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

描述（由申请人提供）：异染色质包括真核生物染色体紧密紧密的重复区域。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中发挥作用，深刻影响人类健康和疾病的表观遗传机制。我们发现分裂酵母异染色质的表观遗传需要RNA干扰（RNAi）来指导组蛋白修饰，这种修饰发生在细胞周期的DNA复制阶段。在分裂酵母中，S.pombe的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的碰撞几乎不可避免。我们提出，RNA干扰促进S期RNA聚合酶（PolII）的释放，允许由前导链DNA聚合酶完成着丝粒DNA复制。DNA聚合酶epsilon直接募集组蛋白修饰Rik1复合体，因此可以随着DNA复制扩散异染色质。在没有RNAi的情况下，停滞的分叉是通过同源重组（HR）修复的，而不需要组蛋白修饰，因此在没有RNAi的情况下，HR是必不可少的。该模型可以解释非编码RNA和DNA复制参与许多表观遗传沉默的例子，包括植物的参数化，以及哺乳动物的印迹和x -失活。S.pombe是细胞周期研究、异色沉默和RNAi的杰出模型系统。我们将研究DNA复制、RNA聚合酶释放、DNA重组和修复在RNAi介导的异染色质组蛋白修饰中的作用。我们将利用异染色质成核和RNAi模型，以及染色体谱和遗传分析来验证我们的假设。我们将以我们最近的研究结果为基础，研究Rik1复合物和着丝粒结合蛋白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. PUBLIC HEALTH RELEVANCE: Epigenetic mechanisms alter gene function independent of DNA sequence, and have profound effects on health and disease. RNA interference impacts these mechanisms by guiding the modification of histones associated with the DNA, ensuring specificity and avoiding inappropriate gene silencing. We have found that replication of the chromosome during cell division occurs at the same time as RNA interference, and that these mechanisms interact to cause silencing. The key molecules involved are conserved from yeast to humans, and are implicated in childhood disease, mental retardation, aging and cancer. Our findings suggest that therapies that target these molecules may also impact gene expression and chromosome organization. We will investigate the underlying mechanism to determine the causes and consequences of RNAi- mediated modification of chromatin during the DNA replication phase of the cell cycle.