RNA quality control factors nucleate Clr4/SUV39H and trigger constitutive heterochromatin assembly.

In eukaryotes, the Suv39 family of proteins tri-methylate lysine 9 of histone H3 (H3K9me) to form constitutive heterochromatin. However, how Suv39 proteins are nucleated at heterochromatin is not fully described. In the fission yeast, current models posit that Argonaute1-associated small RNAs (sRNAs) nucleate the sole H3K9 methyltransferase, Clr4/SUV39H, to centromeres. Here, we show that in the absence of all sRNAs and H3K9me, the Mtl1 and Red1 core (MTREC)/PAXT complex nucleates Clr4/SUV39H at a heterochromatic long noncoding RNA (lncRNA) at which the two H3K9 deacetylases, Sir2 and Clr3, also accumulate by distinct mechanisms. Iterative cycles of H3K9 deacetylation and methylation spread Clr4/SUV39H from the nucleation center in an sRNA-independent manner, generating a basal H3K9me state. This is acted upon by the RNAi machinery to augment and amplify the Clr4/H3K9me signal at centromeres to establish heterochromatin. Overall, our data reveal that lncRNAs and RNA quality control factors can nucleate heterochromatin and function as epigenetic silencers in eukaryotes.

Mathematical model for the role of multiple pericentromeric repeats on heterochromatin assembly.

Although the length and constituting sequences for pericentromeric repeats are highly variable across eukaryotes, the presence of multiple pericentromeric repeats is one of the conserved features of the eukaryotic chromosomes. Pericentromeric heterochromatin is often misregulated in human diseases, with the expansion of pericentromeric repeats in human solid cancers. In this article, we have developed a mathematical model of the RNAi-dependent methylation of H3K9 in the pericentromeric region of fission yeast. Our model, which takes copy number as an explicit parameter, predicts that the pericentromere is silenced only if there are many copies of repeats. It becomes bistable or desilenced if the copy number of repeats is reduced. This suggests that the copy number of pericentromeric repeats alone can determine the fate of heterochromatin silencing in fission yeast. Through sensitivity analysis, we identified parameters that favor bistability and desilencing. Stochastic simulation shows that faster cell division and noise favor the desilenced state. These results show the unexpected role of pericentromeric repeat copy number in gene silencing and provide a quantitative basis for how the copy number allows or protects repetitive and unique parts of the genome from heterochromatin silencing, respectively.

The fission yeast FLCN/FNIP complex augments TORC1 repression or activation in response to amino acid (AA) availability.

The target of Rapamycin complex1 (TORC1) senses and integrates several environmental signals, including amino acid (AA) availability, to regulate cell growth. Folliculin (FLCN) is a tumor suppressor (TS) protein in renal cell carcinoma, which paradoxically activates TORC1 in response to AA supplementation. Few tractable systems for modeling FLCN as a TS are available. Here, we characterize the FLCN-containing complex in Schizosaccharomyces pombe (called BFC) and show that BFC augments TORC1 repression and activation in response to AA starvation and supplementation, respectively. BFC co-immunoprecipitates V-ATPase, a TORC1 modulator, and regulates its activity in an AA-dependent manner. BFC genetic and proteomic networks identify the conserved peptide transmembrane transporter Ptr2 and the phosphoribosylformylglycinamidine synthase Ade3 as new AA-dependent regulators of TORC1. Overall, these data ascribe an additional repressive function to Folliculin in TORC1 regulation and reveal S. pombe as an excellent system for modeling the AA-dependent, FLCN-mediated repression of TORC1 in eukaryotes.

Negative regulation of EGFR signalling by the human folliculin tumour suppressor protein.

Germline mutations in the Folliculin (FLCN) tumour suppressor gene result in fibrofolliculomas, lung cysts and renal cancers, but the precise mechanisms of tumour suppression by FLCN remain elusive. Here we identify Rab7A, a small GTPase important for endocytic trafficking, as a novel FLCN interacting protein and demonstrate that FLCN acts as a Rab7A GTPase-activating protein. FLCN-/- cells display slower trafficking of epidermal growth factor receptors (EGFR) from early to late endosomes and enhanced activation of EGFR signalling upon ligand stimulation. Reintroduction of wild-type FLCN, but not tumour-associated FLCN mutants, suppresses EGFR signalling in a Rab7A-dependent manner. EGFR signalling is elevated in FLCN-/- tumours and the EGFR inhibitor afatinib suppresses the growth of human FLCN-/- cells as tumour xenografts. The functional interaction between FLCN and Rab7A appears conserved across species. Our work highlights a mechanism explaining, at least in part, the tumour suppressor function of FLCN.

