Abstract
The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear 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.
Highlights
The eukaryotic nucleus tends to be organized so that active and inactive sub-nuclear domains are spatially separated [1,2,3,4]
Chromosomes are non-randomly positioned inside cells, and this organization is relevant for genome regulation
We focused on heterochromatic regions on chromosome III—the two telomeres, as well as the silent mating type loci HML and HMR, located on the left and right end of the chromosome, respectively
Summary
The eukaryotic nucleus tends to be organized so that active and inactive sub-nuclear domains are spatially separated [1,2,3,4]. Active genes co-localize in a limited number of transcription factories, while heterochromatic regions are found clustered in silenced nuclear compartments. 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 4–8 clusters at the nuclear periphery [12,13,14,15,16]. 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
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