【作者】 张明；

【导师】 李碧春； 王克华；

【作者基本信息】 扬州大学 ， 农业硕士（专业学位）， 2021， 硕士

【摘要】 原始生殖细胞(Primordial Germ Cells,PGCs)是配子的前体,精子和卵子的祖细胞,负责代与代之间传递遗传和表观遗传信息,确保物种的延续和生存,并且在发育生物学研究、物种保护以及养殖生产领域有着极其重要的意义。鸟类的PGCs因为其独特的发育特点使其成为保护濒危动物和种质资源恢复的有力工具,然而由于体内分离的PGCs数量有限,诱导体系不成熟使其难以大量获取,限制了其应用。其中的关键问题在于影响PGCs发生具体因素仍不明朗,因此科学家迫切需要剖析和阐明PGCs形成过程中的分子调控机制。近年来,研究者们已经发现了 PGCs发生过程中的众多参与调控的基因、细胞因子、信号通路和表观遗传修饰等因素,本课题组前期研究中已经确定BMP4是鸡PGCs形成中的关键基因,并且全基因组存在DNA和H3K4me2的动态变化,但是其中的具体分子调控机制尚不清楚。而随着基因组学的深入研究,人们愈发认识到基因表达模式中存在多种表观遗传现象同时相互作用从而和其他调控元件构成复杂而有序的遗传分子图谱。所以深入探索DNA和H3K4me2这两种不同表观遗传因素对鸡PGCs形成中的关键基因BMP4的影响和理清这些因素与基因的调控元件互作的模式机制有利于人们更加系统全面地认识鸡PGCs的形成机理。为此,本研究根据鸡生殖细胞上的DNA和组蛋白甲基化的动态变化推测表观遗传因素能够调控关键基因参与PGCs的形成,故首先验证DNA甲基化对PGCs形成的影响并且基于课题组前期在鸡胚胎干细胞(Embryonic Stem Cells,ESCs)、和PGCs上H3K4me2的ChIP-seq以及DNA甲基化测序MBD-seq结果筛选确定PGCs形成中的关键基因—BMP4,利用dCas9系统验证DNA甲基化对BMP4的靶向调控作用,进而探索DNA甲基化、H3K4me2和转录因子如何共同调控BMP4转录参与PGCs形成的机制。该研究为阐明PGCs发生过程的表观遗传通路,完善优化PGCs诱导形成体系,推广PGCs的研究应用提供思路和依据。研究结果如下:(1)DNA甲基化调控PGCs的形成通过前期ESCs和PGCs中转录组数据分析DNA甲基化修饰酶的表达变化,确定Dnmt3A和Tet1是影响DNA甲基化的关键修饰酶;分别构建了Dnmt3A和Tet1的Dnmt3A-shRNA1、Dnmt3A-shRNA2和Dnmt3A-shRNA3,以及Tet1-shRNA1、Tet1-shRNA2 和Tet1-shRNA3 干扰载体,转染 DF1 细胞qRT-PCR分析Dnmt3A-shRNA1和Tet1-shRNA3干扰活性最佳(P<0.01),选择作为用于后续验证实验。体内外验证DNA甲基化对PGCs形成的影响,体外实验中干扰Dnmt3A组第4d出现大量类胚体聚团,6d出现大量即将细胞破裂的类胚体,而干扰Tet1第4d和6d都未出现类胚体;Dot-blot检测发现干扰Dnmt3A组甲基化水平显著降低(P<0.05),干扰了 Tet1后DNA甲基化水平小幅上升;qRT-PCR检测干扰Dnmt3A组6d后Cvh、C-kit、BMP4、Blimp1和Lin28a表达显著上升(P<0.05),而干扰了Tet1后各基因组出现显著下调(P<0.05);而流式细胞分析发现干扰Dnmt3A后PGCs形成率显著提升(P<0.05),干扰Tet1后的PGCs形成率显著下调。体内实验中qRT-PCR检测发现干扰Dnmt3A组BMP4、Cvh和C-kit的表达都显著提高(P<0.05),Blimp1和Lin28a的表达极显著上调(P<0.01),干扰Tet1后Cvh、C-kit、BMP4、Blimp1和Lin28a都发生了极显著下调(P<0.01);流式细胞仪分析发现干扰Dnmt3A后PGCs形成效率显著提升(P<0.05),干扰Tet1后PGCs形成效率显著下调(P<0.05)。以上结果表明DNA甲基化抑制PGCs的形成,DNA去甲基化促进PGCs的形成。(2)PGCs形成中DNA甲基化和H3K4me2参与调控BMP4基因对ESCs和PGCs的ChIP-seq以及MBD-seq结果进行联合分析,筛选得到PGCs形成过程中受DNA甲基化和H3K4me2调控的TGF-β通路中关键基因BMP4并作为靶基因研究对象。成功构建pBMP4-EGFP-N1载体,验证具有启动活性;成功构建BMP4不同长度缺失片段启动子-basic载体,双荧光素酶报告系统验证pGL3-613极显著高于pGL3-508和pGL3-281(P<0.01),说明508-613bp为BMP4的核心启动子区。生物信息学方法预测BMP4核心启动子区的CpG岛并通过重亚硫酸盐测序的方法比较在CEFs、ESCs和PGCs中DNA甲基化水平,结果发现ESCs中BMP4启动子区平均DNA甲基化水平显著高于PGCs(P<0.05);基于组蛋白H3K4me2的ChIP-qPCR发现PGCs中BMP4启动子上H3K4me2富集水平显著高于ESCs(P<0.05),并且主要集中于核心启动子区;进一步通过qRT-PCR检测发现PGCs中BMP4和Smad1显著高于ESCs(P<0.05),Bmpr1、Bmpr2和C-kit则极显著高于ESCs(P<0.01);通过Western Blot检测BMP4信号下游磷酸化p-Smad1/3/5复合蛋白在PGCs中显著高于ESCs(P<0.05)。以上结果说明PGCs形成关键基因BMP4在ESCs和PGCs表达上升伴随着启动子区DNA甲基化水平下降和H3K4me2的富集增多。(3)CRISPR-dCas9介导的TET1靶向BMP4启动子DNA去甲基化促进PGCs形成针对BMP4启动子区CpG岛分别设计三对sgRNA序列,连接至pgRNA-humanized载体重组构建成功sgRNA1、sgRNA2和sgRNA3引导载体,与dCas9-TET1CD分别组合转染DF1细胞检测激活BMP4效果,经过qRT-PCR检测发现sgRNA和dCas9-TET1CD载体的组合都能极显著增强BMP4表达(P<0.01),当sgRNA1、2和3混合与dCas9-TET1CD组合共转时BMP4表达达到最高(3.473±0.187),说明多个sgRNA靶定同一个基因可以达到最佳激活效果。在体外ESCs诱导过程中sgRNA1-3和dCas9-TET1CD共转后通过重亚硫酸盐转化测序发现BMP4启动子区甲基化水平显著下降(P<0.05),而通过qRT-PCR检测发现BMP4表达极显著升高(P<0.01),下游信号基因Bmpr1、Smad1表达显著升高(P<0.05),PGCs的标记基因Blimp1和Lin28a表达量也显著升高(P<0.05),而Cvh和C-kit的上调更为显著(P<0.01),而Dnmt3A和Tet1表达无明显差异;Western Blot检测BMP4下游磷酸化p-Smad1/3/5蛋白表达显著上升(P<0.05);流式细胞仪分析检测PGCs的形成率显著提高(P<0.05)。体内0d尿囊腔注射sgRNA1-3和dCas9-TET1CD后通过qRT-PCR检测BMP4和PGCs形成中的标记基因Cvh、C-kit、Blimp1以及Lin28a表达都显著升高(P<0.05),Western Blot检测p-Smad1/3/5蛋白显著增加(P<0.05),流式分析检测发现注射sgRNA1-3和dCas9-TET1CD组的PGCs形成效率显著升高(P<0.05)。