Long Noncoding RNA LncPGCR mediated by TCF7L2 regulates chicken PGCs formation

Background: Several of the thousands of long noncoding RNAs (lncRNAs) have been functionally characterized, yet its specic function and molecular mechanism in the formation of chicken primordial germ cells (PGCs) remains poorly understood. In the present study, we aim to investigate the role of LncPGCR (LncRNA PGCs Regulator) in PGCs formation. Methods: LncPGCR, Cvh, Nanog, C-kit, gga-mir-6577-5p and Btrc expressions were detected by qRT-PCR. The percentage of PGCs cells was detected by ow cytometry, immunocytochemistry, and PAS staining. The interaction of histone acetylation, DNA methylation, transcription factor TCF7L2, and LncPGCR was conrmed by Luciferase reporter assay. The interaction of GAPDH and LncPGCR was measured by RNA pull-down, RIP, and Western blot. Results: We observed the increased expression of LncPGCR in PGCs. It is mainly expressed in the cytoplasm and encodes small peptides. Moreover, over-expression of LncPGCR could promote PGCs formation in vitro and in vivo. Besides, we rst reported that histone acetylation, DNA methylation, and transcription factor TCF7L2 can regulate LncPGCR expression in PGCs. In addition, LncPGCR activates WNT signaling pathways to promote PGCs formation by adsorbing gga-mir-6577-5p, relieving its inhibitory effect on target gene Btrc. Meanwhile, LncPGCR contributed to PGCs formation by increasing the phosphorylation level of GAPDH to activate the TGF-β signal pathway. Conclusion: LncPGCR over-expression promoted ESCs differentiation into PGCs through the potential LncPGCR/miR-6577-5p/BTRC pathway or increasing the phosphorylation level of GAPDH to activate the TGF-β signal pathway.


Background
Primordial germ cells (PGCs) are primordial progenitor cells of germ cells and play a widespread role in the research of the preparation of gonadal chimeras, production of transgenic animals, and genomic imprinting. Moreover, PGC's multidirectional differentiation potential and unique regeneration ability make it one of the most promising seed cells in clinical medicine and tissue engineering research. However, the system for inducing PGCs in vitro is still immature. Due to its complex induction system and low induction e ciency, it is impossible to obtain su cient cells for scienti c research. Over the past decades, mounting evidences have emphasized that the induction of paracrine signaling (Saitou, Barton et al. 2002, Zhao andGarbers 2002), inhibition of somatic fate (Ohinata, Payer et al. 2005), alteration of epigenetic marks (Ancelin, Lange et al. 2006), and maintenance of pluripotency (Kehler, Tolkunova et al. 2004) are important for the formation of PGCs. However, the induction e ciency of PGCs in vitro has not been substantially improved, so we must conduct innovative exploration in the unknown eld to obtain su cient PGCs.
Current evidences demonstrate that LncRNA can participate in cell differentiation by interacting with microRNA(Cesana, Cacchiarelli et al. 2011), recruiting proteins as scaffolds to form complexes (Tsai, Manor et al. 2010), and trapping transcription factors (Guttman, Garber et al. 2010, Mohamed, Gaughwin et al. 2010). Nevertheless, the detail molecular mechanism of LncRNA during ESCs differentiation into PGCs is still sustain unclear. With the availability and rapid development of RNA-seq analysis technology, thousands of LncRNAs have been identi ed in the chicken genome in the past few years (Bao, Wu et al. 2013), and the function of LncRNA in embryonic development and germ cell differentiation has been preliminarily understood, but the speci c regulatory mechanism has not been elucidated.
In this study, LncRNA expression patterns of chicken ESCs, PGCs, and SSCs were detected from singlecell levels by RNA-seq. PGCs-speci c LncRNAs (TCONS_00948124) were identi ed and named LncPGCR (LncRNA PGCs Regulator). LncPGCR is activated by the transcription factor TCF7L2, histone acetylation, and DNA methylation during the formation of PGCs. Moreover, LncPGCR was also shown to regulate the gga-mir-6577-5p targeted gene BTRC by functioning as a competitive endogenous RNA (ceRNA) for ggamir-6577-5p, thereby promoting the expression of BTRC and PGCs formation. At the same time, LncPGCR can interact with GAPDH to activate the TGF-β signal to regulate the formation of PGCs. Here we systematically studied the roles and molecular mechanisms of LncPGCR in PGCs formation.

