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

描述（由申请人提供）：异染色质包含真核细胞染色体的紧密压缩的重复区域。它通过有丝分裂遗传，并在转录沉默，着丝粒规范和基因组完整性中发挥作用，这深刻影响了人类健康和疾病的表观遗传机制。我们发现，裂殖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，这发生在细胞周期的DNA复制阶段。在裂殖酵母中，粟酒裂殖酵母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。我们建议，RNA干扰促进释放RNA聚合酶（PolII）在S期，允许完成着丝粒DNA复制的前导链DNA聚合酶。DNA聚合酶直接募集修饰组蛋白的Rik 1复合物，因此可以随着DNA复制沿着扩散异染色质。在不存在RNAi的情况下，停滞的叉通过同源重组（HR）修复而没有组蛋白修饰，使得HR在不存在RNAi的情况下是必需的。这个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，包括植物中的副突变，以及哺乳动物中的印记和X-失活。粟酒裂殖酵母是细胞周期研究、异染色质沉默和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的模型，以及染色体分析和遗传分析，来验证我们的假设。我们将建立在我们最近的研究结果有关的Rik 1复合物和着丝粒结合蛋白B在DNA复制和修复，以及RNA干扰的作用。 公共卫生相关性：表观遗传机制改变基因功能独立于DNA序列，并对健康和疾病有深远的影响。RNA干扰通过引导与DNA相关的组蛋白的修饰来影响这些机制，确保特异性并避免不适当的基因沉默。我们发现，细胞分裂期间染色体的复制与RNA干扰同时发生，这些机制相互作用导致沉默。从酵母到人类，所涉及的关键分子都是保守的，并且与儿童疾病、智力迟钝、衰老和癌症有关。我们的研究结果表明，针对这些分子的疗法也可能影响基因表达和染色体组织。我们将研究潜在的机制，以确定细胞周期DNA复制期内RNA干扰介导的染色质修饰的原因和后果。

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.