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

Rif1 和 SAF-A 如何重塑染色质以确保有效的 DNA 修复？

• 批准号: 1806189
• 金额: --
• 依托单位: University of Aberdeen
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1806189
• 关键词: How; do; Rif1; SAF; remodel

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.

我们的基因组不断受损，每个细胞每天遭受超过30，000次DNA断裂或损伤。修复必须发生在染色质的背景下，染色质是将DNA包装在细胞核内的核蛋白组装体。染色质结构的调节性变化对于有效的修复是重要的。蛋白质Rif 1已经成为控制DNA修复的关键。Rif 1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。Rif 1还参与将染色质组织成正确大小的环结构域。然而，Rif 1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，Rif 1是一个“蛋白磷酸酶1靶向亚基”，结合蛋白磷酸酶1（PP 1），指导它去磷酸化特定底物。这一发现提出了Rif 1通过介导染色质组分的去磷酸化作用于DNA修复和染色质组织的可能性。在蛋白质组学筛选中，我们鉴定了染色体支架蛋白SAF-A（支架附着因子A;也称为HNRNPU），所述蛋白质在Rif 1-PP 1耗尽后显示出增加的磷酸化。尼克吉尔伯特教授在爱丁堡的实验室表明，SAF-A控制染色质压缩和结构域组织。SAF-A被募集到损伤位点，其耗尽导致DNA修复问题。这个博士项目将测试Rif 1-PP 1通过染色体重塑指导DNA修复的假设，特别是通过去磷酸化染色体支架组件SAF-A来控制染色质压实。该项目解决了三个具体问题：1。Rif 1如何影响修复组件的募集和DNA损伤的解决？为了检查Rif 1对染色质组分的募集和随后的修复的影响，将在永生化的人293细胞中表达内切核酸酶I Ppo 1，以施加受控的DNA损伤（诱导的I-Ppo 1切割人类基因组中的约20个位点）。使用流式细胞术和染色质免疫沉淀，我们将监测招募染色质调节修复组件的损害网站，在控制细胞和细胞耗尽的Rif 1，SAF-A，或两者兼而有之。将通过PCR分析跨断裂位点同时监测DNA修复。本节研究了Rif 1介导的染色质调节剂募集与修复有效性的关系。2. Rif 1是否通过调节SAF-A的染色质致密化活性来指导修复？DNA损伤后，染色质首先浓缩以激活检查点，然后延伸以进行DNA修复。学生将测试Rif 1是否在损伤后使SAF-A去磷酸化以介导这些变化，评估如果Rif 1和SAF-A不存在，染色质致密化和构象如何受到影响。我们将测试不能结合PP 1的Rif 1突变体和在Rif 1/PP 1耗尽最增加的磷酸位点处突变的SAF-A的作用。将使用CRISPR产生突变体，并且将使用蔗糖密度沉降随后深度测序和荧光原位杂交来监测染色质压实。3. Rif 1和SAF-A对建立染色体结构域组织和染色质致密化至关重要吗？我们还将测试Rif 1是否在不受干扰的非DNA损伤条件下通过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域结构和高阶染色体组织的决定因素仍然难以捉摸。通过第1部分和第2部分，学生将通过检查Rif 1和SAF-A对胚胎干细胞染色质组织的影响来验证这一假设。总体而言，该项目为雄心勃勃的学生提供了一个出色的培训机会，通过了解DNA损伤修复如何在体内染色质背景下运作，并研究在发育过程中正常染色质组织的建立，以生物化学研究为基础。