Silent chromatin at the middle and ends: lessons from yeasts

Abstract

Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species.

Figure 1

Variegated expression of a gene on packaging into a heterochromatic structure. (A) Cells expressing the wild-type ADE2 gene from its endogenous, euchromatic locus produce colonies that are white, (B) whereas those lacking the ADE2 gene appear red. (C) Juxtaposition of ADE2 to heterochromatin results in its silencing without changing the underlying coding sequence. Although inherited, the packaging state of ADE2 (euchromatic versus heterochromatic) can switch at a low frequency. This results in a variegating phenotype in a clonal population of cells. An example of a telomeric position effect (TPE) (Gottschling et al, 1990) in S. cerevisiae is shown here.

Figure 2

Silent chromatin assembly in budding and fission yeast. (A) Cis-acting DNA sequences (nucleation sites, yellow boxes) are necessary to nucleate assembly of silent chromatin. Trans-acting proteins that directly bind the nucleation sites are indicated. Nucleation sites at fission yeast centromeres are likely to exist, although they have not been identified to date (yellow boxes). Bidirectional transcription (indicated by black arrows) of cendg/dh/H-like sequences (red boxes) is thought to produce dsRNA, which is processed into siRNAs by the RNAi machinery in S. pombe. siRNAs are required at least for the initiation of heterochromatin assembly at the silent mating-type locus and in addition for the maintenance of heterochromatin at centromeres. (B) Sir3 and Sir4 have dimerization capacity that results in the spread of the SIR complex outward from the nucleation site. Sir3 contributes to the specificity for deacetylated histone tails, whereas Sir4 enhances the affinity of the complex through its ability to bind DNA. Sir2-mediated deacetylation keeps telomeric nucleosomes hypoacetylated creating a high-affinity binding site for Sir3. (C) In S. pombe, the RITS complex promotes Clr4-mediated H3K9 methylation by associating with nascent transcripts through siRNA base pairing, and with methylated H3K9 through the chromodomain of its Chp1 subunit. Low levels of H3K9 methylation are maintained in RNAi mutant cells by a yet to be identified alternative pathway (putative nucleation element, yellow box). Primary siRNAs originating from dsRNA formed by bidirectional transcription of a centromeric sequence could prime further dsRNA synthesis and secondary siRNA generation by recruiting the RDRC complex to the nascent transcript. This would allow the spreading of H3K9me away from the nucleation site. H3K9^me is bound by the chromodomain proteins Chp1, Chp2 and Swi6. The binding of Chp2 to H3K9^me results in the recruitment of the SHREC complex, which in turn deacetylates H3K14. For unknown reasons this reduces RNA Pol II occupancy.

Figure 3

Chromatin-dependent gene silencing mechanisms operate at a transcriptional and/or post-transcriptional level. (A) Silencing of heterochromatin can be achieved by either shutting off transcription (TGS) or by degradation of heterochromatic RNAs (CTGS). In contrast to classic post-transcriptional gene silencing (PTGS), CTGS depends on the status of chromatin from which the gene is transcribed and is therefore referred to as ‘co-transcriptional'. (B) RNAi-mediated degradation of heterochromatic RNAs. Argonaute-containing complexes can be physically linked to heterochromatin through chromodomain proteins. One histone-octamer is shown in grey. The chromodomain protein binds to methylated K9 (orange) of the unstructured N-terminal tail of histone H3. The siRNA (blue) guides Argonaute to the heterochromatic RNA through base-pairing interaction and induces ‘slicing'. (C) Heterochromatic gene silencing mediated by a non-canonical polyA-polymerase and the exosome. RNAs transcribed from heterochromatic regions are identified by Cid14/Trf4 and marked as aberrant with a short polyA tail. This serves as a signal for the exosome to degrade the RNA.

Figure 4

Telomere anchoring and the promotion of TPE in yeast. (A) We show schematically both the silencing-dependent and the silencing- independent pathways of telomere anchoring in S. cerevisiae. The silencing-dependent pathway primarily exploits the integral SIR complex protein, Sir4, and its high-affinity interaction with Enhancer of Silent chromatin 1, which is peripherally associated with the nuclear envelope (NE). Sir4 can also bind yKu, which in turn mediates interaction with telomerase (Schober et al, 2009). Telomerase then binds Mps3, a SUN domain protein that is an integral component of the NE. At telomeres in S phase the yKu–telomerase–Mps3 pathway is sufficient to anchor telomeres in the absence of silent chromatin or Sir4 (Hediger et al, 2002). (B) We show a sequential model for how the binding of telomeres and their sequestration of SIR factors in foci can seed and the establishment of silencing at budding yeast telomeres. We propose that Sir4 is first recruited at the nucleation centre by DNA-binding proteins that can bind Sir4. These include Rap1, ORC, Abf1 and/or yKu. The presence of Sir4 at the locus will then bring it to the nuclear periphery through one of the two Sir4 anchoring pathways (yKu or Esc1) in which the high local concentrations of Sir proteins will help silencing complexes assemble and spread. Once silenced, the repressed telomere can associate with the NE through Esc1, which also increases the local concentration of Sir proteins and reinforces repression with this zone. Importantly, yKu can bind chromosome ends and link them to the nuclear envelope protein, Mps3, in the absence of repression.