Isolation and culture of ESCs, PGCs, and SSCs
The study employed freshly fertilized eggs from Rugao yellow chickens, provided by the Poultry Research Institute of the Chinese Academy of Agricultural Sciences. ESCs were derived from 0-d-old chicken embryos, PGCs from the genital ridge of 4.5-d-old embryos, and SSCs were from testes of 18.5-d-old embryos. Hatching conditions were 37.5°C and 65% relative humidity. For speci c separation and cultivation, refer to Zhang's article ).

Isolation of nuclear and cytoplasmic RNA
Operations were performed according to the manufacturer's protocols of PARIS™ system Protein and RNA Isolation System; add 500 μL Cell Disruption Buffer to every 10 7 PGCs cells, and incubate on ice for 10 mins; After centrifugation at 500 ×g for 5 mins at 4℃, the supernatant is cytoplasm and the precipitate is the nucleus. After the sample is dissolved, RNA extraction is performed.

Preparation of LncPGCR constructs
Three shRNA knockdown target sites were designed and combined to the linear vector pGMLV-SC5. shRNA target sites sequence is shown in Supplementary Table.4. The constructed knockdown vector was transfected with DF1 for activity veri cation, and the most active vector was encapsulated with lentivirus; A pair of primers was designed to amplify the full-length CDS region of LncPGCR. The fragment was cloned into pcDNA3.0 plasmid digested with KpnI and EcoRI to construct an overexpression vector and verify its activity. PCR primers are shown in Supplementary Table.6.

Chicken embryo vascular injection
Under sterile conditions, use tweezers to open a round hole with a diameter of 1-1.5cm in the blunt end of the early embryo eggs (13-17HH) incubated for 48-58h. Find the position of the embryo under a stereomicroscope. Then, using a micropipette, ll the lentiviral vector with a nal polybrene concentration of 8ng / µL or the pcDNA 3.0-PGCR vector wrapped with PEI into glass injection needles and inject them into the blood vessels of chicken embryos; then add 20µL of Streptomyces penicillin vaccinate to the injection site, and nally cross-sealed with medical tape to continue hatching; Chicken embryos were collected at 4.5d and observe the embryos under a stereo uorescence microscope.

Quantitative Real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol (TIANGEN, Beijing, China) and reverse-transcribed into cDNA with the Quantscript RT Kit (TIANGEN, Beijing, China). Gene expression was determined using an ABI PRISM 7500 uorescent quantitative PCR instrument (Applied Biosystems, Carlsbad, California). qRT-PCR primers are shown in Supplementary Table 5, the internal reference gene: β-actin, the number of repetitions: n = 3.

Immunocytochemistry (ICC)
6d ESCs in each group were xed with 4% paraformaldehyde for 30 minutes, and then treated with 0.5% TritonX-100 for 15 minutes. After blocking for 2 hours in the dark, and then added with Cvh antibody.
After incubating at 37 ℃ for 2 hours, overnight at 4℃; Then adding anti-mouse IgG, incubate at 37 ℃ in the dark for 2 hours; Next, it was stained with 5ng / µL DAPI for 15min, and nally, slides were plated with glycerin(50% glycerol, 50% PBS), and the image was sealed. FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) was used to observed the samples.

Flow cytometric
For ow cytometry analysis, the cultures were mildly trypsinized and harvested from 24 well plates. The cells were washed and resuspended in PBS buffer, then add Cvh antibody for labeling, incubate at 4℃ overnight or incubate at 37℃ for 1-2h; Finally, data were analyzed on a Flow cytometer (Becton Dickinson, San Jose, CA) with the Flowjo program.

PAS staining
The para n sections after dewaxing and rehydration were stained according to the PAS / Gycogen Stain kit (D004-1-2) of Nanjing Jiancheng Technology Co., Ltd.

RNA pull-down
Use Ambion's in vitro transcription kit, Maxiscript T7, for in vitro transcription, and perform RNA pull-down experiments according to Thermo's kit instructions to adsorb proteins that interact with LncPGCR; The target proteins were separated on PAGE by vertical electrophoresis and then subjected to silver staining experiments. Design the sense and antisense PCR primers based on the LncPGCR sequence(Supplementary Table 6).

