Abstract
The three-dimensional organization of genomes is dynamic and plays a critical role in the regulation of cellular development and phenotypes. Here we use proximity-based ligation methods (i.e. chromosome conformation capture [3C] and circularized chromosome confrmation capture [4C]) to explore the spatial organization of tRNA genes and their locus-specific interactions with the ribosomal DNA. Directed replacement of one lysine and two leucine tRNA loci shows that tRNA spatial organization depends on both tRNA coding sequence identity and the surrounding chromosomal loci. These observations support a model whereby the three-dimensional, spatial organization of tRNA loci within the nucleus utilizes tRNA gene-specific signals to affect local interactions, though broader organization of chromosomal regions are determined by factors outside the tRNA genes themselves.
Highlights
Structural genome organization is manifested on different levels, such as linear arrays of genes and spatial arrangement of chromosome territories [1]
Fluorescence in situ hybridization (FISH) microscopy has shown that these tRNA genes are clustered throughout the cell cycle, with the assistance of condensin complexes bound at each gene, and that clusters localize to the boundary of the nucleolus in a microtubule-dependent manner [6,7,8]
We use three methods that rely on proximity—Genome Conformation Capture (GCC), chromosome conformation capture (3C), and circularized chromosome conformation capture (4C)—to examine the contributions that tRNA genes make to the positioning of specific loci within the S. cerevisiae nucleus
Summary
Structural genome organization is manifested on different levels, such as linear arrays of genes and spatial arrangement of chromosome territories [1]. Clustering of tRNA genes has been observed in fission yeast [13,14], their subnuclear localization is different from that seen in S. cerevisiae. Proximity-based ligation methodologies, which crosslink spatially adjacent loci, permit investigation of direct physical interactions among genes in greater detail. Two of these techniques, Genome Conformation Capture (GCC) and a variant of HiC, have previously been used to produce a yeast genome contact map [15,16] and confirm microscopy results by showing preferential interactions between tRNA genes [15], consistent with the physical clustering observed using fluorescent microscopy. We use three methods that rely on proximity—GCC, chromosome conformation capture (3C), and circularized chromosome conformation capture (4C)—to examine the contributions that tRNA genes make to the positioning of specific loci within the S. cerevisiae nucleus
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