RNAi-directed assembly of heterochromatin in fission yeast

Heterochromatin is an epigenetically heritable and conserved feature of eukaryotic chromosomes with important roles in chromosome segregation, genome stability, and gene regulation. The formation of heterochromatin involves an ordered array of chromatin changes, including histone deacetylation, histone H3-lysine 9 methylation, and recruitment of histone binding proteins such as Swi6/HP1. Recent discoveries have uncovered a role for the RNA interference (RNAi) pathway in heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and other eukaryotes. Purification of two RNAi complexes, RITS and RDRC, from fission yeast has provided further insight into the mechanism of RNAi-mediated heterochromatin assembly. These discoveries have given rise to a model in which small interfering RNA molecules act as specificity factors that initiate epigenetic chromatin modifications and double strand RNA synthesis at specific chromosome regions.     

芽殖酵母和裂殖酵母中异染色质形成机制

芽殖酵母(Saccharomyces cerevisiae)和裂殖酵母(Schizosaccharomyces pombe)是用来研究异染色质形成、细胞周期、DNA复制等重要细胞功能的理想单细胞真核生物.本文主要介绍这2种酵母中异染色质形成的机制.异染色质是一种抑制基因转录和DNA重组的特殊染色质结构.尽管在芽殖酵母和裂殖酵母中异染色质形成都需要组蛋白修饰,但异染色质建立的机制不同.在芽殖酵母中参与异染色质形成的主要蛋白是Sir1-4蛋白（其中Sir2为组蛋白H3去乙酰化酶）,而组蛋白H3赖氨酸9甲基化酶Clr4和异染色质蛋白Swi6在裂殖酵母异染色质形成中起关键的作用.在这两个酵母中,参与异染色质形成的组蛋白修饰蛋白由DNA结合蛋白招募到异染色质.此外,裂殖酵母也利用RNA干扰系统招募组蛋白修饰蛋白.     

Inputs and outputs for chromatin-targeted RNAi

Plant gene silencing is targeted to transposons and repeated sequences by small RNAs from the RNA interference (RNAi) pathway. Like classical RNAi, RNA-directed chromatin silencing involves the cleavage of double-stranded RNA by Dicer endonucleases to create small interfering RNAs (siRNAs), which bind to the Argonaute protein. The production of double-stranded RNA (dsRNA) must be carefully controlled to prevent inappropriate silencing. A plant-specific RNA polymerase IV (Pol IV) initiates siRNA production at silent heterochromatin, but Pol IV-independent mechanisms for making dsRNA also exist. Downstream of siRNA biogenesis, multiple chromatin marks might be targeted by Argonaute-siRNA complexes, yet mechanisms of chromatin modification remain poorly understood. Genomic studies of siRNA target loci promise to reveal novel biological functions for chromatin-targeted RNAi.     

Phase separation of chromatin and small RNA pathways in plants

Gene expression can be modulated by epigenetic mechanisms, including chromatin modifications and small regulatory RNAs. These pathways are unevenly distributed within a cell and usually take place in specific intracellular regions. Unfortunately, the fundamental driving force and biological relevance of such spatial differentiation is largely unknown. Liquid–liquid phase separation (LLPS) is a natural propensity of demixing liquid phases and has been recently suggested to mediate the formation of biomolecular condensates that are relevant to diverse cellular processes. LLPS provides a mechanistic explanation for the self-assembly of subcellular structures by which the efficiency and specificity of certain cellular reactions are achieved. In plants, LLPS has been observed for several key factors in the chromatin and small RNA pathways. For example, the formation of facultative and obligate heterochromatin involves the LLPS of multiple relevant factors. In addition, phase separation is observed in a set of proteins acting in microRNA biogenesis and the small interfering RNA pathway. In this Focused Review, we highlight and discuss the recent findings regarding phase separation in the epigenetic mechanisms of plants.     

piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans

Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.     

Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes

The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.     

RNA interference in fungi: pathways, functions, and applications

Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora, and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.     

Antiviral silencing in animals

RNA silencing or RNA interference (RNAi) refers to the small RNA-guided gene silencing mechanism conserved in a wide range of eukaryotic organisms from plants to mammals. As part of this special issue on the biology, mechanisms and applications of RNAi, here we review the recent advances on defining a role of RNAi in the responses of invertebrate and vertebrate animals to virus infection. Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been identified. Assays used for the identification of viral suppressors and possible biological functions of both viral miRNAs and suppressors are discussed. We propose that herpes viral miRNAs may act as specificity factors to initiate heterochromatin assembly of the latent viral DNA genome in the nucleus.     

Epigenetic displacement of HP1 from heterochromatin by HIV-1 Vpr causes premature sister chromatid separation

Although pericentromeric heterochromatin is essential for chromosome segregation, its role in humans remains controversial. Dissecting the function of HIV-1-encoded Vpr, we unraveled important properties of heterochromatin during chromosome segregation. In Vpr-expressing cells, hRad21, hSgo1, and hMis12, which are crucial for proper chromosome segregation, were displaced from the centromeres of mitotic chromosomes, resulting in premature chromatid separation (PCS). Interestingly, Vpr displaced heterochromatin protein 1-α (HP1-α) and HP1-γ from chromatin. RNA interference (RNAi) experiments revealed that down-regulation of HP1-α and/or HP1-γ induced PCS, concomitant with the displacement of hRad21. Notably, Vpr stimulated the acetylation of histone H3, whereas p300 RNAi attenuated the Vpr-induced displacement of HP1-α and PCS. Furthermore, Vpr bound to p300 that was present in insoluble regions of the nucleus, suggesting that Vpr aberrantly recruits the histone acetyltransferase activity of p300 to chromatin, displaces HP1-α, and causes chromatid cohesion defects. Our study reveals for the first time centromere cohesion impairment resulting from epigenetic disruption of higher-order structures of heterochromatin by a viral pathogen.