How do Rif1 and SAF-A remodel chromatin to ensure effective DNA repair?

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Our genomes are constantly damaged, each cell suffering upwards of 30,000 DNA breaks or lesions every day. Repair must occur in the context of chromatin, the nucleoprotein assembly that packages DNA within the nucleus. Regulated changes in chromatin structure are important for effective repair.The protein Rif1 has emerged as critical to control DNA repair. Rif1 suppresses inappropriate homologous recombination, ensuring repair of double-stranded DNA breaks by the direct end-joining pathway. Rif1 is also implicated in organising chromatin into correctly sized loop domains. The molecular mechanism through which Rif1 controls DNA repair and chromatin organisation is however still obscure. We recently discovered that Rif1 is a 'Protein Phosphatase 1-targeting subunit', binding Protein Phosphatase 1 (PP1) to direct it to dephosphorylate specific substrates. This discovery raises the possibility that Rif1 acts in DNA repair and chromatin organisation by mediating dephosphorylation of chromatin components. In a proteomic screen for proteins showing increased phosphorylation upon Rif1-PP1 depletion we identified chromosome scaffold protein SAF-A (Scaffold Attachment Factor A; also called HNRNPU). Professor Nick Gilbert's lab in Edinburgh showed that SAF-A controls chromatin compaction and domain organisation. SAF-A is recruited to damage sites and its depletion causes DNA repair problems. This PhD project will test the hypothesis that Rif1-PP1 directs DNA repair through chromosome remodelling, in particular by dephosphorylating chromosome scaffold component SAF-A to control chromatin compaction. Project addresses three specific questions:1. How does Rif1 affect recruitment of repair components and resolution of DNA damage? To examine effects of Rif1 on recruitment of chromatin components and subsequent repair, the endonuclease I Ppo1 will be expressed in immortalised human 293 cells to inflict controlled DNA damage (induced I-Ppo1 cuts around 20 sites in the human genome). Using flow cytometry and chromatin immunoprecipitation, we will monitor recruitment of chromatin-modulating repair components to damage sites, in control cells and cells depleted for Rif1, SAF-A, or both. DNA repair will be simultaneously monitored by PCR analysis across break sites. This section examines Rif1-mediated recruitment of chromatin modulators in relation to repair effectiveness. 2. Does Rif1 direct repair by regulating chromatin compaction activity of SAF-A? Following DNA damage chromatin first condenses to enable checkpoint activation then is subsequently extended for DNA repair to occur. The student will test whether Rif1 dephosphorylates SAF-A after damage to mediate these changes, evaluating how chromatin compaction and conformation are affected if Rif1 and SAF-A are absent. We will test the effect of a Rif1 mutant that cannot bind PP1, and of SAF-A mutated at the phosphosites most increased by Rif1/PP1 depletion). Mutants will be generated using CRISPR, and chromatin compaction will be monitored using sucrose density sedimentation followed by deep sequencing, and by fluorescence in situ hybridisation. 3. Are Rif1 and SAF-A essential to establish chromosome domain organisation and chromatin compaction? We will also test whether Rif1 regulates higher-order chromatin organisation by SAF-A under undisturbed, non-DNA-damaging conditions, an intriguing question as determinants of chromatin domain structure and higher order chromosome organisation in normal cells remain elusive. Informed by Parts 1 and 2, the student will test this hypothesis by examining the effect of Rif1 and SAF-A on chromatin organisation in embryonic stem cells. Overall this project provides an outstanding training opportunity for an ambitious student to build on biochemical studies by understanding how DNA damage repair operates in the in vivo chromatin context, and to investigate establishment of normal chromatin organisation during development.