Survival in Quiescence Requires the Euchromatic Deployment of Clr4/SUV39H by Argonaute-Associated Small RNAs.

Quiescence (G0) is a ubiquitous stress response through which cells enter reversible dormancy, acquiring distinct properties including reduced metabolism, resistance to stress, and long life. G0 entry involves dramatic changes to chromatin and transcription of cells, but the mechanisms coordinating these processes remain poorly understood. Using the fission yeast, here, we track G0-associated chromatin and transcriptional changes temporally and show that as cells enter G0, their survival and global gene expression programs become increasingly dependent on Clr4/SUV39H, the sole histone H3 lysine 9 (H3K9) methyltransferase, and RNAi proteins. Notably, G0 entry results in RNAi-dependent H3K9 methylation of several euchromatic pockets, prior to which Argonaute1-associated small RNAs from these regions emerge. Overall, our data reveal another function for constitutive heterochromatin proteins (the establishment of the global G0 transcriptional program) and suggest that stress-induced alterations in Argonaute-associated sRNAs can target the deployment of transcriptional regulatory proteins to specific sequences.

Nuclear Noncoding RNAs and Genome Stability.

In modern molecular biology, RNA has emerged as a versatile macromolecule capable of mediating an astonishing number of biological functions beyond its role as a transient messenger of genetic information. The recent discovery and functional analyses of new classes of noncoding RNAs (ncRNAs) have revealed their widespread use in many pathways, including several in the nucleus. This Review focuses on the mechanisms by which nuclear ncRNAs directly contribute to the maintenance of genome stability. We discuss how ncRNAs inhibit spurious recombination among repetitive DNA elements, repress mobilization of transposable elements (TEs), template or bridge DNA double-strand breaks (DSBs) during repair, and direct developmentally regulated genome rearrangements in some ciliates. These studies reveal an unexpected repertoire of mechanisms by which ncRNAs contribute to genome stability and even potentially fuel evolution by acting as templates for genome modification.

PRIMED: PRIMEr database for deleting and tagging all fission and budding yeast genes developed using the open-source genome retrieval script (GRS).

The fission (Schizosaccharomyces pombe) and budding (Saccharomyces cerevisiae) yeasts have served as excellent models for many seminal discoveries in eukaryotic biology. In these organisms, genes are deleted or tagged easily by transforming cells with PCR-generated DNA inserts, flanked by short (50-100 bp) regions of gene homology. These PCR reactions use especially designed long primers, which, in addition to the priming sites, carry homology for gene targeting. Primer design follows a fixed method but is tedious and time-consuming especially when done for a large number of genes. To automate this process, we developed the Python-based Genome Retrieval Script (GRS), an easily customizable open-source script for genome analysis. Using GRS, we created PRIMED, the complete PRIMEr D atabase for deleting and C-terminal tagging genes in the main S. pombe and five of the most commonly used S. cerevisiae strains. Because of the importance of noncoding RNAs (ncRNAs) in many biological processes, we also included the deletion primer set for these features in each genome. PRIMED are accurate and comprehensive and are provided as downloadable Excel files, removing the need for future primer design, especially for large-scale functional analyses. Furthermore, the open-source GRS can be used broadly to retrieve genome information from custom or other annotated genomes, thus providing a suitable platform for building other genomic tools by the yeast or other research communities.

Regulation of histone methylation by noncoding RNAs.

Cells can adapt to their environment and develop distinct identities by rewiring their transcriptional networks to regulate the output of key biological pathways without concomitant mutations to the underlying genes. These alterations, called epigenetic changes, persist stably through mitotic or, in some instances, meiotic cell divisions. In eukaryotes, heritable changes to chromatin structure are a prominent, but not exclusive, mechanism by which epigenetic changes are mediated. These changes are initiated by sequence-specific events, which trigger a cascade of molecular interactions resulting in feedback mechanisms, alterations in chromatin structure, histone posttranslational modifications (PTMs), and ultimately establishment of distinct transcriptional states. In recent years, advances in next generation sequencing have led to the discovery of several novel classes of noncoding RNAs (ncRNAs). In addition to their well-established cytoplasmic roles in posttranscriptional regulation of gene expression, ncRNAs have emerged as key regulators of epigenetic changes via chromatin-dependent mechanisms in organisms ranging from yeast to man. They function by affecting chromatin structure, histone PTMs, and the recruitment of transcriptional activating or repressing complexes. Among histone PTMs, lysine methylation serves as the binding substrate for the recruitment of key protein complexes involved in the regulation of genome architecture, stability, and gene expression. In this review, we will outline the known mechanisms by which ncRNAs of different origins regulate histone methylation, and in doing so contribute to a variety of genome regulatory functions in eukaryotes.