Guardians of the genome: Connecting chromatin structure and genome integrity in Saccharomyces cerevisiae

Abstract

Branched DNA structures arise during recombination and during other DNA repair processes such as repair of stalled replication forks. One branched structure, the Holliday Junction (HJ), is a four-way junction that can connect sister chromatids or homologous chromosomes. We show a physical interaction between the HJ-cleaving enzyme Yen1 and Uls1. Uls1 shares homology with the SWI/SNF-family of chromatin remodelers and has a SUMO-targeted ubiquitin ligase activity, but its cellular role remains poorly characterized. SUMOylation of Yen1 strengthened the interaction with Uls1 and both yen1 and uls1 displayed negative genetic interactions with mutations in a gene encoding an alternative HJ-cleaving enzyme MUS81. Hence, Uls1, Yen1 and Mus81 appeared to be partially redundant. A uls1 strain, however, showed minimal changes in nucleosome positioning and occupancy, as determined by a genome-wide micrococcal nuclease sequencing (MNase-Seq) analysis. Together, these results suggested a role of Uls1 in Yen1-mediated HJ resolution rather than SWI/SNF-like chromatin remodeling activity.

Telomeres protect the ends of the linear chromosomes in eukaryotes from both degradation and mistaken recognition as DNA damage. In Saccharomyces cerevisiae, silent information regulator (Sir) proteins silence transcription from telomeres, by forming a higher-order chromatin structure reminiscent of heterochromatin. An MNase-Seq analysis comparing nucleosome occupancy/positioning in a sir2 mutant with a WT strain showed that the histone deacetylase Sir2 was required for stabilizing a nucleosome in subtelomeric X-elements. The Reb1 transcription factor destabilized the same nucleosome, hence competing with Sir2. Transcription at telomeres results in a species of long noncoding RNA called telomeric repeat containing RNAs (TERRAs). Sir2 and Reb1 limited TERRA steady-state levels, by different mechanisms where Reb1 presumably blocked transcription elongation. The transcriptional start sites of TERRAs mapped just centromere proximal to the Sir2-stabilized nucleosome and the nucleosome occupancy of X-elements was dependent on the position close to the telomeres rather than the DNA sequence of the X-element itself.

Further analysis of nucleosome occupancy in the sir2 mutant strain revealed Sir2-stabilized nucleosomes in the intergenic spacer region (IGS) of the ribosomal DNA (rDNA) locus. The lowered nucleosome occupancy in the IGS overlapped with the promoter for a non-coding RNA, which in turn regulates rDNA copy number. The sir2 mutant also displayed indirect effects on nucleosome occupancy at cell-type specific genes.

Together, the results reveal important roles for Sir2 in regulating nucleosome occupancy/positioning and begin exploring links between chromatin structure, TERRA transcription and maintenance of genome stability.