RNAi-directed assembly of heterochromatin in Drosophila

描述(由申请人提供)：通常存在于常染色域的基因在包装成异染色质时被沉默，如位置效应变异(PEV)在果蝇中的例子。导致沉默丧失的功能突变已经确定了异染色质的许多关键成分。 在着丝粒周围区域异染色质的形成似乎依赖于组蛋白去乙酰化，随后组蛋白H3在赖氨酸9位甲基化产生H3-Mk9，以及异染色质蛋白1和2(HP1和HP2)的结合。但是异染色质的形成是如何针对基因组的适当区域的呢？我们最近证明了RNA干扰机制在果蝇中发挥作用，就像之前报道的S.pombe一样。我们观察到，嵌入在异染色质中的转基因的沉默丢失(PEV的抑制)，这是由于piwi、茄子或无家可归者(纺锤体-E)突变的结果，所有这些突变都编码RNAi组件。无家可归者的突变导致H3-Mk9基因的减少，HP1的戏剧性去局部化。我们认为，含有与异染色质中重复元件同源的siRNAs的“靶向复合体”可以指导HP1与这些区域的特异性关联。我们将通过以下方式对这个模型进行测试：1)测试产生上述表型的候选功能突变位点，确定其他RNAi成分和相关的组蛋白修饰酶；2)使用TAP标签方法分离与三种关键蛋白质相关的蛋白质复合体，Homant，PAZ/PIWI结构域蛋白(已知结合siRNA)，以及Sin 3组蛋白修饰复合体的关键成分SDS3，以确定相互作用的模式；以及3)在组织培养细胞中建立这些表型(H3m-K9的丢失，HP1的非定位)的检测方法，以促进对关键因子的无偏见筛选。这些研究的结果将阐明RNAi/异染色质途径，为基因沉默的机制提供新的见解，基因沉默是发育中的一个关键过程，与包括癌症在内的许多健康问题有关。

DESCRIPTION (provided by applicant): Genes normally present in euchromatic domains 0are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by Position Effect Variegation (PEV). Loss-of-function mutations that result in a loss of silencing have identified many critical components of heterochromatin. Heterochromatin formation in pericentric regions appears to be dependent on histone deacetylation, followed by methylation of histone H3 at lysine 9 to generate H3-mK9, and association of Heterochromatin Proteins 1 and 2 (HP1 and HP2). But how is heterochromatin formation targeted to appropriate regions of the genome? We have recently demonstrated that the RNA interference machinery plays a role in Drosophila, as previously reported for S. pombe. We observe that silencing of transgenes embedded in heterochromatin is lost (suppression of PEV) as a result of mutations in piwi, aubergine, or homeless (spindle-E), all of which encode RNAi components. Mutation of homeless leads to a reduction in H3-mK9, and a dramatic delocalization of HP1. We suggest that a "Targeting Complex" containing siRNAs homologous to repetitious elements in heterochromatin can direct specific association of HP1 with these regions. We will test this model by 1) testing candidate loci for loss-of-function mutations that produce the above phenotypes, identifying other RNAi components and histone modification enzymes involved; 2) using a TAP-tag approach to isolate protein complexes associated with three key proteins, HOMELESS, a PAZ/PIWI domain protein (known to bind siRNA), and SDS3, a critical component of the Sin3 histone modification complex, to determine the pattern of interactions; and 3) developing an assay for these phenotypes (loss of H3m-K9, delocalization of HP1) in tissue culture cells to facilitate an unbiased screen for critical factors. The results of these investigations should elucidate the RNAi/heterochromatin pathway, providing new insights into mechanisms of gene silencing, a critical process in development, relevant to many health problems, including cancer.