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

这一建议旨在阐明核内膜上蛋白质复合体的SUMO化控制rDNA重复序列稳定性的机制。保持基因组的完整性本质上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，在第12号染色体上以串联重复的形式组织，每个单位呈现一个几乎相同的序列。由于其重复性，rDNA基因座经常经历同源重组，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(如核仁和着丝粒周围染色中心)之外而抑制重组，反映了由于大量重复序列而导致的基因组不稳定的潜在威胁。因此，为了允许DSB修复重复序列，首先需要将受损的基因座重新定位在异染色域之外，这一过程从酵母到人类都是保守的。然而，单个重复序列是如何从这些异染色质结构中释放出来的，目前还不清楚。在酿酒酵母中，rDNA重复序列通过Cohibin和CLIP(染色质INM蛋白的染色质连接)复合体通过外周拴系限制在核仁内。任何一个复合体的缺失都会破坏rDNA重复序列的稳定性。我们的初步数据表明，NUR1是CLIP的成员之一，它是SUMO化的，特别是在DNA损伤时。值得注意的是，缺乏Nur1的SUMO化导致CLIP和cohibin之间的关联增加，以及rDNA的HR率降低。此外，我们发现CLIP的SUMO化在远缘裂殖酵母中是保守的。通过遗传和生化方法，我们将在本方案中验证这样的假设，即DSB促进CLIP的SUMO化以响应DNA损伤，并触发系链复合体的分解，导致受损的rDNA位点释放并重新定位到核质中，以促进HR的DNA修复。比较两种在系统发育上不同的酵母菌，酿酒酵母和庞氏葡萄球菌，将为保存重复重复序列的这种修复机制提供重要的见解。鉴于核周在异染色质组织中的重要作用，我们的研究有望全面理解在DSB修复和其他与染色质相关的过程中调节异染色质重复序列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.