【作者】 张晓丽；

【导师】 赵小东；

【作者基本信息】 上海交通大学 ， 生物学， 2016， 博士

【摘要】 生殖细胞是遗传信息在代与代之间的传递者,而成熟配子是由生殖干细胞发育而来的,因此研究调控生殖干细胞自我更新和分化的分子机制将有助于我们更好地理解生殖干细胞的基本特性,从而为进一步研究生殖细胞的体外发育及其在生殖医学上的临床应用奠定基础。过去几十年,对于哺乳动物人们普遍接受的一个观点是成体生殖干细胞只存在于雄性动物体内,而雌性哺乳动物因为缺少生殖干细胞,其体内的卵母细胞在胎儿出生之后即不会再增加。然而,近几年的研究发现在小鼠、大鼠和人的成体卵巢内都存在生殖干细胞,且对这些细胞进行了细胞生物学、生化和分子水平的鉴定,但决定该类细胞基本生物学特性的分子调控机制还不是很清楚。细胞命运的决定通常是由遗传调控(即编码在基因组上的启动子和增强子等调控元件)和表观遗传调控(如DNA甲基化和各种组蛋白表观遗传修饰)共同决定的。近几年的研究表明,表观遗传修饰对于调控基因的表达发挥了重要作用,且通过检测表观遗传修饰可以鉴定出基因组上的顺式调控元件。雌性生殖干细胞(FGSC)是一种具有自我更新和单一分化潜能的成体生殖干细胞,为了更好地理解该细胞的基本生物学属性及维持其属性的分子调控机制,本研究以体外培养的小鼠雌性生殖干细胞为材料,利用ChIP-Seq、MethylCap-Seq和RNA-Seq对其进行了组蛋白修饰(H3K4me3,H3K27me3,H3K4me1,H3K27ac)、RNA聚合酶II(RNA Pol II)结合位点、DNA甲基化和基因表达谱系统性的分析。通过与胚胎干细胞(ESC)和原始生殖细胞(PGC)进行比较分析,本研究找出了FGSC特异性的活性增强子序列,发现与这些增强子相关的基因主要参与维持生殖系统的表型。此外,通过分析DNA甲基化修饰对基因表达水平的影响以及对Dnmt1进行的基因沉默实验,本研究发现DNA甲基化对于抑制FGSC中体细胞发育相关基因的表达、维持FGSC的单能性发挥了重要作用。对FGSC和雄性生殖干细胞(MGSC)的DNA甲基化修饰谱进行比较分析的结果显示DNA甲基化对于维持雌性生殖干细胞的性别特征发挥了一定作用。最后,本研究发现Prmt5和Max对于保持FGSC的未分化状态起到了重要作用。综上所述,本研究通过对表观遗传修饰的检测揭示了FGSC特有的染色质状态及其生物学意义,为进一步深入研究FGSC的发生、分化及临床应用提供了一定的理论依据。更多还原

【Abstract】 Because germline stem cells(GSCs)are the sources of mature male and female gametes,which play a key role in genome transmission to the next generation,it is quite necessary to understand the molecular mechanisms of regulating GSCs self-renewal and differentiation.Additionally,the studies of GSCs behavior and gametogenesis in vitro could be very important for clinical application in the field of reproductive medicine.For decades,a widely accepted view in reproductive biology is that germline stem cells exist in adult males and spermatogenesis occurs in the entire adult life.However,the production of oocyte in most female mammals is considered to cease before birth.This doctrine was challenged recent years by studies showing that a population of female germline stem cells(FGSC)exist in adult mouse,rat and human ovaries,which has been detected at cell biology,biological chemistry and molecular biology levels.However,the molecular regulation mechanisms involved in basic biological characteristics of FGSC are unclear.Cell fate determination requires the close coordination between genetic(e.g.,cis-regulatory elements promoters and enhancers)and epigenetic(e.g.,DNA methylation and histone modifications)regulations.Recent reports demonstrated that epigenetic modification play a key role in regulating gene expression and detecting regulatory sequences.FGSCs are unipotent adult stem cells that possess the ability to self-renew while remain capable of giving rise to specialized ovum.However,the underlying molecular mechanisms for the specification of FGSC are not clear.With the aim to address this question,this study performed ChIP-Seq,MethylCap-Seq and RNA-Seq to detect the profiling of histone modifications(H3K4me3,H3K27me3,H3K4me1,H3K27ac),RNA polymerase II,DNA methylation and gene expression using mouse FGSC cultured in vitro.Through comparisons to primordial germ cells(PGC)and embryonic stem cells(ESC),this work revealed that FGSC characterized with an unique chromatin signature at distal regulatory elements,which distinguishes FGSC from PGC and ESC.Gene functional annotation reveals that genes related with FGSC-specific regulatory elements are highly involved in maintaining reproductive system phenotypes.Moreover,gene expression analysis and the test of Dnmt1 knockdown showed that DNA methylation play an important role in repressing somatic programme and maintaining unipotency of FGSC by inhibiting the activities of development related genes.When compared with male germline stem cells(MGSC)this study revealed that DNA methylation is potentially involved in maintaining sexual identity of FGSC.Additionally,the results of RNA interference showed that Prmt5 and Max are implicated in FGSC identity by repressing expression of genes linked to differentiation.Together,this study reveals that the molecular chromatin signatures of FGSC play a key role in maintaining FGSC-specific biological characteristics.Moreover,this work provides resources for further analysis of generation and differentiation of FGSC and establishes a preliminary foundation for clinical application of FGSC.更多还原