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Currie, Mark

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Currie

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Mark

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Currie, Mark

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2016 - 20172

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Author's Publications: search.filters.isAuthorOfPublication.21ca4793-9a1c-4b08-b9b3-23bf156ebaa8

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    Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    (2017) Jih, Gloria; Iglesias, Nahid; Currie, Mark; Bhanu, Natarajan V.; Paulo, Joao; Gygi, Steven; Garcia, Benjamin A.; Moazed, Danesh
    Heterochromatic DNA domains play important roles in regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway1–9. Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and tri-methylation (H3K9me2 and H3K9me3, respectively) and heterochromatin formation10–17. H3K9me is in turn required for recruitment of RNAi to chromatin to promote sRNA amplification11,15,18. Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast S. pombe homolog of mammalian SUV39H H3K9 methyltransferases, we designed active site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing (CTGS) at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defense.
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    Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes
    (eLife Sciences Publications, Ltd, 2016) Behrouzi, Reza; Lu, Chenning; Currie, Mark; Jih, Gloria; Iglesias, Nahid; Moazed, Danesh
    Heterochromatin is a conserved feature of eukaryotic chromosomes with central roles in regulation of gene expression and maintenance of genome stability. Heterochromatin formation involves spreading of chromatin-modifying factors away from initiation points over large DNA domains by poorly understood mechanisms. In Saccharomyces cerevisiae, heterochromatin formation requires the SIR complex, which contains subunits with histone-modifying, histone-binding, and self-association activities. Here, we analyze binding of the Sir proteins to reconstituted mono-, di-, tri-, and tetra-nucleosomal chromatin templates and show that key Sir-Sir interactions bridge only sites on different nucleosomes but not sites on the same nucleosome, and are therefore 'interrupted' with respect to sites on the same nucleosome. We observe maximal binding affinity and cooperativity to unmodified di-nucleosomes and propose that nucleosome pairs bearing unmodified histone H4-lysine16 and H3-lysine79 form the fundamental units of Sir chromatin binding and that cooperative binding requiring two appropriately modified nucleosomes mediates selective Sir recruitment and spreading. DOI: http://dx.doi.org/10.7554/eLife.17556.001