RNA immunoprecipitation (RIP) assays
RIP assays were performed using a GAPDH Anti-Phosphoserine (SPM101, Abcam), and operations were performed according to the manufacturer's protocols of RiboCluster Pro lerTM (RN1001, MBL)RIP-Assay kit. Western Blot was used to detect the expression of the phosphorylation level of GAPDH on the extracted cellular protein samples after RNA enrichment.

Data analysis
Relative gene expression was calculated using the 2 −ΔΔCt method after PCR. All experiments were performed in triplicate, and the data are expressed as mean ± standard error. Signi cant differences between the groups were determined with two-sample t-tests in SPSS 18.0. (*, P<0.05, signi cant difference.**, P<0.01, extremely signi cant difference).GraphPad Prism7 software was used for mapping.

Results
3.1 LncPGCR, a PGCs speci c LncRNA in Chickens, mainly expressed in the cytoplasm We analyzed RNA-seq data for lncRNAs from chicken ESCs, PGCs, and SSCs. Results showed that more than 75% of DELs were enriched in biological processes ( Figure 1A). RT-PCR revealed that the expression of PGCs speci cally expressed LncRNA was consistent with the sequencing results, indicating that the sequencing results were accurate and available(Supplementary Figure 1A). GO and KEGG enrichment analysis PGCs speci cally expressed lncRNA(Supplementary Figure 1B,C), and excavated lncRNAs that are simultaneously enriched in the differentiation of ESCs into germ cells and germ cells differentiationrelated signaling pathways. We noticed that LncRNA (TCONS_00948124) is speci cally expressed in PGCs (Table 1). Then, we assessed LncPGCR expression in chicken tissues.Results showed that LncRNA was signi cantly higher in chicken gonads than in other tissues (Supplementary Figure 1D). These data demonstrated high LncPGCR expression was closed related to the development of PGCs, so we named it LncPGCR (LncRNA PGCs Regulator).
LncRNA exhibits distinct molecular mechanisms and functions due to their different expression positions. We found that LncPGCR was expressed in both the nucleus and the cytoplasm of the PGCs, but the expression level in the cytoplasm was signi cantly higher than that in the nucleus, which indicated that the mechanism of LncPGCR action mainly existed post-transcriptional level ( Figure 1B). There are four ORFs in the body region of LncPGCR, of which ORF-2 encodes a small peptide, which may be involved in the formation of PGCs in this way ( Figure 1C). These results indicate that LncPGCR is a gene that is highly expressed in the cytoplasm of PGCs and encodes a protein.

LncPGCR promotes ESCs differentiation into PGCs in vitro
Then, we investigated the role of LncPGCR on PGCs growth. We transfected the LncPGCR overexpression/knockdown vectors based on the RA induction model and observed the morphology of ESCs cells in each group on the 2nd and 4th days. We noticed that in the normal induction model, the cells started to appear Embryoid Body (EB) on the 2nd day, and the EB began to increase and grow on the 4th day. After over-expression of LncPGCR, large EB appeared on the 2nd day, and small gaps began to appear on the edge of the embryoid body of type 4d. However, knockdown with LncPGCR delayed the appearance of EB and did not appear on the 4th day ( Figure 2A). Meanwhile, the expression of Nanog decreased signi cantly after over-expression of LncPGCR (P <0.05), while the expression of cvh and c-kit increased signi cantly (P <0.01); Reciprocally, knockdown of LncPGCR decreased cvh and c-kit expression and increased the expression of Nanog( Figure 2B). Also, immunocytochemical and ow cytometry analysis con rmed that LncPGCR can promote the formation of PGCs ( Figure 2C, D). These data demonstrated that LncPGCR can promote the formation and development of PGCs by inhibiting cell totipotence.