以上结果说明靶向BMP4启动子区DNA去甲基化可以增加BMP4表达,促进PGCs的形成。(4)DNA甲基化和组蛋白甲基化拮抗调节BMP4信号参与PGCs的形成通过生物信息学预测BMP4启动子区潜在转录因子LIN54、ZEB1和NFAT5,并通过构建缺失结合位点的核心启动片段连接Basic载体,转染DF1细胞后通过双荧光素酶报告系统检测发现只有在缺失了 ZEB1的结合位点之后启动活性极显著降低(P<0.01),说明ZEB1是BMP4核心启动子区域的关键转录因子。构建Zeb1的过表达载体之后转染DF1细胞,qRT-PCR检测过表达Zeb1后的Zeb1和BMP4表达量显著上升(P<0.05),并且Zeb1在体内0-6d的过程中表达逐渐升高;而oe-Zeb1与核心启动片段basic载体共转DF1细胞,双荧光素酶检测BMP4核心启动子片段的启动活性显著升高(P<0.05),以上结果表明Zeb1作为BMP4转录因子可促进其转录表达。成功构建Dnmt3A和Tet1的过表达载体,与组蛋白去甲基化酶LSD1过表达载体一起共转染DF1细胞后,48h后双荧光素酶报告系统检测发现启动活性极显著下调(P<0.01),接着转染Tet1和Zeb1的过表达载体96h后再次检测启动活性发生了显著上调(P<0.05)。结果说明DNA甲基化和H3K4去甲基化能够抑制BMP4转录活性,而在DNA去甲基化和转录因子Zeb1能够挽救和增强BMP4的转录活性。ChIP-qPCR检测PGCs细胞发现ZEB1和H3K4me2在BMP4启动子的核心区域都存在富集,干扰了 Dnmt3A时,H3K4me2的富集水平和ZEB1的结合都发生了显著的增加(P<0.05),CpG岛的DNA甲基化水平显著下降(P<0.05);而在干扰了组蛋白甲基化酶MLL2后BMP4启动子区的H3K4me2的富集水平和ZEB1的结合都发生了显著的减少(P<0.05),DNA甲基化水平发生了显著上升(P<0.05)。结果表明DNA去甲基化可以增加BMP4的核心启动子区里H3K4me2的富集和转录因子ZEB1的结合,而H3K4me2去甲基化能够相应的减少H3K4me2和ZEB1的富集和结合,并且DNA甲基化和H3K4me2的富集水平呈拮抗关系,为下一步深入探索表观遗传通路在PGCs形成的具体机制奠定了良好基础。更多还原

【Abstract】 Primordial Germ Cells(PGCs)are the precursors of gametes,the progenitor cells of sperm and eggs,responsible for the transmission of genetic and epigenetic information between generations,ensuring the continuity and survival of species,and in developmental biology,the field of species protection and cultivation and aquaculture production has extremely important research significance.Because of their unique developmental characteristics,bird PGCs have become a powerful genetic engineering tool for protecting endangered animals and transplanting in vitro.However,due to the limited number of PGCs isolated in vivo,the immature induction system makes it difficult to obtain in large quantities,limiting its application.The key issue is that the specific factors affecting the occurrence of PGCs are still unclear.Therefore,scientists urgently need to analyze and clarify the molecular regulation mechanism in the formation of PGCs.In recent years,researchers have discovered many genes,factors,signal pathways,and epigenetic modifications involved in the process of PGCs.The previous research of this group has determined that BMP4 genes are PGCs and H3K4me2 is in the formation of PGCs.With the in-depth study of genomics,people have increasingly realized that there are multiple epigenetic phenomena in gene expression patterns,while interactions and other regulatory elements constitute a complex and orderly genetic molecular map.Therefore,in-depth exploration of the influence of different epigenetic factors on the key genes in the formation of PGCs and a clear understanding of the mode of interaction between these factors and gene regulatory elements are conducive to a more systematic and comprehensive understanding of the formation mechanism of PGCs.Therefore,this study first verified the influence of DNA methylation on the formation of PGCs and was based on the ChIP-seq and DNA methylation sequencing MBD-seq of H3K4me2 on chicken embryonic stem cells(ESCs)and PGCs in the early stage of the research group.The results screened the key gene BMP4 in the