RNAi and Epigenetic Control of Higher-Order Chromatin Assembly

高阶染色质组装的 RNAi 和表观遗传控制

• 批准号: 8349035
• 负责人: shivinder s grewal
• 金额: $ 109.25万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349035
• 关键词: Affect; Antisense RNA; Beryllium; Biochemical; Cell Cycle; Cell physiology; Cells; Chromatin; Chromatin Modeling; Chromosome Structures; Complex; Coupled; Development; Double-Stranded RNA; Ensure; Epigenetic Process; Fission Yeast; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomics; Heterochromatin; Histone H2A; Histone H3; Investigation; Large-Scale Sequencing; Light; Link; Location; Lysine; Maintenance; Maps; Measures; Mediating; Messenger RNA; Modeling; Modification; Pattern; Play; Process; Proteins; RNA Interference; RNA Polymerase II; Regulation; Resolution; Role; S Phase; Small Interfering RNA; Small RNA; Structure; Therapeutic; Transcript; Variant; Work; cancer therapy; human disease; insight; interest; novel; prevent

The dynamic regulation of higher-order chromosome structure governs diverse cellular processes ranging from stable inheritance of gene expression patterns to other aspects of global chromosome structure essential for preserving genomic integrity. Our earlier studies revealed that RNA interference (RNAi), whereby double-stranded RNAs silence cognate genes, plays a critical role in targeting of heterochromatin, a specialized form of chromatin that can inhibit transcription and recombination across large chromosomal domains, to specific locations in the fission yeast Schizosaccharomyces pombe genome. Subsequent genetic and biochemical investigations identified RNAi-induced transcriptional gene silencing (RITS) complex that provided direct link between small RNAs and heterochromatin formation. These studies also uncovered surprising interdependency between heterochromatin and RNAi mechanism and led to the discovery of an elegant self-reinforcing RNAi loop mechanism that ensures both transcriptional and post-transcriptional silencing in cis. In this loop mechanism, RNAi machinery operates as a stable component of the heterochromatic domains via tethering of RNAi complexes (such as RITS) to heterochromatin marks (including histone H3 methylated at lysine 9) to destroy repeat transcripts that escape heterochromatin-mediated transcriptional silencing. The processing of transcripts by RNAi machinery generate small interfering RNAs (siRNAs) that are utilized for further targeting of heterochromatin complexes, so the mechanism continues. We have extended these analyses to gain insights into full spectrum of target sequences across the entire S. pombe genome affected by the RNAi and heterochromatin machineries. In a comprehensive study, we developed a high-resolution map of the heterochromatin distribution across the entire S. pombe genome. These analyses together with mapping of RNAi components and large scale sequencing of siRNAs associated with an RNAi effector RITS complex, involved in heterochromatic silencing, have yielded novel insights into the epigenetic profile of this model eukaryotic genome. In an interesting new development, our recent work suggests that heterochromatic structures are dynamically regulated during the cell cycle. In particular, heterochromatic repeat elements are transcribed during a brief window during the S-phase. Importantly, we have discovered that the transcription of repeats by RNA polymerase II is coupled to the recruitment of heterochromatin complexes, supporting a prominent role for transcriptional machinery in determining the epigenetic makeup of the genome. In another surprising finding, we have recently discovered that low levels of heterochromatin factors localize broadly across euchromatic regions containing genes and cooperate with RNAi machinery to regulate expression of RNA polymerase II transcripts across large portions of the genome. In particular, we have found that heterochromatin/RNAi factors prevent accumulation of potentially deleterious antisense RNAs. Heterochromatin and RNAi factors are partially redundant in this regard with a histone H2A variant H2A.Z. Loss of Clr4/Suv39h-containing heterochromatin silencing complex or an Argonaute protein alone has little effect on antisense transcript levels, but cells lacking either of these factors and H2A.Z show markedly increased levels of antisense RNAs that are normally degraded by the exosome. These analyses suggest that in addition to performing other functions, heterochromatin and RNAi factors cooperate with H2A.Z to suppress antisense transcripts, which has important implications for diverse chromosomal processes.

高阶染色体结构的动态调节支配着从基因表达模式的稳定遗传到保持基因组完整性所必需的全局染色体结构的其他方面的各种细胞过程。我们早期的研究表明，RNA干扰（RNAi），即双链RNA沉默同源基因，在靶向异染色质中起着关键作用，异染色质是一种特殊形式的染色质，可以抑制跨大染色体结构域的转录和重组，在裂殖酵母裂殖酵母基因组中的特定位置。随后的遗传和生物化学研究确定了RNAi诱导的转录基因沉默（RITS）复合物，该复合物提供了小RNA和异染色质形成之间的直接联系。这些研究还揭示了异染色质和RNAi机制之间令人惊讶的相互依赖性，并导致发现了一种优雅的自增强RNAi环机制，该机制确保了顺式转录和转录后沉默。在这种环机制中，RNAi机制作为异染色质结构域的稳定组分，通过将RNAi复合物（如RITS）拴系到异染色质标记（包括在赖氨酸9处甲基化的组蛋白H3）上来破坏逃避异染色质介导的转录沉默的重复转录物。通过RNAi机制加工转录物产生小干扰RNA（siRNA），其用于进一步靶向异染色质复合物，因此该机制继续。我们已经扩展了这些分析，以深入了解整个S。通过RNAi和异染色质机制影响粟酒基因组。在一项全面的研究中，我们开发了一个高分辨率的异染色质分布图在整个S。粟酒基因组这些分析与RNAi组分的作图和与RNAi效应子RITS复合物相关的siRNA的大规模测序一起，参与异染色质沉默，产生了对该模型真核基因组的表观遗传谱的新见解。在一个有趣的新发展，我们最近的工作表明，异染色质结构在细胞周期的动态调节。特别地，异染色质重复元件在S期期间的短暂窗口期间被转录。重要的是，我们已经发现，RNA聚合酶II的重复序列的转录与异染色质复合物的募集相结合，支持转录机制在决定基因组的表观遗传组成中的重要作用。在另一个令人惊讶的发现中，我们最近发现，低水平的异染色质因子广泛地定位于含有基因的常染色质区域，并与RNAi机制合作来调节RNA聚合酶II转录物在基因组的大部分中的表达。特别地，我们已经发现异染色质/RNAi因子防止潜在有害的反义RNA的积累。异染色质和RNAi因子在这方面与组蛋白H2 A变体H2A.Z部分冗余。含有Clr 4/Suv 39 h的异染色质沉默复合物或单独的Argonaute蛋白的损失对反义转录物水平几乎没有影响，但是缺乏这些因子和H2A.Z中的任一种的细胞显示出通常被外泌体降解的反义RNA的水平显著增加。这些分析表明，除了执行其他功能，异染色质和RNAi因子与H2A.Z合作，以抑制反义转录本，这对不同的染色体过程具有重要意义。