Structural genome organization is manifested on different levels, such as linear arrays of genes and spatial arrangement of chromosome territories 
. Recent studies have implicated interactions that form between genomic loci in the regulation of genes 
and of cellular processes such as development 
Examination of the spatial organization of gene families can provide insight into how position relates to evolutionary or functional imperatives. The largest family of co-regulated genes in the eukaryotic genome is the RNA polymerase III (Pol III)-transcribed tRNA gene family. The budding yeast Saccharomyces cerevisiae
has 274 tRNA genes that are dispersed throughout the linear maps of the 16 chromosomes. 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 
. Condensin has also been localized to the nucleolar ribosomal DNA (rDNA) repeats, and mutants of condensin affect proper compaction of the rDNA repeats 
. Clustering of tRNA genes has also been observed in fission yeast 
, although their subnuclear localization is different from that seen in S. cerevisiae
. Proximity-based ligation methodologies, which cross-link spatially adjacent loci, now 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 
and confirm microscopy results by showing preferential interactions between tRNA genes 
, consistent with the physical clustering observed using fluorescent microscopy.
Since the localization of a large number of dispersed genes to a single subnuclear region necessarily requires a vast rearrangement of the genome, it is of interest to investigate whether individual tRNA gene associations are a controlling influence on the overall organization of the genome, or merely serve as non-specific “fasteners,” providing some level of local condensation, while global organization is determined by other factors. Here 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.