formation of PGCs,and used the dCas9 system to verify the targeted regulation of DNA methylation on BMP4,and then explored how DNA methylation,H3K4me2 and transcription factors jointly regulate BMP4 transcription and participate in the mechanism of PGCs.This study provides ideas and basis for elucidating the epigenetic pathways of PGCs,perfecting and optimizing the formation of PGCs,and promoting the research and application of PGCs.The results of the study are as follows:(1)DNA methylation regulates the formation of PGCs.Dnmt3A-shRNAl,Dnmt3A-shRNA2 and Dnmt3A-shRNA3,as well as Tet1-shRNA1,Tet1-shRNA2 and Tet1-shRNA3 interference vectors were constructed,respectively,and transfected into DF1 cells for qRT-PCR analysis of Dnmt3A-shRNAl and Tet1-shRNA3 have the best interference activity(P<0.01),and they are selected for subsequent verification experiments.The effect of DNA methylation on the formation of PGCs was verified in vivo and in vitro.In in vitro experiments,a large number of dark aggregates appeared in the interference Dnmt3A group on the 4th day,and a large number of embryoid bodies that were about to rupture on the 6th day,while the interference Tet1 did not appear on the 4th and 6th days.Embryoid body;Dot-blot detection found that the methylation level of interference Dnmt3A group was significantly reduced(P<0.05),and DNA methylation level increased slightly after interference with Tet1;qRT-PCR detection interference Dnmt3A group 6d after Cvh,C-kit the expression of BMP4,Blimp 1,and Lin28a increased significantly(P<0.05),and the genomes were significantly down-regulated after interference with Tetl(P<0.05);and flow cytometry analysis found that the formation rate of PGCs increased significantly after interference with Dnmt3A(P<0.05),The formation rate of PGCs after interference with Tet1 was significantly reduced.In vivo experiments,qRT-PCR detection revealed that the expressions of BMP4,Cvh and C-kit in the interference Dnmt3A group were significantly increased(P<0.05),and the expressions of Blimpl and Lin28a were extremely significantly up-regulated(P<0.01).After interference with Tet1,the expressions of Cvh and C-kit were significantly increased(P<0.05).kit,BMP4,Blimpl and Lin28a all had extremely significant down-regulation(P<0.01);flow cytometry analysis found that the formation efficiency of PGCs was significantly increased after interference with Dnmt3A(P<0.05),and the formation efficiency of PGCs was significantly down-regulated after interference with Tet1(P<0.05).The above results indicate that DNA methylation inhibits the formation of PGCs,and DNA demethylation promotes the formation of PGCs.(2)DNA methylation and H3K4me2 participate in the regulation of BMP4 genes in the formation of PGCs.Joint analysis of the ChIP-seq and MBD-seq results of ESCs and PGCs was carried out to screen the key pathways that are regulated by DNA methylation during the formation of PGCs-TGF-β/BMP signaling pathway,and use the key gene BMP4 as the target gene research object.The pBMP4-EGFP-N1 vector was successfully constructed to verify that it has priming activity;the BMP4 promoter-basic