线虫体细胞和生殖细胞命运的决定与维持需要细胞自我吞噬，LSY-2和SUMO化修饰通路的协同作用

【作者】 严立波；

【导师】 张宏；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 在大部分有性生殖的动物中,生殖细胞会在胚胎发育的早期就同其它的体细胞分离开。很多生物,包括秀丽隐杆线虫和果蝇,生殖细胞的形成过程中,卵母细胞特异的生殖细胞质会分布到下一代的原始生殖细胞中。秀丽隐杆线虫早期胚胎发育过程中,体细胞和生殖细胞各自的前体细胞的建立需要母性遗传而来的蛋白,比如PIE-1和MEX-1的作用。而维持体细胞生殖细胞命运的差异则需要多个染色质重塑复合物的活性,包括lin-35 Rb通路和NURD复合物,在这些基因的突变体中,体细胞会异位表达生殖细胞的特性。生殖细胞体细胞的差异是怎么建立起来得?除了PIE-1和MEX-1是否还有其它的关键因子或者信号通路参与了这一过程?在体细胞中,lin-35Rb通路和NURD复合物是如何定位到它们的靶基因(生殖细胞特异的基因)上去,并如何维持这些基因的抑制状态得?对这些关于动物发育过程中决定和维持体细胞和生殖细胞命运的机制,我们还知之甚少。为了研究体细胞和生殖细胞的命运是如何被决定和维持得,我们做了一个全基因组的RNA干扰筛选,去寻找在体细胞里有P颗粒(生殖细胞的标记物)的报告基因,pgl-1∷gfp,异位表达的突变体。我们发现,细胞自我吞噬(autophagy)通路基因的突变体的体细胞在胚胎发育早期以及幼虫早期,会有P颗粒的异位聚集。这显示在秀丽隐杆线虫中,基础水平的细胞自我吞噬作用参与了体细胞中对生殖细胞特异的基因产物的降解过程。细胞自我吞噬是真核生物中主要的降解途径之一,而且是唯——个可以降解整个细胞器的途径。细胞自我吞噬广泛地存在于从酵母到人类的真核生物中。但是细胞自我吞噬确切的分子机制和生理功能还不是很清楚,现在我们也缺乏方便可靠的工具来研究细胞自我吞噬。以前一般认为细胞自我吞噬是一种没有选择性的降解过程,但是,最近越来越多的证据证明,细胞自我吞噬可能可以选择性得降解底物,至少在一些情况下是有选择性得。但是细胞自我吞噬是如何来选择底物得,还不清楚。发现新的更多的被细胞自我吞噬降解的底物,会有助于我们对选择性细胞自我吞噬的分子机制以及功能的了解。我们的研究表明,细胞自我吞噬作用在秀丽隐杆线虫胚胎发育过程中,选择性地降解体细胞中的P颗粒,细胞自我吞噬作用通过这种方式参与了秀丽隐杆线虫体细胞生殖细胞命运差异的建立过程。这一发现不但提示我们细胞自我吞噬作用在动物发育过程中的一个重要的生理学作用,而且给我们提供了一个很好的秀丽隐杆线虫细胞自我吞噬的标记物—pgl-1∷gfp,pgl-1∷gfp可以用来监测线虫中的细胞自我吞噬作用。如果细胞自我吞噬作用正常,体细胞中就没有pgl-1∷gfp颗粒状的聚集,如果细胞自我吞噬作用被破坏了,在胚胎发育早期的体细胞中就会有P颗粒的聚集。因此我们可以用GFP来监测秀丽隐杆线虫中的细胞自我吞噬作用。这样,我们就可以把强大的遗传筛选模型动物——秀丽隐杆线虫和方便的检测方法pgl-1∷gfp结合起来,用于研究细胞自我吞噬作用的分子机制和调控机理,及其生理作用。我们还发现锌指蛋白LSY-2和SUMO修饰通路的突变体的体细胞表现出了许多生殖细胞的特性,比如有P颗粒的表达。而且,在体细胞命运的维持过程中,lsy-2和lin-35 Rb通路的基因有相互促进的作用。我们进一步发现LSY-2中的一个碱性结构域能够直接结合RNA,这显示在体细胞中,RNA可能参与了抑制生殖细胞特异基因的表达。我们的研究揭示了在线虫体细胞和生殖细胞的命运决定和维持过程,需要细胞的自我吞噬,染色质重塑复合体,RNA结合蛋白LSY-2和翻译后的SUMO修饰通路的协同作用。更多还原

