Characterization of the Role of the RPA135-IGS1 Interaction in the Stability of Ribosomal DNA in Saccharomyces cerevisiae

Abstract

The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. The O’Sullivan laboratory previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA repeats (IGS1-rDNA) and the intergenic sequence between the locus encoding the second largest RNA polymerase I (RNAP I) subunit and a lysine tRNA gene (RPA135-tK(CUU)P). Considering the functional association of the loci involved in the RPA135-IGS1 interaction and the lack of information of this type of structure, I set out to test the hypothesis that the interaction represented a feedback loop. I used quantitative chromosome conformation capture (q3C) in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. The tK(CUU)P locus was involved in stabilizing the RPA135-IGS1 interaction. I also demonstrated that the RPA135-IGS1 interaction is dependent on the inactive rDNA repeats and the Msn2 protein. Surprisingly, I found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. I conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and IGS1-rDNA loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. My results provide evidence that chromosomal interactions involve composite DNA elements and affirm the importance of linear and threedimensional genome organization in genome function. This study is the first to characterize a biological function for an inter-chromosomal interaction.