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

我们的基因组不断受到破坏，每个细胞每天遭受超过30,000次DNA断裂或损伤。修复必须在染色质的背景下发生，染色质是将DNA包装在细胞核内的核蛋白组装。染色质结构的调节变化对于有效的修复是重要的。Rif1蛋白已经成为控制DNA修复的关键。Rif1抑制不适当的同源重组，确保通过直接末端连接途径修复双链DNA断裂。Rif1还参与将染色质组织成大小合适的环状结构域。然而，Rif1控制DNA修复和染色质组织的分子机制仍然不清楚。我们最近发现Rif1是一个‘蛋白磷酸酶1靶向亚基’，结合蛋白磷酸酶1(PP1)来指导它去磷酸化特定的底物。这一发现增加了Rif1通过介导染色质成分的去磷酸化而在DNA修复和染色质组织中发挥作用的可能性。在蛋白质组学筛选显示Rif1-PP1耗尽时磷酸化增加的蛋白质中，我们确定了染色体支架蛋白SAF-A(支架附着因子A；也称为HNRNPU)。尼克·吉尔伯特教授在爱丁堡的实验室表明，SAF-A控制着染色质的压缩和结构域的组织。SAF-A被招募到损伤部位，其枯竭会导致DNA修复问题。这个博士项目将检验这样的假设，即Rif1-PP1通过染色体重塑指导DNA修复，特别是通过去磷酸化染色体支架成分SAF-A来控制染色质紧凑。该项目解决了三个具体问题：1.Rif1如何影响修复组件的招募和DNA损伤的解决？为了检测Rif1对染色质成分的募集和随后的修复的影响，将在永生化的人类293细胞中表达I PPO1内切酶，以造成受控的DNA损伤(诱导I-PPO1切割人类基因组中约20个位置)。使用流式细胞术和染色质免疫沉淀，我们将监测染色质调节修复成分在损伤部位的募集，在对照细胞和/或Rif1和/或SAF-A缺失的细胞中。DNA修复将通过跨断裂位点的聚合酶链式反应分析进行同步监测。本节研究Rif1介导的染色质调节剂的招募与修复效果的关系。2.Rif1是否通过调节SAF-A的染色质紧致活性而直接修复？DNA损伤后，染色质首先浓缩以激活检查点，然后扩展以进行DNA修复。学生将测试Rif1是否在损伤后使SAF-A去磷酸化以调节这些变化，评估如果Rif1和SAF-A缺失时染色质紧致和构象如何受到影响。我们将测试不能与PP1结合的Rif1突变体和SAF-A在Rif1/PP1耗尽增加最多的磷酸盐位点突变的效果。突变将使用CRISPR产生，染色质紧凑将使用蔗糖密度沉积随后的深度测序和荧光原位杂交进行监测。3.Rif1和SAF-A是建立染色体结构域组织和染色质紧凑所必需的吗？我们还将测试Rif1是否在未受干扰的、非DNA损伤的条件下通过SAF-A调节高阶染色质组织，这是一个有趣的问题，因为正常细胞中染色质结构和高阶染色体组织的决定因素仍然难以捉摸。根据第1部分和第2部分的知识，学生将通过检查Rif1和SAF-A对胚胎干细胞染色质组织的影响来检验这一假设。总体而言，这个项目为有抱负的学生提供了一个极好的培训机会，通过了解DNA损伤修复如何在体内染色质环境中运行来建立生物化学研究，并调查发育过程中正常染色质组织的建立。

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.