Transposons, RNA Interference and Heterochromatin

描述（由申请人提供）：细胞学上，异染色质被定义为差异染色的浓缩染色体区域，而分子上，它对染色体分离，重组和基因表达有广泛的影响。转座因子是大多数真核生物中异染色质的组成部分，但它们在染色质修饰中的作用尚不清楚。我们将以拟南芥（Arabidopsis thaliana）和裂变酵母S. pombe为模型系统，研究异染色质通过转座子介导的基因调控使基因沉默的观点。我们将使用基因组平铺微阵列来检测拟南芥4号染色体上的DNA和组蛋白修饰以及转录。我们已经证明，染色质重塑突变体DNA甲基化1 （ddml）的减少对分离的异染色质转座子负载区域中赖氨酸4和赖氨酸9上甲基化的组蛋白h3的相对分布有主要影响。我们将使用正向和反向诱变来识别对不同类型转座子具有相似作用的新位点。在裂变酵母中，我们已经证明了异色着丝粒重复序列是转录的，并且转录物是RNA干扰的底物。RNAi突变体在着丝粒沉默和功能上存在缺陷，并且它们不能将组蛋白H3甲基赖氨酸9（异染色质的标志）招募到着丝粒重复序列中。我们将在裂变酵母中研究这种效应的分子和细胞学基础。初步数据表明，这一机制在拟南芥中是保守的。我们将使用每个系统来探索异染色质在染色体生物学中的古老作用。

DESCRIPTION (provided by applicant): Cytologically, heterochromatin is defined as condensed chromosomal regions that are differentially stained, while molecularly, it has a broad impact on chromosome segregation, recombination and gene expression. Transposable elements are an integral part of heterochromatin in most if not all eukaryotes, but their role in chromatin modification is poorly understood. We will examine the proposition that heterochromatin silences genes by virtue of transposon mediated gene regulation, using Arabidopsis thaliana and the fission yeast S. pombe as model systems. We will use genomic tiling microarrays to assay DNA and histone modification, as well as transcription, along Arabidopsis chromosome 4. We have demonstrated that the chromatin remodeling mutant decrease in DNA methylation 1 (ddml) has major effects on the relative distribution of histoneH3 methylated on lysine 4 and lysine 9 in an isolated, transposon-laden region of heterochromatin. We will use forward and reverse mutagenesis to identify novel loci that have similar effects on different classes of transposons. In fission yeast, we have shown that heterochromatic centromere repeats are transcribed, and that the transcripts are substrates for RNA interference. Mutants in RNAi are defective in centromere silencing and function, and they fail to recruit histone H3 methyl lysine 9 (a hallmark of heterochromatin) to the centromeric repeats. We will investigate the molecular and cytological basis for this effect in fission yeast. Preliminary data suggests this mechanism is conserved in Arabidopsis. We will use each system to explore the ancient role of heterochromatin in chromosome biology.