboratory of Animal Breeding Reproduction and Molecular Design for expressed in PGC. Therefore, we speculated that C2EIP is Jiangsu Province, College of Animal Science and Technology, Yangzhou likely a novel key genetic marker for PGC generation and University, Yangzhou, Jiangsu 225009, P.R. China 2Animal & Avian Sciences, University of Maryland, Baltimore, MD 20741, USA differentiation. This hypothesis is supported by earlier Edited by A Stephanou. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Zuo et al. Cell Death and Disease (2018) 9:497 Page 2 of 12 studies showing that CRISPR/Cas9-mediated knockout of intowell-cultured DF-1 cells and indirect immuno- C2EIP in chick embryos significantly limited their devel- fluorescence experiments were performed usingthe poly- opment13 and reduced PGC generation efficiency, and clonal antibody. The results showed that C2EIP was indeed C2EIP overexpression promoted PGC generation. The expressed in the cytoplasm (Fig. 1g), and similar results GST pull-down assay showed that C2EIP interacted with were also found in PGC (Supplementary Figure 1e). Our PTCH2, an important membrane protein of the Hedge- results suggested that C2EIP was a PGC-specificgenethat hog (HH) signaling pathway, which can upregulate germ could encode protein. cell differentiation and PGC generation15. However, whether C2EIP function is associated with HH signal C2EIP is highly expressed in pgc via promoter and histone activation remains unknown. Thus, we investigated the modification ubiquitination and dephosphorylation status of PTCH2 in Because we observed C2EIP more highly expressed in response to different expression levels of C2EIP. We PGC than in ESC or SSC, we speculated that the activity found that C2EIP overexpression induced ubiquitinatio- of the C2EIP promoter changes during PGC generation, nand dephosphorylation of PTCH2 and upregulated PGC which leads to differential gene expression in PGC com- generation by releasing SMO protein and activating the pared to ESC and SSC. Therefore, we identified the pro- HH signaling pathway. We also determined that C2EIP moter region of the C2EIP gene based on the original expression was regulated by transcription factor STAT1 promoter TATAbox and CAATbox and cloned the C2EIP as well asmethylation and acetylation patterns. In sum- promoter fragment to replace the pEGFP-N1 promoter in mary, we conclude that C2EIP is a novel, PGC-specific a pC2EIP-EGFP recombinant vector. The constructed marker gene that regulates PGCgeneration in vivo and pC2EIP-EGFP vector (Fig. 2a) can express green fluor- in vitro. escent protein in DF-1 cells (Supplementary Figure 2a), confirming the C2EIP promoter activity. Results Studies have shown that epigenetic factors (DNA C2EIP encodes a cytoplasmic protein that is differentially methylation, histone acetylation, and transcription fac- expressed in PGC tors) affect promoter activity. We predicted, by bioinfor- PGC are the ancestral cells of male germ cells16,17,and matics analysis, that the cloned C2EIP promoter fragment our research group has previously studied their migration, does not contain CpG islands but does contain a small colonization, and differentiation18. Bioinformatic analyses number of CG loci, suggesting that the C2EIP gene pro- revealed that C2EIP is located on the second chromosome moter activity is not likely to be affected by DNA and contains a 648-bp coding sequence (CDS) that encodes methylation. To verify this hypothesis, we linked the 215 amino acids with a relative molecular weight of 24.26 C2EIP promoter fragment into the pGL3-Basic vector to kD. C2EIP is a hydrophilic, non-secretory protein situated construct the pGL3-787 recombinant vector, which we mainly in the cytoplasm that does not include a signal transfected into DF-1 cells that we treated with or without peptide sequence or special structural domain. Previous 10 μmol/L DNA methylation inhibitor (5-Zacd). The cells RNA-seq results showed that expression of C2EIP (XM- were collected for the dual luciferase reporter assay, which 001233327.1, Chr2) is PGC-specific (Figs. 1a, b) (Supple- showed that the inhibition of methylation resulted in mentary Figure 1a, b)13. High-throughput sequencing significant upregulation (37.24 ± 3.24) of C2EIP promoter results showed that the expression of C2EIP in PGCwas activity compared to it without 5-Zacd treatment(23.41 ± significantly higher than that in ESC and SSC, in which 2.13), indicating that although the C2EIP promoter region there was little to no C2EIP expression (Fig. 1c). Western does not contain a CpG island, DNA methylation can be blotting and qRT-PCR showed that expression of C2EIP in mediated by the CG loci and affects promoter activity PGC was higher than in ESC and SSC (Figs. 1d, e), a finding (Fig. 2b). C2EIP gene expression was detected in cultured supported by in vitro retinoic acid (RA) induction experi- PGC after treatment with 5-Zacd, which significant ments19,20 that determined C2EIP expression was upregu- increased C2EIP expression, confirming the luciferase lated 5.5-fold compared to non-RA-induced cells (Fig. 1d). assayresults (Supplementary Figure 2b).While screening The stop codon of C2EIP was mutated, and a C2EIP- for other epigenetic factors affecting the activity of the EGFP-N1 fusion expression vector was constructed to C2EIP promoter, it was found that treatment of pGL3- confirm that C2EIP successfully encoded protein in DF-1 787-transfected DF-1 cells with10 μmol/L of the histone cells (Fig. 1f) (Supplementary Figure 1c). To further deacetylase inhibitor TSA significantly upregulatedC2EIP investigate the subcellular localization of C2EIP proteins, promoter activity (68.47 ± 3.11)compared to it without
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