LncCPSET1 Cooperates With Histone H3K4 Methylase MLL2/COMPASS To Promote The Formation Of Chicken PGCs

Primordial germ cells（PGCs）are the progenitor cells of sperm and oocytes,dedicated to passing on genetic information to offspring.There are similarities and differences in the migration process of PGCs in different species.PGCs eventually migrate through the dorsal mesentery into the gonad,while the approach to reach the dorsal mesentery varies among species.Unlike by active migration across the hindgut epithelium to the dorsal mesentery（in Drosophila and mice,etc.）,avian PGCs migrate passively to the dorsal mesentery through the vasculature.This unique migration route makes bird PGCs a good material for genetic resource conservation and preparation of bioreactors.Among them,chicken has become the best choice to study the formation and application of PGCs due to the advantages of low cost,high yield and easy operation.However,in practice,the limited number of PGCs in vivo results in a lower proportion of donor-derived offspring in germline chimeric chickens.Even reducing the number of acceptors PGCs cannot solve this problem,so it is urgent to find new ways to increase the number of PGCs.The method of simulating the specialization process of PGCs in vitro is beneficial to study the mechanism of its occurrence,increasing the number of PGCs from a genetic point of view.Key regulatory genes（Blimp1,Prdm14,and Sox17,etc.）,BMP and WNT signal transduction systems,and cytokines（RA,SCF,and bFGF,etc.）were found to determine the specialization of PGCs.With the deepening of researches,epigenetic reprogramming has also become an important factor affecting the growth and development of PGCs.However.The roles of a large number of non-coding RNAs occupying genomic positions and highly conserved histone modifications in the formation of chicken PGCs are still unclear,and the interaction mechanism between epigenetic modifications is also unknown.In this study,based on the positive regulatory effect of H3K4me2 on the formation of chicken PGCs found earlier,CHIP-seq was used to deeply analyze the enrichment pattern and transcriptional regulation pattern of H3K4me2 in PGCs.Combined with the previously discovered lncCPSET1（chicken-PGCs-specifically-expressed transcript 1,also known as lncBMP4）,we investigated the effect of the combination of lncRNA and H3K4me2 on the formation of chicken PGCs.At the same time,from the pre-transcriptional level,we tried to explore how H3K4me2 competes with DNA methylation to regulate the transcription of lncCPSETl;from the post-transcriptional level,we tried to explore how H3K4me2 cooperates with lncCPSET1 to regulate the transcription of key genes in PGCs.This study provides a theoretical basis for studying the mechanism of chicken PGCs from the perspective of epigenetics,improving the regulatory network of PGCs,and increasing the induction efficiency of PGCs in vitro.Furthermore,it provides a reference for studying the regulation of gene transcription by multiple epigenetic modifications.The research results are as follows:（1）H3K4me2 is necessary for the formation of chicken PGCs.Based on the previous study that H3K4me2 promotes the formation of chicken PGCs.Embryonic stem cells（ESCs）.PGCs and Spermatogonial stem cells（SSCs）were collected for H3K4me2 CHIP-seq sequencing.The analysis found that the peak of H3K4me2 was mainly enriched in ESCs and scattered in PGCs,then regained in SSCs.MACS analysis of the length of H3K4me2 binding to DNA sequences（width of peak peak）showed that the peak width in ESCs is mainly concentrated in 200～1000 kb,H3K4me2 is concentrated in 300～500 kb in PGCs,and peak is concentrated in 300～1000 kb in SSCs.Peak enrichment fold showed that ESCs had the largest number of peaks,and the enrichment fold was 5-10 times.The number of peaks and reads decreased in PGCs,and rebounded in SSCs.It suggested that H3K4me2 presents dynamic changes in three kinds of cells.The enrichment analysis of H3K4me2 in genome-widefunctional regions demonstrated that peaks in ESCs were concentrated in TSS±3kb,peaks in PGCs were enriched at both ends of TSS loci,tended to be evenly distributed in TSS±5kb,and SSCs were enriched in TSS±3kb region.The amount of H3K4me2 summits enrichment was less in PGCs than in ESCs and SSCs.The results of genomic region distribution showed that the