vector with different length deletion fragments was successfully constructed,and the dual luciferase reporter system verified that pGL3-613 was significantly higher than pGL3-508 and pGL3-281(P<0.01),indicating that 508-613bp is the core promoter region of BMP4.Bioinformatics methods predict the CpG islands in the core promoter region of BMP4 and compare the DNA methylation levels in CEFs,ESCs and PGCs by bisulfite sequencing.The results show that the average DNA methylation levels in the BMP4 promoter region in ESCs Significantly higher than PGCs(P<0.05);ChIP-qPCR based on histone H3K4me2 found that the enrichment level of H3K4me2 on the BMP4 promoter in PGCs was significantly higher than that of ESCs(P<0.05),and it was mainly concentrated in the core promoter region;further passed qRT-PCR detection revealed that BMP4 and Smad1 in PGCs were significantly higher than ESCs(P<0.05),and Bmprl,Bmpr2 and C-kit were significantly higher than ESCs(P<0.01);Western Blot was used to detect phosphorylation p-downstream of BMP4 signal Smad1/3/5 complex protein was significantly higher in PGCs than ESCs(P<0.05).The above results indicated that the expression of BMP4,a key gene for PGCs formation,increased in ESCs and PGCs,along with the decrease of DNA methylation level in the promoter region and the increase of H3K4me2 enrichment.(3)CRISPR-dCas9-mediated TET1 targeting BMP4 promoter DNA demethylation promotes the formation of PGCs.Three pairs of sgRNA sequences are designed respectively for the CpG islands in the BMP4 promoter region,and they are connected to the pgRNA-humanized vector to construct sgRNAl,sgRNA2,and sgRNA2.The sgRNA3 guide vector and dCas9-TET1CD were combined to transfect DF1 cells to detect the effect of BMP4 activation.After qRT-PCR detection,it was found that the combination of sgRNA and dCas9-TET1CD vector could significantly enhance the expression of BMP4(P<0.01).When mixed with 3 and dCas9-TET1CD,the expression of BMP4 reached the highest(3.473±0.187),indicating that multiple sgRNAs targeting the same gene can achieve the best activation effect.In the process of ESCs induction in vitro,sgRNA1-3 and dCas9-TET1CD were co-transformed and the BMP4 promoter region methylation level was significantly decreased by bisulfite conversion sequencing(P<0.05),while the expression of BMP4 was found to be extremely low by qRT-PCR.Significantly increased(P<0.01),the expression of downstream signaling genes Bmprl and Smad1 increased significantly(P<0.05),the expression of PGCs marker genes Blimpl and Lin28a also increased significantly(P<0.05),while Cvh and C-kit The up-regulation of PGCs was more significant(P<0.01);Western Blot detected a significant increase in the expression of phosphorylated p-Smad1/3/5 downstream of BMP4(P<0.05);the formation rate of PGCs was significantly increased(P<0.05).The expression of marker genes Cvh,C-kit,Blimpl and Lin28a in the formation of BMP4 and PGCs were significantly increased by qRT-PCR after injection of sgRNAl-3 and dCas9-TET1CD into the allantoic cavity on day 0(P<0.05),Western Blot detection The p-Smad 1/3/5 protein increased significantly(P<0.05).Flow cytometric analysis revealed that the formation efficiency of PGCs in the group injected with sgRNA1-3 and dCas9-TET1CD was significantly increased(P<0.05).The above results indicate that DNA demethylation targeting the BMP4 promoter region can increase BMP4 expression and promote the formation of PGCs.