Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

Dekker J (2009) Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions. PLoS Genet 5(5): e1000478. doi:10.1371/journal.pgen.1000478 Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long- Range Interactions Adriana Miele 0 Kerstin Bystricky 0 Job Dekker 0 Wendy A. Bickmore, Medical Research Council Human Genetics Unit, United Kingdom 0 1 Program in Gene Function and Expression, University of Massachusetts Medical School , Worcester , Massachusetts, United States of America, 2 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School , Worcester , Massachusetts, United States of America, 3 Laboratoire de Biologie Mole culaire Eucaryote (LBME), University of Toulouse , Toulouse, France, 4 UMR5099 , Centre National de la Recherche Scientifique , IFR109, Toulouse , France The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic subnuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions. - Funding: This work was supported in part by a grant from the National Institutes of Health (HG003143) to JD. KB acknowledges financial support from the University Paul Sabatier, Toulouse, and the ANR (project JC05-42116) and the Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The eukaryotic nucleus tends to be organized so that active and inactive sub-nuclear domains are spatially separated [14]. For instance, active genes co-localize in a limited number of transcription factories, while heterochromatic regions are found clustered in silenced nuclear compartments. Examples of the latter are found in Drosophila melanogaster and Arabidopsis thaliana where the large heterochromatic regions encompassing the centromeres associate to form a single chromocenter, and in mammalian cells where centromeres cluster in a small number of foci [57]. In most cases heterochromatin is found clustered near the nuclear envelope [1,8,9]. In the yeast Saccharomyces cerevisiae, heterochromatin is found at and near the 32 telomeres, and at the two silent mating type loci, HML and HMR, located near the left and right telomere of chromosome III, respectively [10,11]. These 34 loci co-localize in 48 clusters at the nuclear periphery [1216]. A similar phenomenon is observed in Schizosaccharomyces pombe in which the heterochromatic centromeres, telomeres, and mating type loci cluster in silent foci at the nuclear periphery [17]. Heterochromatic clusters are thought to represent nuclear subcompartments that are enriched in silencing proteins, while the rest of the nucleus is depleted in such factors [14,18,19]. Although the importance of association of genes with silent compartments in the process of silencing is well established, the mechanisms that drive these interactions are poorly understood. Formation of heterochromatin at HM loci has been characterized in detail (for reviews see [11,20,21]). Silencing at HML and HMR requires cis-acting silencer elements [11]. Protein complexes, such as Rap1p and the Origin Recognition Complex (ORC), bind to these silencer elements and help recruit Silent Information Regulator (Sir) proteins. Sir1p associates with Orc1p. Subsequently, Sir4p is recruited to the silencers via its interaction with Rap1p and Sir1p. Sir4p likely recruits Sir2p and is also required to recruit Sir3p to the silencer. Sir2p is a NAD-dependent histone deacetylase that deacetylates H4 K16 at nearby nucleosomes, which provides a binding site for additional SIR2-4 complexes [22,23]. This positive feedback loop allows spreading of the SIR24 complex throughout the mating type loci, resulting in positioned nucleosomes and gene silencing throughout the region [24,25]. Thus, histones and appropriate nucleosome assembly contribute to formation of heterochromatin, perhaps due to the fact that binding and spreading of the Sir complex occurs through direct interactions with histones. In addition, genetic evidence indicates that the histone chaperone Asf1p and the CAF-1 and HIR nucleosome assembly complexes have partially overlapping Chromosomes are non-randomly positioned inside cells, and this organization is relevant for genome regulation. Spatial clustering of heterochromatic loci provides a striking example of nuclear compartmentalization. In S. cerevisiae, the presence of heterochromatic sub-nuclear domains has been well established, but their mechanisms of formation are not fully understood. Here, we analyzed the DNA elements and protein complexes that are critical for formation of heterochromatic clusters. We focused on heterochromatic regions on chromosome IIIthe two telomeres, as well as the silent mating type loci HML and HMR, located on the left and right end of the chromosome, respectively. We employed live cell 3-D imaging and chromosome conformation capture (3C) and found that these loci specifically interact most prominently near silencer elements that flank the loci. Analysis of a panel of mutants showed that complexes involved in silencing are also involved in long-range interactions. Interestingly, we find that heterochromatic interactions are mechanistically distinct from silencing and independent of tethering to the nuclear periphery. Our results indicate that formation of heterochromatic clusters depends on correctly (...truncated)