In Vivo Dynamics of Swi6 in Yeast: Evidence for a Stochastic Model of Heterochromatin

Abstract

The mechanism for transcriptional silencing of pericentric heterochromatin is conserved from fission yeast to mammals. Silenced genome regions are marked by epigenetic methylation of histone H3, which serves as a binding site for structural heterochromatin proteins. In the fission yeast Schizosaccharomyces pombe, the major structural heterochromatin protein is Swi6. To gain insight into Swi6 function in vivo, we have studied its dynamics in the nucleus of living yeast. We demonstrate that, in contrast to mammalian cells, yeast heterochromatin domains undergo rapid, large-scale motions within the nucleus. Similar to the situation in mammalian cells, Swi6 does not permanently associate with these chromatin domains but binds only transiently to euchromatin and heterochromatin. Swi6 binding dynamics are dependent on growth status and on the silencing factors Clr4 and Rik1, but not Clr1, Clr2, or Clr3. By comparing the kinetics of mutant Swi6 proteins in swi6؊ and swi6 ؉ strains, we demonstrate that homotypic protein-protein interactions via the chromoshadow domain stabilize Swi6 binding to chromatin in vivo. Kinetic modeling allowed quantitative estimation of residence times and indicated the existence of at least two kinetically distinct populations of Swi6 in heterochromatin. The observed dynamics of Swi6 binding are consistent with a stochastic model of heterochromatin and indicate evolutionary conservation of heterochromatin protein binding properties from mammals to yeast.In eukaryotic cells, genomes exist in the form of chromatin. Morphological studies have described two major types of chromatin: euchromatin corresponds to loosely packed chromatin, where most active genes are transcribed, whereas heterochromatin consists of condensed, predominantly transcriptionally repressed chromatin (48). Heterochromatin is molecularly characterized by a high density of nucleosomes containing histone H3 methylated on lysine 9 (H3-K9) (5, 27). In humans, H3-K9 methylation is mediated by methyltransferases Suv39h1 and Suv39h2 (43). The presence of methylated H3-K9 creates a specific binding site for one of the major heterochromatin proteins, HP1 (heterochromatin protein 1) (5,27,37,43). HP1 was originally identified in Drosophila melanogaster as a suppressor of variegation, and it has been shown to modify mammalian position effect variegation in a dose-dependent manner (11,19). The role of HP1 does not appear to be limited to heterochromatin since HP1 also represses, and in some cases activates, euchromatic genes (24, 28). Consistent with its primary role in the formation and maintenance of heterochromatin, HP1 is predominantly localized in heterochromatin domains (12, 33).The system of repression involving methylation of H3-K9 as a mark which is recognized by a structural chromatin protein is conserved from mammalian cells to Schizosaccharomyces pombe. The fission yeast homologues of Suv39h and HP1 are Clr4 and Swi6, respectively (25,30,47). Swi6 binds to three transcriptionally silent heterochromatic regions, the mating ty...

Elimination of shelterin components bypasses RNAi for pericentric heterochromatin assembly

“…We suspect that the ability of pot1D and tpz1D to bypass dcr1D is due to the loss of telomere and subtelomere sequences, which consequently affects silencing at telomeres. Such a model is consistent with recent imaging and biochemical studies showing that HP1/ Swi6 proteins are highly mobile on chromatin (Cheutin et al 2003(Cheutin et al , 2004Festenstein et al 2003;Sadaie et al 2008).…”

“…H3K9me allows for the binding of heterochromatin protein 1 (HP1), which compacts chromatin through its self-association and the recruitment of other chromatin-modifying activities (Grewal and Jia 2007). Although heterochromatin has traditionally been considered silent, stable, and static, recent biochemical and imaging studies show that the binding of HP1 proteins to heterochromatin regions is surprisingly dynamic (Cheutin et al 2003(Cheutin et al , 2004Festenstein et al 2003;Sadaie et al 2008). Moreover, although heterochromatin normally excludes the transcriptional machinery, the DNA repeats within heterochromatin are transcribed, and the transcripts are processed by the RNAi pathway into siRNAs, which target histone-modifying complexes to repetitive DNA elements (Moazed, 2009;Lejeune and Allshire 2011;Castel and Martienssen 2013).…”

Distinct modes of DNA accessibility in plant chromatin

“…However, several studies have now suggested that this view might be an oversimplification. Not only do various high-molecular weight probes and proteins readily enter heterochromatin [10][11][12][13][14] , but also transcription of repetitive sequences by specific RNA polymerases is required for methylation of H3K9 and heterochromatin assembly 43 . It is possible that bursts of transcription occur particularly during S-phase offering an explanation to reconcile the conflicting concepts of transcriptional repression and active transcription in heterochromatin [44][45][46] .…”

“…The view of higher-order structure as a regulator of accessibility, however, has been challenged by measurements of diffusional motilities of nuclear proteins. Proteins can readily diffuse even into highly condensed heterochromatin [10][11][12] , and inert diffusion probes of the size of large protein complexes readily entered condensed chromatin 13,14 . Similarly, a recent study of genome-wide DNA accessibility in Drosophila using M.SssI methylation footprinting questions the view of greatly reduced accessibility of heterochromatin: DNA in chromatin carrying H3K9me2 seems to be as accessible as the rest of the genome 15 .…”

Methylation-independent Binding to Histone H3 and Cell Cycle-dependent Incorporation of HP1β into Heterochromatin

“…Recently published FRAP data on HP1-gfp transfected cells have shown that all forms of HP1-gfp exhibit rapid dynamics, with the bulk of the fluorescence returning to the bleached area within seconds (36,45,47,48). To find out whether this holds in our experimental system, we performed FRAP and inverse FRAP studies on transiently transfected HeLa cells.…”