(4)DNA methylation and histone methylation antagonizes the regulation of BMP4 signaling and participates in the formation of PGCs.The potential transcription factors LIN54,ZEB1 and NFAT5 in the promoter region of BMP4 are predicted by bioinformatics,and the basic vector is connected by constructing a core promoter fragment lacking the binding site After transfection of DF1 cells,the dual luciferase reporter system detected that the activation activity was significantly reduced only after the binding site of ZEB1 was deleted(P<0.01),indicating that ZEB1 is a key transcription factor in the core promoter region of BMP4.The Zeb1 overexpression vector was constructed and transfected into DF1 cells.The expression of Zebl and BMP4 increased significantly after the overexpression of Zeb1 by qRT-PCR(P<0.05),and the expression of Zebl gradually increased during the 0-6d in vivo;and The oe-Zeb1 and the core promoter fragment basic vector were co-transfected into DF1 cells.The promoter activity of the BMP4 core promoter fragment detected by dual luciferase was significantly increased(P<0.05).The above results indicate that Zeb1 as a BMP4 transcription factor can promote its transcriptional expression.The overexpression vector of Dnmt3A and Tet1 was successfully constructed and co-transfected with the histone demethylase LSD1 overexpression vector to DF1 cells.After 48 hours,the dual luciferase reporter system detected that the activation activity was extremely significantly down-regulated(P<0.01)After transfection with the overexpression vectors of Tetl and Zebl for 96 hours,it was tested again that the activation activity was significantly up-regulated(P<0.05).The results show that DNA methylation and H3K4 demethylation can inhibit the transcription activity of BMP4,while the DNA demethylation and transcription factor Zeb1 can rescue and enhance the transcription activity of BMP4.ChIP-qPCR detection of PGCs cells found that both ZEB1 and H3K4me2 were enriched in the core region of the BMP4 promoter.When Dnmt3A was interfered,the enrichment level of H3K4me2 and the binding of ZEB1 both increased significantly(P<0.05),and CpG islands The level of DNA methylation of the DNA was significantly decreased(P<0.05);and after interference with the histone methylase MLL2,the enrichment level of H3K4me2 in the promoter region of BMP4 and the binding of ZEB1 were significantly reduced(P<0.05)),the level of DNA methylation increased significantly(P<0.05).The results show that DNA demethylation can increase the enrichment of H3K4me2 in the core promoter region of BMP4 and the binding of transcription factor ZEB1,while H3K4me2 demethylation can correspondingly reduce the enrichment and binding of H3K4me2 and ZEB1,and DNA methylation was antagonistic to the enrichment level of H3K4Me2.更多还原