【Abstract】 Germ cells are set aside from other somatic cells at early embryonic stage in most sexually reproducing animals and formation of germ cells in many organisms, including in C.elegans and Drosophila,involves the segregation of specialized germ plasma from oocyte to germ precursor cells.Establishment of somatic and germ precursor cells during early embryogenesis in C.elegans requires maternal distributed proteins,such as PIE-1 and MEX-1,while maintenance of the soma/germ distinction requires the activities of multiple chromatin remodeling complexes, including the 1in-35 Rb pathway and the NURD complex,whose mutants lead to the ectopic expression of germline traits by somatic cells.How the germ/soma distinction is established? Whether other critical factors or pathways are involved in this process besides PIE-1 and MEX-1? How the lin-35 Rb pathway and the NURD complex are targeted to and the repressive state of germline specific genes is maintained in somatic cells? So,the mechanisms that specify and maintain the distinct somatic and germ cell fates during animal development are poorly understood.To understand how the somatic and germ cell fate is specified and maintained, we screened a library of bacterial clones expressing dsRNA designed to individually inactivation of 16,540 genes(targeting about 86%of the genome) for mutants with ectopic expression of the P granule specific reporter,pgl-1::gfp,in somatic cells. In this study,we show that a basal level of autophagy is required for eliminating the germ cell-specific gene products in somatic cells in C.elegans. Mutations in the autophagy pathway lead to the accumulation of germ P granules in various somatic tissues during embryogenesis and early larvae.Autophagy is one of the major degradative pathways in eukaryotic cells,and it is the only one with the capacity to degrade entire organelles.This process is ubiquitous among eukaryotes and has been discovered from yeast to man.But the exact molecular mechanism and the physiological function of autophagy are still unclear and we lack convenient tool to answer these questions.It is generally believed that autophagy is a non-selective,bulk degradation system of long-lived proteins and organelles.However,this general belief has been challenged recently.Emerging evidence indicates that autophagic degradation is highly selective,at least in certain cases.Right now,how autophagy specifically selects cargo is still largely unknown.Identification of more selectively degraded cargoes will help determine the molecular basis of the selective action and functions of autophagy.In our study we found that C.elegans P granules were selectively degraded in somatic cells in early embryo by autophagy.Mutations in the autophagy pathway lead to the accumulation of germ P granules in various somatic tissues during embryogenesis and early larvae.So this discovery provides a good marker(pgl-1::gfp) to monitor autophagy process in C.elegans.If function of autophagy machinery is normal,pgl-1::gfp will not accumulated in somatic tissues in embryo and early larvae. If the autophagy machinery was disrupted,pgl-1::gfp would be accumulated in somatic tissues.So we can use GFP to monitor autophagy in C.elegans easily.We found that mutations in lsy-2,encoding a zinc finger protein,result in various somatic cells adopting characteristics of germ cells including ectopic expression of P granules and this transformation requires the activitieds of multiple chromatin remodeling complexes.Moreover,lsy-2 functions synergistically with the lin-35 Rb pathway in specifying larval development and the maintaining the soma/germ distinctions.We further show that a basic domain in LSY-2 directly binds to RNA,suggesting a role for RNA in repressing germ cell-specific genes in somatic cells.We also found that mutations in sumoylation pathway,including ubc-9, uba-2/aos-1 and smo-1,lead to the expressin of germ cell fate by somatic cells.Our analysis reveal that the establishment and maintenance of the soma/germ distinction in C.elegans requires the concerted action of autophagy,chromatin remodeling complexes,the RNA binding protein LSY-2 and the post-translational sumoylation pathway.That autophagy pathway involes in soma/germ distinction provides a convenient tool(pgl-1::gfp) to monitor autophagy in C.elegans.更多还原