Transposons, RNA Interference and Heterochromatin

描述（由申请人提供）：在细胞学上，异染色质被定义为差异染色的浓缩染色体区域，而在分子上，异染色质对染色体分离、重组和基因表达具有广泛影响。转座因子是大多数真核生物异染色质的组成部分，但其在染色质修饰中的作用知之甚少。我们将利用拟南芥和裂殖酵母S. pombe作为模型系统。我们将使用基因组平铺微阵列来分析DNA和组蛋白修饰，以及转录，沿沿着拟南芥4号染色体。我们已经证明，染色质重塑突变体减少DNA甲基化1（ddml）的组蛋白H3甲基化赖氨酸4和赖氨酸9在一个孤立的，转座子负载区域的异染色质的相对分布有重大影响。我们将使用正向和反向诱变来鉴定对不同类别的转座子具有相似作用的新位点。在分裂酵母中，我们已经表明异染色质着丝粒重复序列被转录，并且转录物是RNA干扰的底物。RNAi中的突变体在着丝粒沉默和功能上有缺陷，并且它们不能将组蛋白H3甲基赖氨酸9（异染色质的标志）募集到着丝粒重复序列中。我们将研究分裂酵母中这种效应的分子和细胞学基础。初步数据表明，这种机制是保守的拟南芥。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.