Dissecting RNAi-mediated Heterochromatin Formation

描述（由申请人提供）：异染色质最初定义为在间期保持高度浓缩的核DNA位点。忠实的染色体分离和基因组稳定性需要异染色质，这证明了组装这些基因座的至关重要性;事实上，Suv 39 h基因敲除小鼠（异染色质形成所需的一种成分）增加了肿瘤风险。最近的数据表明，RNAi机制是异染色质化所必需的，其机制尚不清楚。我们已经设计了实验，将解决这个问题，在遗传上易处理的裂变酵母，S。粟球。我们打算确定（1）RNAi是否以顺式或反式或两者介导异染色质形成，（2）异染色质靶向是否需要靶基因座的转录，（3）RNAi介导的异染色质形成在不存在dsRNA的情况下是否是可遗传的，（4）RNAi介导的异染色质形成是否导致异染色质扩散，和（5）RNAi和异染色质指定机器的组件的组装顺序。此外，我们设计了一种新的遗传筛选，以分离出RNAi介导的异染色质形成所需的新因子。

DESCRIPTION (provided by applicant): Heterochromatin was originally defined as loci of nuclear DNA that remain highly condensed during interphase. Faithful chromosome segregation and genome stability requires heterochromatin, demonstrating the critical importance of assembling these loci; in fact, knockout mice for Suv39h, a component required for heterochromatin formation, have increased tumor risks. Recent data indicates that the RNAi machinery is required for heterochromatization by an as yet unknown mechanism. We have designed experiments that will address this problem in the genetically tractable fission yeast, S. pombe. We intend to determine (1) whether RNAi mediates heterochromatin formation in cis or in trans or both, (2) whether heterochromatin targeting requires transcription of the target locus, (3) whether RNAi-mediated heterochromatin formation is heritable in the absence of dsRNA, (4) whether RNAi-mediated heterochromatin formation results in heterochromatin spreading, and (5) the order of assembly of components of the RNAi and heterochromatin-specifying machinery. Furthermore, we have designed a novel genetic screen to isolate novel factors that are both required for and disrupt RNAi-mediated heterochromatin formation.