enrichment of H3K4me2 in promoter region in PGCs was lower than that in ESCs and SSCs.Analysis of the enrichment changes of H3K4me2 in the promoter region showed that compared with ESCs,274 new peaks appeared,and 1094 peaks were lost in PGCs.Compared with PGCs,306 new peaks appeared and 214 were lost in SSCs.Among them,67.9%of H3K4me2-enriched genes showed peak loss in PGCs（compared to ESCs）,including Cvh,Blimp1,Lin28,C-kit,Alpl,Bmp4,Bmpr1b,Wnt5a,Tcf7l2 associated with PGCs,indicating that the dynamic expression of H3K4me2 is involved in cell lineage determination.Further in-depth analysis of the width of H3K4me2 enrichment illustrated that Narrow region（2001000 bp）enrichment mostly existed in PGCs,and differentially peaks were also more enriched in the narrow region.Venn analysis of genes in the narrow region and genes found by RNAseq showed that 151 genes（including Kit,Alpl,Cvh,Blimp1,Lin28,Bmp4,and Bmpr1b）were expressed in PGCs,with high RNA expression by RPKM analysis,GO and KEGG pathway analysis indicated that in the narrow region,genes were enriched in more signaling pathways,including 362 genes and 11 signaling pathways（Wnt,transforming growth factor-β,etc）.The BMP4 signal was enriched in H3K4me2 narrow region in PGCs.During the induction of PGCs-like,H3K4me2 activated the expression of key signaling molecules Bmp4,Bmpr1a,Bmpr2,Id2,Smad5 and C-kit in the BMP4 signaling pathway（P<0.01）by qRT-PCR.Therefore,Narrow H3K4me2 is an epigenetic factor necessary for the formation of PGCs by activating the expression of PGCs-specific genes and signaling pathways.（2）lncCPSET1 cooperates with H3K4me2 to regulate PGCs formation.15 differential lncRNAs related to germ cell differentiation were screened out by previous RNAseq sequencing analysis in ESCs,PGCs and SSCs.In this study,the lncRNA TCONS₀0874170 was defined as lncCPSET1（chicken PGCs specifically expressed transcript 1）.lncCPSET1 was specifically expressed in PGCs（P<0.05）by qRT-PCR.Homology alignment of transcripts using NONCODE suggested that lncCPSET1 is highly conserved in chicken.ESCs were transfected with histone methylase Mll2 interference vector and lncCPSET1 alone and in combination,respectively in vitro.The results showed that the number of embryoid bodies was reduced by interfering with Mll2/lncCPSET1 alone;qRTPCR supported that after interfering with lncCPSET1 and Mll2 at the same time,the expression of PGCs marker gene Blimp1 was significantly decreased（P<0.01）,and the germ layer marker genes Gata6 and Eomes were significantly decreased（P<0.05）.The results of flow cytometry and indirect immunofluorescence showed that the number of PGCs-like cells was inhibited.Conversely,simultaneous interference with histone demethylase Lsdl and overexpression of lncCPSET1 significantly increased the number of PGCs-like（P<0.05）.The lncCPSET1 interference vector and the Mll2 interference vector were injected into chicken embryos.The results of qRT-PCR showed that the expressions of reproductive marker genes Ddx4 and Blimp1 were significantly reduced（P<0.01）.Flow cytometry analysis showed that the number of PGCs was significantly decreased（P<0.05）.Conversely,simultaneously interfering with Lsdl and overexpressing lncCPSET1,the number of PGCs significantly increased in vivo（P<0.05）.The above results indicated that lncCPSET1 combined with H3K4me2 synergistically promoted the formation of PGCs in vitro and in vivo.（3）H3K4me2 cooperates transcription factor Jun to promote the transcription of lncCPSET1.In order to investigate the regulatory mode between H3K4me2 and lncCPSET1,this study firstly investigated the regulation of H3K4me2 during the transcription of lncCPSET1.Western blot showed that after interfering with Mll2 in PGCs,the level of H3K4me2 decreased;after interfering with Lsd1,the expression level of H3K4me2 increased.qRT-PCR results illustrated that interference with Mll2,the expression of lncCPSET1 was significantly inhibited（P<0.05）;on the contrary,interfering with Lsd1 significantly increased the level of lncCPSET1（P<0.05）.The pGL3-lncCPSET1 promoter dual-luciferase reporter recombinant vector and the Mll2/Lsd1 interference vector were co-transfected into DF1 cells.The detection of dual-luciferase activity showed that the activity of the lncCPSET1 promoter was significantly reduced by interfering with Mll2（P<0.01）,while lncCPSET1 