Transposons, RNA Interference and Heterochromatin

描述（申请人提供）：异染色质最早是在植物中发现的，但广泛存在于真核生物中，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（TEs）是异染色质的主要组成部分，可通过组蛋白和DNA修饰进行转录沉默，由拟南芥中染色质重构物DDM1及其哺乳动物同源物Lsh1引导，两者在生殖细胞中均高水平表达。我们发现拟南芥的TEs在花粉粒的营养核中被激活，这是一种最终分化的“支持”细胞，在受精前完全包围精子细胞。在WT花粉粒和ddm1突变株中，来自着丝粒反转录转座子的24nt小RNA被大量21-22nt小RNA所取代。DDM1在营养细胞核中不表达，这解释了为什么在花粉和DDM1中都发生了类似的TEs和21nt小rna的再激活。我们将研究这些新的21nt siRNA通过从周围的花粉粒转运来引导精子细胞中任何活性成分的沉默。提出的机制让人想起哺乳动物种系中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ddm1突变体中一些TE衍生的sirna靶向特异性基因进行甲基化，导致外胚轴；外显子在ddm1突变体中是众所周知的，但在癌细胞中也是如此，其中TEs被表观遗传激活，肿瘤抑制基因被沉默。我们将结合正向和反向遗传筛选进一步剖析这一途径，并希望揭示异染色质和TEs在动物和植物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：异染色质在几乎所有高等真核生物中由转座子和卫星重复序列组成，但其功能仍然是一个谜。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21nt小RNA，在那里转座子被积极转录。这些小RNA在整个植物的染色质重塑基因突变体中被发现，DNA甲基化1减少（DDM1）。我们将研究这些小RNA的生物发生，以及它们沉默精子细胞中的转座子的命题，让人想起动物种系中的piwi RNA。除了转座元件小rna外，ddm1背景中还出现了21nt个与甲基化基因（表等位基因）匹配的小rna。这些基因sRNAs可能是ddm1中散发性基因甲基化（外等位基因）的原因。癌细胞在DNA甲基化方面也有类似的变化，这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.