RNAi-dependent formation of heterochromatin and its diverse functions

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Expression profiling of eukaryotic genomes has revealed widespread transcription outside the confines of protein-coding genes, leading to production of antisense and non-coding RNAs (ncRNAs). Studies in Schizosaccharomyces pombe and multicellular organisms suggest that transcription and ncRNAs provide a framework for the assembly of heterochromatin, which has been linked to various chromosomal processes. In addition to gene regulation, heterochromatin is crucial for centromere function, cell fate determination as well as transcriptional and posttranscriptional silencing of repetitive DNA elements. Recently, heterochromatin factors have been shown to suppress antisense RNAs at euchromatic loci. These findings define conserved pathways that probably have major impact on the epigenetic regulation of eukaryotic genomes.

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Formation of heterochromatin: a role for the RNAi machinery

The core of heterochromatin assembly pathway found in Drosophila and mammals is conserved in S. pombe [5]. It involves posttranslational modifications of histones and a common set of structural proteins. In addition to histone deacetylation, heterochromatin assembly requires methylation of histone H3 at lysine 9 (H3K9me) that provides binding site for the HP1 family chromodomain proteins [12]. In S. pombe, Chp1, Chp2, and Swi6 bind H3K9me through their chromodomain. Methylation of H3K9 is

S-phase transcription of centromeric repeats and heterochromatin assembly

An important aspect of RNAi-mediated heterochromatin assembly is that target repeats need to be transcribed. In addition to generating siRNA precursors, Pol II transcription appears to play a more direct role in heterochromatin assembly [28, 31•]. Mutations in Pol II subunits impair heterochromatic assembly [32•, 33•]. Heterochromatic silencing also requires splicing factors known to associate with elongating Pol II [34, 35]. An apparent paradox is that Pol II must access sequences assembled

Spreading and maintenance of heterochromatin

Heterochromatin nucleated at specific sites can spread to the surrounding regions. This process, impacted by the chromosomal context [44], involves a complex interplay between histone-modifying activities and proteins that bind to modified histones [5]. Spreading of heterochromatin has been reported to require RNAi [45]. RITS subunit Tas3, that connects RNAi and heterochromatin factors [46, 47], contains an alpha helical motif, which oligomerises to promote cis spreading of RITS at centromeres [

Posttranscriptional and transcriptional heterochromatic silencing

Heterochromatin spreading has been described in different species. However, the biological significance of this conserved feature of heterochromatin has not been extensively studied. Mapping of heterochromatin-associated factors by ChIP in S. pombe has unraveled a new theme in which heterochromatin serves as a recruiting platform for factors involved in different chromosomal processes [11]. Both posttranscriptional and transcriptional silencing activities rely on heterochromatin for their

Heterochromatin-independent targeting of HDAC activities

RNAi and heterochromatin while dramatically affecting the silencing of centromeric repeats have only minor impact on retrotransposons and their remnants dispersed across S. pombe genome [58]. As retrotransposons also pose threat to genome stability, additional mechanisms must exist to silence these elements. An emerging theme is that a ‘toolkit’ of repressors are targeted to different classes of repeats by distinct recruitment mechanisms (Figure 2a). Similar to HP1 targeting HDACs to

Antisense suppression by heterochromatin and RNAi factors

Recent studies have revealed a surprising function for RNAi and heterochromatin factors in suppressing antisense transcripts at euchromatic loci [62, 63]. Components of Clr4 complex can be detected at euchromatic genes albeit at low levels. Deletions of Clr4 (or its interacting Rik1) and Ago1 cause upregulation of readthrough antisense RNAs at convergent genes. Heterochromatin might facilitate localization of cohesin that in turn promotes transcription termination [62]. Antisense suppression

Heterochromatin and genome stability

Heterochromatin-mediated repression of transcription and recombination is essential for maintaining genomic integrity [9]. Indeed, when null alleles of heterochromatin factors are combined with mutations in recombination/repair proteins, double mutants show severe growth defects [64•]. HP1 proteins recruit a variety of effectors essential for genome stability. Loading of Hsk1-Dbf4 kinase essential for initiation of DNA replication at centromeres and the mat locus requires Swi6 [65]. In

Relationship of heterochromatic silencing in S. pombe to other systems

Heterochromatin formation in S. pombe shares many parallels with epigenetic silencing in other systems, although different lineages have emphasized different aspects of heterochromatin regulation. Small RNA-based mechanisms play prominent roles in chromatin modifications in plants, ciliates, C. elegans and Drosophila [70, 71, 72, 73]. Tetrahymena selectively eliminates parts of its genome when it produces actively transcribed macronucleus. This process requires RNAi and small RNAs that are

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

I thank F. Reyes-Turcu, M. Zofall, N. Ashourian, N. Komissarova and H. Cam for helpful comments. Research in Grewal laboratory is supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

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