Exploring the role of SUMOylation of the CLIP (chromatin linkage of INM protein) complex in rDNA tethering and maintenance

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This proposal aims to elucidate the mechanisms by which SUMOylation of a protein complex at the inner nuclear membrane controls the stability of rDNA repeats. Maintaining genome integrity is essentially for cellular viability. In Saccharomyces cerevisiae, the rDNA locus consists of approximately 150 copies, organized in tandem repeats on chromosome XII, each unit presenting a near-identical sequence. Due to its repetitive nature, the rDNA locus often undergoes homologous recombination (HR), making it one of the most unstable regions in the genome. While the primary function of HR is to maintain genome integrity by repairing DNA double-strand breaks (DSBs), uncontrolled recombination can cause deleterious effects to the cell.In many cellular systems, recombination is suppressed by excluding the HR machinery from heterochromatic structures, such as the nucleolus and pericentromeric chromocenters, reflecting the potential threat of genome instability due to the large number of repeats. Thus, in order to allow DSB repair of repetitive sequences, the damaged locus needs first to be relocalized out of the heterochromatic domain, a process that is conserved from yeast to humans. However, how individual repeats are released from these heterochromatin structures remains poorly understood.In S. cerevisiae, the rDNA repeats are constrained within the nucleolus through peripheral tethering via the cohibin and the CLIP (chromatin linkage of INM protein) complexes. The absence of either complex destabilizes rDNA repeats. Our preliminary data indicate that Nur1, one of the members of CLIP, is SUMOylated, especially upon DNA damage. Notably, the absence of Nur1 SUMOylation results in an increased association between CLIP and cohibin as well as a decreased HR rate of rDNA. Moreover, we find that SUMOylation of CLIP is conserved in the distantly related yeast Schizosaccharomyces pombe. Through genetic and biochemical approaches, we will test in this proposal the hypothesis that DSBs promote SUMOylation of CLIP in response to DNA damage and trigger the disassembly of the tethering complex, resulting in the release and relocalization of the damaged rDNA locus into the nucleoplasm to promote DNA repair by HR. Comparing two phylogenetically different yeast species, S. cerevisiae and S. pombe, will provide important insights into the conservation of this repair mechanism of repetitive repeats. Given the prominent role of the nuclear periphery in heterochromatin organization, our proposed research is expected to provide a comprehensive understanding of crucial mechanisms regulating the spatial distribution of heterochromatic repeats during DSB repair and other chromatin-related processes.