LncPGCR promotes ESCs differentiation to PGCs in vivo
To further verify the function of LncPGCR in the formation of PGC, we injected the PGCR-sh2 lentiviral vector and pcDNA3.0-PGCR vector into the embryonic blood vessels after 2.5 days of chicken embryo hatching and continued to hatch. Collecting chicken embryos from the knockdown group when it hatched to 4.5d found that it could excite green uorescence, indicating that the injected knockdown vector could effectively integrate into the chicken embryo genome ( Figure 3A). The formation of PGCs in the genital ridge was signi cantly reduced after knockdown LncPGCR through para n section observation ( Figure  3B). At the same time, qRT-PCR showed that after knocking down LncPGCR, the expression of Nanog in the genital ridge of chicken embryos at 4.5d increased signi cantly (P<0.01), while the expression of cvh and c-kit decreased signi cantly (P<0.01) ( Figure 3C); ow cytometry analysis con rmed that the formation of PGCs was suppressed after knockdown LncPGCR (1% ± 0.19) (Figure 3D), which was consistent with the results of induction in vitro. In summary, LncPGCR has a positive regulatory effect on the formation of chicken PGCs.

Epigenetic factors regulate differential expression of LncPGCR
LncPGCR played a vital role in the formation of PGC. Therefore, we set out to determine the regulators of LncPGCR. Firstly, we cloned the promoter of LncPGCR and replaced the CMV promoter of pEGFP-N1 ( Figure 4A), and found that it had promoter activity after transfection into DF-1 (Supplementary Figure  2A); Dual-luciferase assay revealed that the -1033 ~ -661bp region was the core active region of the LncPGCR promoter( Figure 4B).
To further explore the transcriptional regulation mechanism of the LncPGCR, we rst used The JASPAR database to predict the -1033 ~ -661bp region and found that there is a binding site for the transcription factor TCF7L2 ( Figure 4C). The dual-luciferase assay revealed that the TCF7L2 binding site mutation signi cantly reduced the LncPGCR promoter activity (P<0.01), indicating that the transcription factor TCF7L2 is a positive regulator of the LncPGCR promoter ( Figure 4D), and the expression of TCF7L2 was up-regulated during the induction of ESCs into PGCs in vitro ( Figure 4E), suggesting that TCF7L2 is a key factor for the speci c expression of LncPGCR in PGCs.
Epigenetics is essential for regulating gene transcription (Sewack, Ellis et al. 2001, Takeshima, Wakabayashi et al. 2014). Deacetylation and methylation of chromatin could silence the expression of genes. Dual-luciferase assay veri ed that 5-Azadc and TSA can signi cantly increase the LncPGCR promoter activity(P<0.01)( Figure 4F). Collectively, these data demonstrate that the speci c expression of LncPGCR in PGCs is regulated by DNA methylation, histone acetylation, and the transcription factor TCF7L2.
3.5 LncPGCR facilitates the formation of PGCs partially through sponging gga-mir-6577-5p, and then upregulating Btrc Bioinformatics analysis of the sequence of LncPGCR found its target gene Btrc. The interaction between miRNA and LncRNA is a classic regulation mode, which is widely involved in various life activities. We further identi ed 3 miRNAs with common binding targets between LncPGCR and Btrc ( Table 2). Based on their binding site information and bioinformatics scores, we chose gga-mir-6577-5p for veri cation. qRT-PCR showed that LncPGCR can promote the expression of Btrc, while gga-mir-6577-5p can inhibit its expression ( Figure 5A, B), con rming that Btrc is a common target gene of LncPGCR and gga-mir-6577-5p.
We noticed that gga-mir-6577-5p also inhibited the expression of LncPGCR ( Figure 5C), suggesting that there is a potential CeRNA regulation mode among LncPGCR, gga-mir-6577-5p, and Btrc. Strikingly, when overexpressed LncPGCR in systems gga-mir-6577-5p inhibit Btrc expression, we found that Btrc expression rose back ( Figure 5D). These results indicated that LncPGCR can promote the formation of PGCs by competitively adsorbing gga-mir-6577-5p to release Btrc.

The complex of LncPGCR-GAPDH activates TGF-β signaling to promote the formation of PGCs
Interactions between RNA and protein is one of the decisive factors for the realization of cellular physiological processes (Lukong, Chang et al. 2008, Castello, Fischer et al. 2013. To nd proteins that interact with LncPGCR, we screened 23 protein interaction pairs through RNA pull-down combined with LC-MS / MS mass spectrometry, including GAPDH, KRT5, KRT19, LOC776816, etc. Among them, the interaction proteins GAPDH attracted our attention (Table 3, Figure 6A). Researches report that GAPDH can negatively regulate PKM2 to affect TGF-β signaling pathway transduction (Hjerpe, Brage et al. 2013, Yanling 2014). Moreover, our previous studies have also con rmed that the TGF-β signaling pathway can promote the formation of PGCs (Zuo, Jin et al.). Then the question arises: what role does LncPGCR play in the transduction of the TGF-β signaling pathway? In this study, RIP and Western Blot were performed, and GAPDH protein phosphorylation was inhibited after knockdown LncPGCR ( Figure 6B). These results indicate that LncPGCR can induce the phosphorylation of GAPDH to activate the TGF-β signal to regulate the formation of PGCs.