promoter activity was significantly increased（P<0.01）with interference of Lsdl,indicating that H3K4me2 regulates lncCPSET1 transcription through Mll2/Lsd1.The lncCPSET1 promoter was further divided into four fragments,and CHIP-qPCR showed that H3K4me2 was extremely enriched in the lncCPSET1 promoter region（P<0.01）,and the enrichment in P1 fragment in PGCs was significantly higher than that in ESCs and SSCs（P<0.01）.After interfering with Mll2,the enrichment of H3K4me2 on the P1 fragment was significantly decreased（P<0.01）;while after interfering with Lsd1,the enrichment of H3K4me2 was significantly increased（P<0.05）.In order to identify other possible epigenetic modifications in the promoter region of lncCPSET1,bioinformatics analysis of the promoter region revealed that there is a CpG island with a length of 199 bp and 7 methylation sites in this region.The P1 fragment has both H3K4me2 enrichment and CpG islands.Bisulfite sequencing showed that DNA methylation on the P1 fragment was 77.1%in ESCs,67.1%in PGCs,and 87.1%in SSCs.The working concentration of DNA methylase inhibitor 5’aza was 10 μM by Edu assay.The DF1 cells and PGCs-like treated with 5’aza were collected.qRT-PCR showed that 5’aza significantly inhibited the expression of lncCPSET1（P<0.05）.To investigate how dual epigenetic modifications（DNA methylation and H3K4me2）regulate lncCPSET1 expression,transcription factors were introduced as observation objects.Transcription factor prediction was performed on the promoter P1 fragment of lncCPSET1 through the JASPAR online website,and 79 potential transcription factors were subjected to GO annotation and KEGG enrichment analysis,and three candidate transcription factors,JUN,TCF3 and CREB1,were screened.Overexpression vectors of three transcription factors were successfully constructed and their activity was verified.It was found that only JUN could significantly increase the activation activity and mRNA level of lncCPSET1 by dual-luciferase reporter system and qRT-PCR（P<0.01）.Furthermore,it was found by CHIP-qPCR that JUN could bind to the P1 fragment of the promoter region of lncCPSET1 in PGCs.qRT-PCR showed that overexpression of Jun also significantly increased the expression of lncCPSET1 in the in vitro induction system（P<0.01）.Then,dual luciferase reporter system and qRT-PCR indicated that the increase of H3K4me2 significantly enhanced the transcriptional regulation of lncCPSET1 by JUN with interference of Lsd1 and overexpression of Jun.CHIP-qPCR showed that the binding of Jun and lncCPSET1 promoter regions was significantly increased after Lsd1 interference.This indicated that H3K4me2 can promote transcription factor JUN to bind to the lncCPSET1 promoter to regulate its expression.However,qRT-PCR and dual-luciferase reporter vector detection showed that 5’aza significantly inhibited the positive transcriptional regulation of lncCPSET1 by JUN（P<0.01）.Furthermore,the reasons why low levels of DNA methylation inhibited the transcriptional effect of JUN were analyzed.qRT-PCR illustrated that although low DNA methylation significantly promoted the mRNA level of Jun（P<0.01）,it had no significant effect on the protein level of JUN;CHIP-qPCR showed that DNA methylation also did not affect the binding of JUN to the lncCPSET1 promoter region.To explore whether low DNA methylation affects the level of H3K4me2,Western blot and CHIPqPCR showed that low DNA methylation significantly reduced the level of H3K4me2（P<0.05）without affecting its enrichment in lncCPSET1 promoter fragment 1.Next,in order to investigate which epigenetic modification dominates the expression of lncCPSET1,DF1 and PGCs were simultaneously transfected with Lsd1 interference vector and 5’aza.Dualluciferase reporter and qRT-PCR indicated that lncCPSET1 transcript levels were significantly increased with the treatment of 5’aza and Lsd1 interference simultaneously（P<0.05）,accompanied by a significant increase in the enrichment of H3K4me2 and JUN in the lncCPSET1 promoter region（P<0.05）.On this basis,overexpression of Jun at the same time,dual luciferase reporter,qRT-PCR and CHIP-qPCR showed that the transcription of lncCPSET1 was significantly increased（P<0.01）.The above results indicated that compared with DNA methylation,H3K4me2 dominates activation of lncCPSET1 by the transcription factor JUN.