Continuous requirement for the Clr4 complex but not RNAi for centromeric heterochromatin assembly in fission yeast harboring a disrupted RITS complex
| Autor: | Alaa Shaban, Janet F. Partridge, Kevin M. Creamer, Olivia L. George, Sreenath Shanker, Godwin Job |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Cancer Research
Chromatin Immunoprecipitation Heterochromatin Chromosomal Proteins Non-Histone Centromere Cell Cycle Proteins Biology QH426-470 Molecular Biology/Histone Modification Methylation Histones 03 medical and health sciences 0302 clinical medicine RNA interference Genetics and Genomics/Epigenetics Histone methylation Schizosaccharomyces Genetics Heterochromatin assembly Molecular Biology/Chromatin Structure Molecular Biology Genetics (clinical) Ecology Evolution Behavior and Systematics 030304 developmental biology 0303 health sciences Methyltransferase complex Lysine fungi Molecular Biology/Chromosome Structure RNA-Binding Proteins Histone-Lysine N-Methyltransferase Methyltransferases Argonaute Cullin Proteins Molecular Biology/Centromeres Genetics and Genomics/Chromosome Biology Argonaute Proteins Mutation Heterochromatin protein 1 RNA Interference Molecular Biology/RNA-Protein Interactions Schizosaccharomyces pombe Proteins Carrier Proteins 030217 neurology & neurosurgery Research Article |
| Zdroj: | PLoS Genetics, Vol 6, Iss 10, p e1001174 (2010) PLoS Genetics |
| ISSN: | 1553-7404 1553-7390 |
| Popis: | Formation of centromeric heterochromatin in fission yeast requires the combined action of chromatin modifying enzymes and small RNAs derived from centromeric transcripts. Positive feedback mechanisms that link the RNAi pathway and the Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in requirements for H3K9 methylation for full siRNA production and for siRNA production to achieve full histone methylation. Nonetheless, it has been proposed that the Argonaute protein, Ago1, is the key initial trigger for heterochromatin assembly via its association with Dicer-independent “priRNAs.” The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. Here we exploit an assay for heterochromatin assembly in which loss of silencing by deletion of RNAi or Clr-C components can be reversed by re-introduction of the deleted gene. We showed previously that a mutant version of the RITS complex (Tas3WG) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits maintenance of heterochromatin, but prevents its formation when Clr4 is removed and re-introduced. Here we show that the block occurs with mutants in Clr-C, but not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, play a more critical role in assembly when the integrity of RITS is disrupted. Consistent with previous reports, cells lacking Clr-C components completely lack H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low levels of H3K9me2. Further supporting the existence of RNAi–independent mechanisms for establishment of centromeric heterochromatin, overexpression of clr4+ in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants display indistinguishable low levels of H3K9me2 (in contrast to a previous report) challenge the model that priRNAs trigger heterochromatin formation. Instead, our results indicate that RNAi cooperates with RNAi–independent factors in the assembly of heterochromatin. Author Summary Centromeres are the chromosomal regions that promote chromosome movement during cell division. They consist of repetitive DNA sequences that are packaged into heterochromatin. Disruption of centromeric heterochromatin leads to chromosome loss that can result in miscarriages and genetic disorders. We have sought to define the precise steps leading to heterochromatin assembly using fission yeast as the model system. To accomplish this we employed our novel Tas3WG mutant strain that can propagate preassembled heterochromatin but cannot support its de novo establishment. Current models suggest that small RNAs initiate heterochromatin assembly by targeting the RNAi machinery and subsequently the Clr-C chromatin-modifying complex to the centromere. Here, we demonstrate that transient depletion of components of the RNAi pathway that generate or bind small RNAs does not perturb heterochromatin assembly in our Tas3WG strain. Instead, transient depletion of the Clr-C complex blocks heterochromatin assembly, suggesting a critical role for continuous Clr-C activity during heterochromatin assembly in Tas3WG cells. We have directly tested whether Clr-C can target centromeres when expressed in cells deficient for RNAi and Clr-C. We find that RNAi–independent recruitment of Clr-C can occur and likely contributes to the critical initiating mechanisms of heterochromatin assembly. |
| Databáze: | OpenAIRE |
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