Discussion
In this study, we identi ed a key LncRNA (LncPGCR) in promoting ESCs differentiate into PGCs. Here, we showed that LncPGCR was highly expressed in gonads tissues, and higher LncPGCR expression was closed associated with PGCs formation. We also con rmed that LncPGCR can promote PGC formation in vivo and in vitro. Further, our data also imply that LncPGCR' roles in promoting PGCs formation are through sponging gga-mir-6577-5p, and then activating Btrc. Besides, LncPGCR can regulate the phosphorylation level of GAPDH protein and activate TGF-β signal to regulate the formation of PGCs.
Epigenetic modi cation can regulate differential expression of LncPGCR. Previous studies have shown that histone acetylation can change the structure of nucleosomes, keep the chromatin conformation open, and promote the binding of transcription factors to chromosomal DNA, which is conducive to gene transcription and expression. And DNA methylation can inhibit gene expression by changing chromatin structure (Ng and Bird 1999). Here, we nd that reducing the level of DNA methylation or increasing the level of histone acetylation can promote the transcription of LncPGCR, which is consistent with previous studies. What is interesting to us is that the transcription factor TCF7L2 can target the regulation of LncPGCR expression. Transcription Factor 7-like-2 TCF7L2 , also known as T-cell transcription factor 4 (TCF-4), is a transcription factor containing a DNA-binding domain. Previous studies have shown that TCF7L2 is an important component of the Wnt signaling pathway (Kwak, Cho et al.). Moreover, our previous studies have veri ed that Wnt signaling pathways are involved in regulating the differentiation of ESCs to SSCs . It was also found that TCF7L2 can affect the formation of PGCs as a key transcription factor downstream of the Wnt signaling pathway(data not published), suggesting that LncPGCR is regulated by WNT signals to participate in the formation of PGCs.
LncRNA can be used as a molecular sponge of endogenous miRNA, and its molecular regulatory mechanism through binding to miRNA is the ceRNA mechanism (Sun, Nie et al. 2016), this mechanism has received the attention of academic circles since it was proposed and it represents a new gene expression regulation model. Wang found that linc-ROR can regulate human ESC maintenance and differentiation, and subsequent sequence and function analysis found that because linc-ROR shared miRNA response elements with core transcription factors such as Oct4, Sox2, and Nanog, Linc-ROR acts as a sponge during ESC cell differentiation, preventing these core transcription factors from being miRNAmediated inhibition (Wang, Xu et al.). Here, we found that LncPGCR showed similar functions, which could adsorb gga-mir-6577-5p, improve Btrc expression during PGCs formation.
Mostly lncRNAs function by interacting with corresponding RNA-binding proteins, such as lncRNA TERRA through interaction with hnRNPA1 to achieve cell immortality(Oliva-Rico and Herrera 2017). In our study, we found that LncPGCR can effectively regulate the phosphorylation level of GAPDH protein. Furthermore, it was found that GAPDH can negatively regulate PKM2 to affect the transduction of the TGF-β signaling pathway, and the TGF-β signaling pathway can promote the differentiation of ESCs to PGCs (Zuo, Jin et al.). These ndings suggest that LncPGCR can participate in the development of PGCs by regulating the TGF-β signaling pathway. The above results indicate that LncPGCR may exert various molecular mechanisms and play a key role in PGCs development.

Conclusion
In summary, LncPGCR acts as an adsorption sponge to release the inhibitory effect of gga-mir-6577-5p on BTRC. At the same time, the interaction between LncPGCR and its recruited protein GAPDH activates the TGF-β signal and promotes PGCs production. The results of this study elaborated on the function and molecular mechanism of LncPGCR in the formation of PGCs and provided a theoretical basis for further improving the molecular network of LncRNA regulating PGCs formation.