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

本研究旨在阐明蛋白质复合物在核膜内的SUMO化控制rDNA重复序列稳定性的机制。维持基因组的完整性主要是为了细胞的生存能力。在酿酒酵母中，rDNA基因座由大约150个拷贝组成，在染色体XII上以串联重复序列组织，每个单元呈现几乎相同的序列。由于其重复性，rDNA基因座经常发生同源重组（HR），使其成为基因组中最不稳定的区域之一。虽然HR的主要功能是通过修复DNA双链断裂（DSB）来维持基因组的完整性，但不受控制的重组会对细胞造成有害影响。在许多细胞系统中，通过将HR机制排除在异染色质结构（如核仁和近着丝粒染色中心）之外来抑制重组，反映了由于大量重复而导致的基因组不稳定性的潜在威胁。因此，为了允许重复序列的DSB修复，受损的基因座首先需要重新定位到异染色质结构域之外，这是从酵母到人类的保守过程。然而，如何从这些异染色质结构中释放单个重复序列仍然知之甚少。在酿酒酵母中，rDNA重复序列通过cohibin和CLIP（染色质连接INM蛋白）复合物通过外周束缚被限制在核仁内。任何一种复合物的缺失都会使rDNA重复序列不稳定。我们的初步数据表明，Nur1，CLIP的成员之一，是SUMO化，特别是在DNA损伤。值得注意的是，Nur1 SUMO化的缺乏导致CLIP和cohibin之间的关联增加以及rDNA的HR率降低。此外，我们发现SUMO化的CLIP是保守的远亲酵母裂殖酵母粟酒裂殖酵母。通过遗传和生物化学方法，我们将测试在这个建议的假设，DSB促进SUMO化的CLIP响应DNA损伤，并触发解体的拴系复合物，导致在释放和重新定位受损的rDNA基因座到核质，以促进DNA修复的HR。cerevisiae和S. pombe，将提供重要的见解，保护这种修复机制的重复序列。鉴于异染色质组织的核周边的突出作用，我们提出的研究有望提供一个全面的了解，在DSB修复和其他染色质相关的过程中调节异染色质重复序列的空间分布的关键机制。

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.