Transposons, RNA Interference and Heterochromatin

描述（由申请人提供）：异染色质首先在植物中发现，但在真核生物中广泛存在，并在染色体结构、着丝粒功能、基因表达和转座子沉默中起重要作用。转座因子（Transposable elements，TE）是异染色质的主要组成部分，通过组蛋白和DNA的修饰，在拟南芥中的染色质重塑基因DNA甲基化减少1（DDM 1）及其哺乳动物同源基因Lsh 1的指导下，受到转录沉默的影响，这两个基因在生殖细胞中都有高水平的表达。我们已经发现拟南芥中的TE在花粉粒的营养核中被激活，花粉粒是一种在受精前完全包围精细胞的终末分化的“支持”细胞。在WT花粉粒和ddm 1突变体植物中，来自着丝粒反转录转座子的24 nt小RNA被大量21- 22 nt小RNA取代。DDM 1在营养核中不表达，这解释了为什么TE和21 nt小RNA的类似再活化在花粉和ddm 1中发生。我们将研究这些新的21 nt siRNA通过从周围的花粉粒转运来指导精子细胞中任何活性元件沉默的命题。所提出的机制让人想起哺乳动物生殖系中piRNA介导的TE调节，以及纤毛虫中微核和大核之间的相互作用。花粉和ddm 1突变体中的一些TE衍生的siRNA靶向特定基因的甲基化，导致表观等位基因;表观等位基因在ddm 1突变体中是众所周知的，但在癌细胞中也是如此，其中TE表观遗传地被激活，而肿瘤抑制基因被沉默。我们将使用正向和反向遗传筛选的组合来进一步剖析这一途径，并希望阐明异染色质和TE在动物和植物中RNAi介导的沉默，染色体组织和生殖细胞分化中的保守作用。公共卫生相关性：异染色质是由转座子和卫星重复序列组成的，在几乎所有的高等真核生物中都存在，但其功能至今仍是一个谜。在拟南芥中，我们发现插入异染色质的反转录转座子在花粉粒的营养核中产生大量的21 nt小RNA，在那里转座子被活跃地转录。这些小RNA在整个植物中的染色质重塑DNA甲基化减少1（DDM 1）的突变体中发现。.我们将研究这些小RNA的生物起源，以及它们沉默精子细胞中转座子的命题，让人想起动物生殖系中的piwi RNA。除了转座因子小RNA外，与甲基化基因（表观等位基因）匹配的21 nt小RNA也出现在ddm 1背景中。这些基因sRNA可能负责ddm 1中的零星基因甲基化（表观等位基因）。癌细胞在DNA甲基化方面也有类似的变化，可能使用相同的机制。

DESCRIPTION (provided by applicant): Heterochromatin was first discovered in plants, but is widespread among eukaryotes and has important roles in chromosome structure, centromere function, gene expression and transposon silencing. Transposable elements (TEs) are major constituents of heterochromatin and are subject to transcriptional silencing through histone and DNA modification, guided by the chromatin remodeler DECREASE IN DNA METHYLATION1 (DDM1) in Arabidopsis, and its mammalian homolog Lsh1, both of which are expressed at high levels in germ cells. We have found that TEs in Arabidopsis are activated in the vegetative nucleus of the pollen grain, a terminally differentiated "support" cell that completely encloses the sperm cells before fertilization. In WT pollen grains and in ddm1 mutant plants, 24nt small RNA from centromeric retrotransposons are replaced by large amounts of 21-22nt small RNAs. DDM1 is not expressed in the vegetative nucleus, explaining why similar reactivation of TEs and 21nt small RNAs occur in both pollen and ddm1. We will investigate the proposition that these novel 21nt siRNA guide silencing of any active elements in sperm cells via transport from the surrounding pollen grain. The proposed mechanism is reminiscent of piRNA-mediated TE regulation in the mammalian germline, and of interactions between micronuclei and macronuclei in ciliates. Some TE- derived siRNAs in pollen and ddm1 mutants target specific genes for methylation resulting in epialleles; epialleles are well known in ddm1 mutants, but also in cancer cells, in which TEs are activated epigenetically and tumor suppressor genes are silenced. We will use a combination of forward and reverse genetic screens to further dissect this pathway, and hope to shed light on the conserved role of heterochromatin and TEs in RNAi-mediated silencing, chromosome organization and germ cell differentiation in animals and plants. PUBLIC HEALTH RELEVANCE: Heterochromatin is composed of transposons and satellite repeats in almost all higher eukaryotes, but its function remains a mystery. In Arabidopsis we have found that retrotransposons inserted into heterochromatin generate large amounts of 21nt small RNA in the vegetative nucleus of pollen grains, where transposons are actively transcribed. These small RNA are found throughout the plant in mutants of the chromatin remodeler Decrease in DNA methylation 1 (DDM1). . We will investigate the biogenesis of these small RNAs, and the proposition that they silence transposons in sperm cells, reminiscent of piwi RNA in animal germline. In addition to the transposable element small RNAs, 21nt small RNAs matching methylated genes (epialleles) arise in the ddm1 background. These genic sRNAs may be responsible for sporadic gene methylation (epialleles) in ddm1. Cancer cells have similar changes in DNA methylation, which may use the same mechanism.