RNAi-mediated Targeting of Hetorochromatin Assembly

描述(申请人提供)：这项工作的长期目标是了解RNA干扰(RNAi)途径如何调节分裂酵母中的异染色质基因沉默。RNAi是一种广泛存在的沉默机制，在转录后和转录水平都有作用，由双链RNA分子触发，这些双链RNA分子被加工成小干扰RNA(称为siRNAs)。SiRNAs通过效应复合体引导互补的靶核酸失活。我们的实验室已经纯化了一个这样的效应复合体，它直接或间接地靶向DNA，以组装表观遗传的异染色质结构域。这种复合体，我们称之为RITS(RNA诱导的转录基因沉默的启动)，包含Ago1，Chp1和Tas3蛋白，物理上将RNAi途径与异染色质联系起来。Ago1亚基在酵母和人类之间是保守的，它的同源物形成了另一种效应器复合体的核心亚单位，该复合体使用siRNAs靶向mRNA以使其失活。Chp1是一种异染色质结构蛋白，与赖氨酸9甲基化的组蛋白H3有关，组蛋白H3是异染色质的保守标志。此外，该复合体包含与异染色质组装处的着丝粒DNA重复序列匹配的siRNA。这项建议的目标是了解RITS是如何针对特定的染色体区域进行异染色质失活的，并对RNAi介导的异染色质组装的机制进行生化剖析。 RNAi和异染色质蛋白的保守性表明，这里为裂解酵母复合体开发的原理将适用于其他环境。对RNAi在表观遗传染色质结构域组装中的作用的基本了解不仅将为理解该过程如何失败提供一个框架，而且还将为设计基于干预的治疗策略提供底物和知识。

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how the RNA interference (RNAi) pathway regulates heterochromatic gene silencing in fission yeast. RNAi is a wide spread silencing mechanism that acts at both the posttranscriptional and transcriptional levels and is triggered by double stranded RNA molecules that are processed to small interfering RNAs (called siRNAs). SiRNAs guide the inactivation of complementary target nucleic acids by effector complexes. Our laboratory has purified one such effector complex that either directly or indirectly targets DNA for assembly of epigenetic heterochromatin domains. This complex, which we have termed RITS (RNA-induced [nitiation of Transcriptional Gene Silencing), contains the Ago1, Chp1, and Tas3 proteins and physically links the RNAi pathway to heterochromatin. The Ago1 subunit is conserved from yeast to human and its homologs form the core subunits of another type of effector complex that uses siRNAs to target mRNA for inactivation. Chp1 is a heterochromatin structural protein that associates with lysine 9-methylated histone H3, a conserved marker of heterochromatin. In addition, the complex contains siRNAs that match centromeric DNA repeats where heterochromatin is assembled. The goals of this proposal are to understand how RITS targets specific chromosome regions for heterochromatic inactivation and to perform a biochemical dissection of the mechanism of RNAi-mediated heterochromatin assembly. The conservation of RNAi and heterochromatin proteins suggests that the principles developed here for the fission yeast complexes will apply in other settings. A basic understanding of the role of RNAi in assembly of epigenetic chromatin domain will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention.