The Nse1p RING domain is required for maintaining transcriptional silencing and genomic organization in Saccharomyces cerevisiae

Abstract

Chromatin is a dynamic structure that reorganizes to support proper transcription, replication, repair, and segregation. Principal chromosomal constituents that shape chromosomes are the SMC (Structural Maintenance of Chromosomes) complexes. The Saccharomyces cerevisiae genome codes for six SMC proteins, which form three complexes named Cohesin, Condensin, and the Smc5/6 complex. The Smc5/6 complex is an essential, evolutionarily conserved multi-subunit protein complex known for its role in replication, DNA damage repair, and maintenance of rDNA and telomere length. In budding yeast, the Smc5/6 complex is an octameric complex comprising Smc5-Smc6 (heterodimers) and six non-SMC elements, Nse1-Nse6. The Smc5/6 complex is the only SMC complex in which two of the non-SMC elements/subunits have catalytic activity. The Nse1 and the Nse2 subunits contain a RING (Really Interesting New Gene) Zn-finger-like motif. The RING domain of the Nse1p contains eight conserved metal-binding residues that coordinate two zinc atoms in a cross-brace conformation. The RING domain of the Nse1p subunit is characteristic of E3 Ubiquitin ligases and is essential for survival in the presence of external genotoxic stress. The Smc5/6 complex binds to the centromeres, pericentromeric regions, chromosome arms, and near telomeres. A disruption in the activity of the Smc5/6 complex affects several critical cellular processes.

In Part I of the study, I have investigated the role of the Nse1p RING domain in the maintenance of silencing in budding yeast. In Saccharomyces cerevisiae, transcriptional silencing occurs at the silent mating-type loci (HMLα and HMRa, or the HM loci), telomeres, and the ribosomal DNA (rDNA) tandem array. Our observations show that TPE (Telomere Position Effect) is impaired, and subtelomeric genes are de-repressed in the nse1 RING domain mutants. The mutants defective in the Nse1p RING domain also display de-repression of long non-coding RNAs transcribed from telomeres, called Telomere repeat-containing RNA (TERRA). The telomeric association of the Smc5/6 and the SIR complexes are impaired in the nse1 mutants. Despite reduced SIR complex association at telomeres, the genetic interaction analysis with sir2Δ revealed that Nse1p-mediated repression is partially independent of the SIR complex. Interestingly, genetic interaction analysis of rpd3Δ, hda1Δ, and rif1Δ with the nse1C274A mutant revealed partial rescue of subtelomeric silencing. Moreover, mutations in the Nse1p RING domain resulted in moderately reduced telomere tethering to the nuclear envelope and telomere clustering, indicating a defect in telomere organization.

In Part II of my study, I have established the requirement of the functional Nse1p RING domain in regulating the chromosomal association of the Smc5/6 complex and the Cohesin complex in budding yeast. Our observations highlight a distinct enrichment of the Smc5/6 complex at the centromeres in nse1 mutants. Furthermore, the association of the Cohesin complex with the tested Cohesin binding sites is also elevated in the nse1 mutants. The nse1 mutants also display a delay in BrdU incorporation, representing delayed replication. The rDNA copy number decreases in the nse1 mutants. The nse1 RING mutants show severe cohesion defects at the CEN and arm locus. We observed that the mutants of the Smc5/6 complex confer Benomyl sensitivity. Our results from genetic interaction analysis of the Smc5/6 complex hypomorphs with the SAC (Spindle Assembly Checkpoint) and SPoC (Spindle Position Checkpoint) components reveal a functional link of the Smc5/6 complex with these pathways.

To summarize, we show that the Nse1p RING domain can influence the chromatin association of the Smc5/6 and Cohesin complexes in Saccharomyces cerevisiae. Perturbations in the RING domain of Nse1p lead to the de-repression of telomere proximal genes, partially independently of the SIR complex, possibly by virtue of its requirement in telomere tethering and clustering. In addition, the nse1 mutants display rDNA defects and impairment of cohesion.