RNAi, Histone Modification and the DDB1/CPSF-Like Complex Rik1

描述(申请人提供)：异染色质由真核细胞染色体紧密致密的重复区域组成。它通过有丝分裂遗传，在转录沉默、着丝粒规范和基因组完整性方面发挥作用，从而深刻影响人类健康和疾病的表观遗传机制。我们发现，分裂酵母中异染色质的表观遗传需要RNA干扰(RNAi)来指导组蛋白修饰，组蛋白修饰发生在细胞周期的DNA复制阶段。在分裂酵母中，S.pombe着丝粒重复序列具有小RNA簇和复制起点的交替排列，这使得转录和复制机制几乎不可避免地发生碰撞。我们认为，在S期，RNA干扰促进了RNA聚合酶的释放，使领先的链DNA聚合酶完成着丝粒复制。DNA聚合酶epsilon直接招募组蛋白修饰的Rik1复合体，因此可以在DNA复制的同时传播异染色质。在没有RNAi的情况下，停滞的叉子可以通过同源重组(HR)修复，而不需要组蛋白修饰，所以在没有RNAi的情况下，同源重组是必不可少的。这个模型可以解释许多表观遗传沉默的例子中非编码RNA和DNA复制的参与，包括植物中的参数突变，以及哺乳动物中的印记和X失活。S.pombe是研究细胞周期、异染色质沉默和RNAi的优秀模型系统。我们将研究DNA复制、RNA聚合酶释放、DNA重组和修复在RNAi介导组蛋白异染色质修饰中的作用。我们将利用异染色质成核和RNAi模型，以及染色体图谱和遗传分析来检验我们的假设。我们将基于我们最近的结果，即Rik1复合体和着丝粒结合蛋白B在DNA复制和修复以及RNA干扰中的作用。

DESCRIPTION (provided by applicant): Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. It is inherited through mitosis and has roles in transcriptional silencing, centromere specification and genome integrity, which profoundly impact epigenetic mechanisms in human health and disease. We have found that the epigenetic inheritance of heterochromatin in fission yeast requires RNA interference (RNAi) to guide histone modification, which occurs during the DNA replication phase of the cell cycle. In the fission yeast S.pombe centromeric repeats have an alternating arrangement of small RNA clusters and origins of replication that makes collision of the transcription and replication machineries all but inevitable. We propose that RNA interference promotes release of RNA polymerase (PolII) during S phase, allowing completion of centromeric DNA replication by the leading strand DNA polymerase. DNA Polymerase epsilon directly recruits the histone-modifying Rik1 complex and so can spread heterochromatin along with DNA replication. In the absence of RNAi, stalled forks are repaired by homologous recombination (HR) without histone modification, so that HR is essential in the absence of RNAi. This model may explain the participation of non-coding RNA and DNA replication in many examples of epigenetic silencing, including paramutation in plants, and imprinting and X-inactivation in mammals. S.pombe is an outstanding model system for cell cycle research, heterochromatic silencing, and RNAi. We will examine the roles of DNA replication, RNA Polymerase release, DNA recombination and repair in heterochromatic histone modification mediated by RNAi. We will utilize models of heterochromatic nucleation and RNAi, as well as chromosome profiling and genetic analysis, to test our hypothesis. We will build on our recent results concerning the roles of the Rik1 complex and Centromere-binding protein B in DNA replication and repair, as well as RNA interference.