RNAi and Epigenetic Control of Higher-Order Chromatin Assembly

高阶染色体结构的动态调节控制着多种细胞过程，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。我们早期的研究表明，RNA 干扰 (RNAi)，即双链 RNA 沉默同源基因，在异染色质（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）靶向裂殖酵母裂殖酵母基因组中的特定位置方面发挥着关键作用。随后的遗传和生化研究发现了 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我强化 RNAi 环机制，该机制确保顺式转录和转录后沉默。在这种环机制中，RNAi 机制通过将 RNAi 复合物（例如 RITS）束缚到异染色质标记（包括在赖氨酸 9 处甲基化的组蛋白 H3）上，作为异染色质结构域的稳定组件发挥作用，以破坏逃避异染色质介导的转录沉默的重复转录本。 RNAi 机器对转录本的处理会产生小干扰 RNA (siRNA)，用于进一步靶向异染色质复合物，因此该机制仍在继续。我们扩展了这些分析，以深入了解受 RNAi 和异染色质机制影响的全谱靶序列。在一项综合研究中，我们绘制了整个粟酒裂殖酵母基因组中异染色质分布的高分辨率图。这些分析连同 RNAi 成分的图谱和与 RNAi 效应子 RITS 复合物相关的 siRNA 的大规模测序，参与异染色质沉默，已经对该模型真核基因组的表观遗传谱产生了新的见解。在一项有趣的新进展中，我们最近的工作表明异染色质结构在细胞周期中受到动态调节。特别是，异色重复元件在 S 期的短暂窗口内被转录。重要的是，我们发现RNA聚合酶II对重复序列的转录与异染色质复合物的招募相结合，支持转录机制在确定基因组表观遗传组成中的重要作用。在另一个令人惊讶的发现中，我们发现低水平的异染色质因子广泛分布在含有基因的常染色质区域，并与 RNAi 机制合作调节大部分基因组中 RNA 聚合酶 II 转录物的表达。特别是，我们发现异染色质/RNAi 因子可以防止潜在有害的反义 RNA 的积累。在这方面，异染色质和 RNAi 因子对于组蛋白 H2A 变体 H2A.Z 来说是部分冗余的。含有 Clr4/Suv39h 的异染色质沉默复合物或单独的 Argonaute 蛋白的丢失对反义转录物水平几乎没有影响，但缺乏这些因子和 H2A.Z 中的任何一个的细胞显示出通常被外泌体降解的反义 RNA 水平显着增加。这些分析表明，除了执行其他功能外，异染色质和 RNAi 因子还与 H2A.Z 合作抑制反义转录本，这对不同的染色体过程具有重要意义。在另一个重要发现中，我们发现了一种新的异染色质组装途径，该途径依赖于转录和 RNA，但不需要 RNAi 机制。我们发现，兼性异染色质是在配子发生所需的基因上建立的（在营养细胞中受到抑制），并且其形成依赖于保守的RNA降解因子，包括参与转录物多腺苷酸化的蛋白质复合物和降解基因转录物的外泌体。重要的是，通过该途径形成的异染色质是响应诱导配子发生的信号而受到调节的。最近，我们研究了细胞如何区分不同类型的 RNA 分子并将它们的加工与异染色质的建立联系起来。我们发现了与不同因子（包括剪接因子）相关的核心机制，以介导 mRNA、ncRNA 和内含子的靶向，从而在整个基因组的特定位点组装异染色质结构域。这些开创性的研究为理解高等真核生物中更复杂的调控网络（包括涉及多梳沉默的调控网络）铺平了道路，并为理解基因组大规模重编程以响应通过异染色质修饰而发生的发育和环境线索奠定了基础。

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 the RNAi-induced transcriptional gene silencing (RITS) complex that provides a 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 generates 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 the full spectrum of target sequences affected by the RNAi and heterochromatin machineries. In a comprehensive study, we developed a high-resolution map of 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 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. In another important finding, we have discovered a novel heterochromatin assembly pathway that relies on transcription and RNAs but does not require RNAi machinery. We have discovered that facultative heterochromatin is established at genes required for gametogenesis (which are repressed in vegetative cells), and that its formation is dependent on conserved RNA degradation factors, including a protein complex involved in polyadenylation of transcripts and the exosome that degrade gene transcripts. Importantly, heterochromatin formation by this pathway is modulated in response to signals that induce gametogenesis. Most recently, we have investigated how the cell distinguishes different types of RNA molecules and links their processing to heterochromatin establishment. We discovered core machinery that associates with different factors (including splicing factors) to mediate targeting of mRNA, ncRNA and introns to assemble heterochromatin domains at specific sites throughout the genome. These groundbreaking studies have paved the way for understanding the more complex regulatory networks at work in higher eukaryotes including that involve polycomb silencing, and has provided a foundation for understanding the large scale reprogramming of the genome in response to developmental and environmental cues that occur through modifications of heterochromatin.