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

• Applicant: Professor Dr. Sigurd Braun
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 401430508

• Final Report Year: 2023

Final Report Abstract

Eukaryotic genomes contain large amounts of “fragile” regions, where DNA breaks frequently occur. Those fragile sites are often characterized by the presence of repetitive sequences. Repair of these DNA sequences can lead to genetic rearrangements through a repair mechanism known as homologous recombination (HR). Uncontrolled HR is often associated with neurodegenerative diseases and cancer. Therefore, repetitive DNA is often shielded from repair factors by sequestration into specific compartments like the nucleolus that contains hundreds of repeats of the ribosomal DNA (rDNA). But what happens if rDNA repeats are damaged? Previous studies have shown that damaged rDNA repeats can be transiently released from the nucleolus to allow access to the DNA repair machinery. However, the molecular mechanism that triggers the release has remained unknown. During this project, we unveiled critical steps of the molecular mechanism by which rDNA repeats are released to the nucleoplasm during DNA damage. In the model organism budding yeast (Saccharomyces cerevisiae), the rDNA repeats are tethered to the inner nuclear membrane and sequestered inside the nucleolus through the interaction of two protein complexes: the cohibin complex, which binds to rDNA repeats, and the CLIP complex, which is part of the inner nuclear membrane. Together, they form the rDNA tethering complex. Using a combination of biochemistry, microscopy and yeast genetics, we showed that multiple steps trigger the rDNA release from the nuclear envelope into the nucleoplasm. First, DNA damage results in the phosphorylation of the CLIP complex. This modification promotes the addition of another molecular ‘tag’ known as Small Ubiquitin-like Modifier, or SUMO, on both protein complexes involved in tethering. Proteins earmarked by SUMO are recognized by distinct molecular machineries, for instance the AAA ATP-dependent chaperone Cdc48 (p97 in mammals). Through its adaptor protein Ufd1, Cdc48 binds to the SUMOylated tethering complex and breaks up the interaction between its partners in an ATP-dependent manner. This causes the release of damaged rDNA repeats from the nucleolus into the nucleoplasm, promoting their DNA repair by the HR machinery. Notably, we demonstrated that nucleolar rDNA release in mammalian cells also requires the activity of Cdc48/p97, implying that this repair mechanism is conserved in higher eukaryotes. These findings provide crucial insights into the dynamic regulation of DNA damage repair. Noteworthy, while yeast cells deficient in rDNA tethering show hyper-recombination and loss of individual rDNA repeats reminiscent of cancer, we found that cells that cannot untether rDNA from the nuclear envelope are not viable. This implicates that rDNA untethering happens frequently in eukaryotic cells, illustrating the importance of this regulatory mechanism.

Publications

• Neutral epigenetic inheritance: being prepared for future generations. Nature Structural & Molecular Biology, 26(6), 391-392.
  Capella, Matías & Braun, Sigurd
  
• ESCRT recruitment by the S. cerevisiae inner nuclear membrane protein Heh1 is regulated by Hub1-mediated alternative splicing. Journal of Cell Science, 133(24).
  Capella, Matías; Martín, Caballero Lucía; Pfander, Boris; Braun, Sigurd & Jentsch, Stefan
  
• ESCRTing Heterochromatin Out of the Nuclear Periphery. Developmental Cell, 53(1), 3-5.
  Capella, Matías & Braun, Sigurd
  
• Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase. Nature Communications, 12(1).
  Capella, Matías; Mandemaker, Imke K.; Martín, Caballero Lucía; den, Brave Fabian; Pfander, Boris; Ladurner, Andreas G.; Jentsch, Stefan & Braun, Sigurd
  
• The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery. Nature Structural & Molecular Biology, 29(9), 910-921.
  Martín, Caballero Lucía; Capella, Matías; Barrales, Ramón Ramos; Dobrev, Nikolay; van Emden, Thomas; Hirano, Yasuhiro; Suma, Sreechakram Vishnu N.; Fischer-Burkart, Sabine; Kinugasa, Yasuha; Nevers, Alicia; Rougemaille, Mathieu; Sinning, Irmgard; Fischer, Tamás; Hiraoka, Yasushi & Braun, Sigurd