Nuclear small RNA control of the germ to soma transition

 描述（由申请人提供）：配子中所含的染色体与受精后仅几次细胞分裂的体细胞中所含的染色体非常不同。配子染色体是转录不可接近的，而体细胞染色体在与活跃转录基因相关的特定位置显示可接近的染色质。受精后转录允许染色质的这些特定区域的建立对于早期胚胎发生及以后是必不可少的，但是直接参与这一过程的调节因子的身份及其分子作用机制都没有很好地理解。 直接介导体细胞染色体修饰的序列特异性建立过程的候选因子类别是Argonaute/Piwi蛋白质家族的核定位成员。这些蛋白质与小RNA结合，使它们能够通过碱基配对特异性识别和调节互补核酸序列。核定位的Piwi蛋白对于抑制各种无脊椎动物和脊椎动物物种的生殖细胞核中的转座DNA元件的动员是必不可少的，但它们可能在受精前后的配子中具有额外的功能。在大多数实验系统中，将这些功能与转座子抑制分离是具有挑战性的，这进一步使对其功能的详细理解变得复杂。 纤毛四膜虫嗜热细胞是一个有用的系统，为研究核小RNA的功能，在胚芽到索马过渡，多个，独立的核Piwi引导的途径调节转座子阻遏和其他功能，在有性生殖。四膜虫蛋白Twi 1 p通过建立抑制性组蛋白修饰来抑制转座子，而Twi 8 p的生物学作用和功能在很大程度上是未知的。我的工作已经证明Twi 8 p对受精后的事件是必不可少的，它装载了来自基因组位点的小RNA，这些小RNA引起非受精性的发育。 Twi 8 p相关的小RNA基因座（TASLs）。我建议Twi 8 p指导建立体细胞染色体修饰通过TASLs在有性生殖过程中。我将使用四膜虫中开发的强大分子生物学工具来确定这些修饰是什么以及是什么蛋白质建立它们。我还将确定在有性生殖过程中的TASLs的生物学作用，通过特异性地破坏它们在亲本或合子基因组中的作用，并表征分子缺陷。通过利用在四膜虫中建立的用于小RNA基因调控的独特工具和技术，我将有助于我们对这些在无脊椎动物和脊椎动物中至关重要的保守过程的理解。

 DESCRIPTION (provided by applicant): The chromosomes contained in gametes are very different from the chromosomes contained in somatic cells just a few cell divisions following fertilization. Gametic chromosomes are transcriptionally inaccessible, while somatic chromosomes exhibit accessible chromatin in specific locations linked to actively transcribed genes. Establishment of these specific regions of transcriptionally permissive chromatin following fertilization is essential for early embryogenesis and beyond, but the neither the identities of the regulatory factors that directly participate in this process nor their molecular mechanisms of action are well understood. A candidate class of factors that directly mediate the process of sequence-specific establishment of somatic chromosome modifications are nuclear localized members of the Argonaute/Piwi family of proteins. These proteins associate with small RNAs, which enable them to specifically recognize and regulate complementary nucleic acid sequences by base pairing. Nuclear localized Piwi proteins are essential for repressing the mobilization of transposable DNA elements in the nuclei of germ cells of a wide variety of invertebrate and vertebrate species, but it is likely that they have additional function in gametes both before and after fertilization. Disentangling these functions from transposon repression is challenging in most experimental systems, which further complicates attaining a detailed understanding of their function. The ciliate Tetrahymena thermophila is a useful system for the study of nuclear small RNA functions during the germ to soma transition, as multiple, separate nuclear Piwi-guided pathways regulate transposon repression and other functions during sexual reproduction. The Tetrahymena Piwi protein Twi1p represses transposons by establishing repressive histone modifications, while the biological role and function of Twi8p is largely unknown. My work has demonstrated that Twi8p is essential for events that follow fertilization and that it is loaded with small RNAs derived from genomic loci that give rise to non coding RNAs, termed Twi8p-associated small RNA loci (TASLs). I propose that Twi8p guides the establishment of somatic chromosome modifications through TASLs during sexual reproduction. I will determine what these modifications are and what proteins establish them using the powerful molecular biology tools developed in Tetrahymena. I will also determine the biological role of the TASLs during sexual reproduction by specifically disrupting them in either the parental or zygotic genome and characterizing the molecular defects. By leveraging the unique tools and techniques established in Tetrahymena for small RNA gene regulation, I will contribute to our understanding of these conserved processes that are essential in invertebrates and vertebrates alike.