（4）Screening of candidate key genes co-regulated by lncCPSET1 and H3K4me2.In view of the findings that lncCPSET1 and H3K4me2 synergistically regulate PGCs formation,this study further explored the target genes co-regulated by them.PGCs were collected after overexpressing of lncCPSET1.qRT-PCR showed that the expression of lncCPSET1 was significantly increased（P<0.01）.PGCs obtained from three independent experiments were pooled for transcriptome sequencing analysis.Volcano plots and MA plots showed that overexpression of lncCPSET1 resulted in a large number of differentially expressed genes.GO analysis of differential genes demonstrated that a large number of genes were concentrated in reproductive process,metabolic process,nuclear process and signal transduction process.COG analysis found that overexpression of lncCPSET1 affects transcription and translation processes.KEGG annotation analysis showed that differential genes were widely enriched inoxidative phosphorylation metabolism,P450 and ECM pathways,and were involved in signal transduction,signal molecule interaction,transcription,cell proliferation and apoptosis.Combined transcriptome sequencing and H3K4me2 CHIP-seq analysis,Venn analysis showed that there were 495 genes positively regulated by lncCPSET1 and differentially enriched with H3K4me2.GO annotation and KEGG enrichment analysis revealed that candidate genes were concentrated in biological processes such as JNK cascade regulation,cell fate determination,Wnt signaling,and ERK1/ERK2 cascade regulation.Molecular functional annotation found that candidate genes were enriched in RNA binding and DNA binding functions,and widely enriched in Wnt signaling and Tgf-β signaling pathways.The target genes co-regulated by lncCPSET1 and H3K4me2 were further screened.The differential genes in Wnt signal and Tgf-β were subjected by heat map analysis,and 4 genes related to cell differentiation were screened out in each pathway.Based on the prediction of histone methylase enrichment in the promoter region,combined with the results of qRT-PCR,Fzd2,Id1,Id4 and Bmp4 were finally determined as candidate genes for subsequent verification.（5）lncCPSET1 as MLL2/COMPASS scaffold regulates Bmp4 expression.In order to further explore the mechanism by which lncCPSET1 and H3K4me2 cooperatively regulate the expression of target genes,this study investigated the regulatory mode between lncCPSET1 and the histone methylase complex（MLL2/COMPASS）.Successfully constructed eukaryotic overexpression recombinant tag vectors pcDNA3.1-Ash21-FLAG,pcDNA3.1-Dpy30-HIS,pcDNA3.1-Wdr5-GST,and pcDNA3.1-Rbbp5-MYC for histone methylase COMPASS（ASH2L,DPY30,WDR5 and RBBP5）.The expression of mRNA and tagged protein was verified by qRT-PCR and Western blot.In order to confirm that the four histone methylases exist in the form of complexes in chicken,Co-IP was conducted and revealed that ASH2L,DPY30 and WDR5 can bind to each other,while the binding between RBBP5 and DPY30 and WDR5 is weak.qRT-PCR showed that after overexpression of the four histone methylases,only Ash2l and significantly increased the expression of Bmp4（P<0.05）.After overexpression of lncCPSET1,qRT-PCR demonstrated that the expression of Fzd2,Id1,Id4 and Bmp4 in PGCs-like and PGCs were significantly increased（P<0.01）.Therefore,Bmp4 was identified as a target gene regulated by lncCPSET1 and MLL2/COMPASS.In order to study the regulation mode between lncCPSET1 and histone methylases,we first overexpressed lncCPSET1,qRT-PCR showed that the expression of Dpy30 was significantly increased（P<0.05）.Co-IP suggested that neither interference or overexpression of lncCPSET1 affected the binding between complexes,but interference with lncCPSET1 inhibited the protein expression of DPY30.Furthermore,RIP experiments revealed that lncCPSET1 could bind to DPY30.CHIP-qPCR showed that DPY30 was enriched in the Bmp4 promoter.The above results indicated that lncCPSET1 could regulate Bmp4 expression by binding with DPY30-mediated histone methylase complex to target the promoter.In conclusion,H3K4me2 is enriched in the promoter region through the Narrow region（200-1000 bp）in PGCs,which promotes the activation of JUN in the promoter to lncCPSET1 expression;after transcription,lncCPSET1 binds to DPY30 to target promoter of Bmp4.By this way,lncCPSET1 and H3K4me2 synergistically promote the formation of PGCs in vitro and in vivo.