RNAi-directed assembly of heterochromatin in Drosophila

描述（由申请人提供）：通常存在于常染色质结构域0中的基因在包装到异染色质中时沉默，如果蝇中的位置效应杂色（PEV）所示。导致沉默丧失的功能丧失突变已经鉴定出异染色质的许多关键组分。 臂间区异染色质的形成似乎依赖于组蛋白脱乙酰化，然后是组蛋白H3在赖氨酸9处的甲基化以产生H3-mK 9，以及异染色质蛋白1和2（HP 1和HP 2）的缔合。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，正如以前报道的S。粟酒我们观察到，由于piwi，aubergine或homeless（spindle-E）中的突变，嵌入异染色质中的转基因沉默丢失（PEV抑制），所有这些突变都编码RNAi组分。homeless的突变导致H3-mK 9的减少，以及HP 1的显著离域。我们认为，含有与异染色质中重复元件同源的siRNA的“靶向复合物”可以指导HP 1与这些区域的特异性结合。我们将通过1）测试产生上述表型的功能丧失突变的候选基因座，鉴定其他RNAi组分和涉及的组蛋白修饰酶; 2）使用TAP标签方法分离与三个关键蛋白HOMELESS、PAZ/PIWI结构域蛋白相关的蛋白质复合物（已知结合siRNA）和SDS 3（Sin 3组蛋白修饰复合物的关键组分），以确定相互作用的模式;和3）开发用于组织培养细胞中这些表型（H3 m-K9的缺失、HP 1的离域）的测定法，以促进关键因子的无偏筛选。这些研究的结果应该阐明RNAi/异染色质途径，为基因沉默机制提供新的见解，基因沉默是发育中的一个关键过程，与许多健康问题，包括癌症有关。

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.