去甲基化酶TET1调控山羊妊娠早期胎盘发育的分子机制研究

Survival of a newborn is affected especially by sufficiency of the placenta. The placenta is of utmost importance for intrauterine fetal development and growth. Deregulation of placentation can lead to adverse outcomes for both mother and fetus. Ten-Eleven-Translocation 1 (TET1), which can oxidize 5-methylcytosine is one of the significant factors both in placental development and epigenetic regulation. However, the molecular mechanism of TET1 regulation on placenta development during early pregnancy is still unclear. To characterize early fetal placental development, gravid uterine tissues will be collected from pregnant does of Dazu Black goat from day 15 to 35 after mating. Determination of cell proliferation wil be based on Ki67 protein immunodetection, global methylation is based on 5-methyl-cytosine (5mC) expression and mRNA expression for TET1 immunolocalization and on mRNA expression of several factors involved in the regulation of angiogenesis in placenta. Then we will investigate the effects of TET1 on proliferation of placental trophoblast cells and scan the signaling by ChIP-seq and ChIP-qPCR. Thirdly, we will find the interaction of TET1 and some activating transcription factors by immunoblotting, GST pull-down and Co-IP experiments. Finally, we will investigate the genetic relations of the polymorphisms of TET1 gene candidate genes with the placental traits. The research findings should have important theoretical and practical significance in further goat breeding and production.

羊繁殖性能的发挥，不仅体现在产羔率上，还体现在羔羊品质和成活率上。胎儿的妊娠维持、发育甚至初生羔羊品质和成活率都依赖于早期正常的胎盘发育。TET1作为细胞分化、胎盘/胎儿发育的重要调控因子，介导的甲基化动态变化对细胞分化和维持有关键作用，但其调控胎盘发育的分子机制研究尚是空白。鉴于此，本项目利用国家级畜禽遗传资源大足黑山羊，通过TET1的定性和定量研究，分析其在妊娠早期的动态表达模式；采用ChIP-seq和Co-IP等手段筛选鉴定TET1表观调控的靶基因；借助体外动物胎盘模型，通过基因芯片分析筛选出TET1表观调控胎盘发育的主要信号通路分子；进一步对TET1及其靶基因重要突变位点的多态性与山羊胎盘性状进行关联分析，明确TET1对妊娠早期胎盘发育的影响，筛选其调控胎盘发育的下游信号通路和靶基因，揭示TET1介导的去甲基化与胎盘发育之间的表观遗传学关系，为羊的分子育种及相关应用研究奠定基础。

山羊的妊娠维持、发育甚至初生羔羊品质和成活率都依赖于早期正常的胎盘发育。TET1作为细胞分化、胎盘/胎儿发育的重要调控因子，介导的甲基化动态变化对细胞分化和维持有关键作用。本项目为探究TET1调控山羊妊娠早期胎盘发育的分子机制，（1）扩增获得了大小为7056bp的山羊TET1基因cDNA全长序列CDS序列，并进行了生物信息学分析，明晰了TET1基因在20d、25d、30d、60d、90~120d和150d的山羊胎儿胎盘发育中的动态表达模式，发现TET1基因在山羊胎盘滋养层组织特异性高表达，调控5mC/5hmC平衡，与子叶滋养层组织性状存在显著正相关关系；（2）建立了成熟完善的山羊胎盘原代滋养层细胞（GTCs）体外培养体系，明确了TET1与靶基因对山羊胎盘滋养层细胞生长的影响；（3）发现TET1分子可调控Wnt信号通路上游SFRP5和DKK3甲基化水平，影响GTCs迁移和增殖功能；进一步发现TET1可调控MMP2、MMP9、TIMP1和TIMP2表达水平，进而影响滋养层细胞迁移和侵袭能力；（4）筛选获得包括RASGRP3分子在内的山羊妊娠早期胎盘滋养层功能关键基因与信号通路，RASGRP3分子在山羊妊娠早期胎盘滋养层特异性表达，并与胎盘子叶性状相关；RASGRP3基因通过激活RAP1、促进PI3K/AKT/mTOR通路的磷酸化水平，调控山羊胎盘滋养层细胞的增殖、凋亡、迁移和侵袭功能；（5）TET1及其靶基因重要突变位点的多态性与山羊胎盘性状进行关联分析，筛选出了多个与山羊胎盘性状相关的TET1基因SNP位点，其中SNP_585和SNP_657与山羊子叶密度存在显著相关关系；明确了TET1和RASGRP3对山羊妊娠早期胎盘发育的影响，筛选其调控胎盘发育的下游信号通路和靶基因，揭示了TET1 介导的去甲基化与山羊胎盘发育之间的表观遗传学关系，为羊的分子育种及相关应用研究奠定了基础。发表论文26篇，其中SCI收录论文10篇；申请国家发明专利2项，授权2项；出版著作3部；项目组晋升副教授3名、入选人才计划1人；培养研究生12名（其中博士2名）。举办学术会议1次；参加国内外学术会议23人次。研究计划全部完成，并达到预期目标。