Cohesin and chromosome translocation

粘连蛋白和染色体易位

• 批准号: 10315206
• 负责人: Susannah Rankin
• 金额: $ 3.66万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10315206
• 关键词: Acetyltransferase; Affect; CRISPR/Cas technology; Cell Cycle Progression; Cell Line; Cell model; Cells; Cellular biology; Chromatin; Chromosomal Rearrangement; Chromosomal translocation; Complex; DNA; DNA Damage; DNA Double Strand Break; Disease; Doctor of Philosophy; Environment; Enzymes; Frequencies; Genes; Genetic; Genome; Goals; Laboratories; Latino; Malignant Neoplasms; Measures; Microscopy; Modification; Monitor; Parents; RNA Interference; Regulation; Signal Transduction; Site; Students; System; Training Support; cohesin; cohesion; disadvantaged background; experimental study; genome integrity; inhibitor/antagonist; interest; parent grant; prevent; protein complex; repaired; response; tumorigenesis

This supplement is to support the training of Jeff Schoen, a Latino student from a disadvantaged background, as he pursues a PhD in Cell Biology in the Rankin laboratory. The parent grant includes studies of how the cohesin complex is regulated in response to cell cycle progression through modification by the Esco acetyltransferase enzymes. The Rankin lab has had a long interest in cohesin regulation, in particular in vertebrate-specific elaborations of cohesion control, and how cohesion is modified in response to DNA damage. The goal of this supplemental project is to determine how cohesin prevents translocation. Translocations form when DNA double strand breaks are inappropriately repaired to unrelated DNA fragments, resulting in gross genome rearrangement. Translocation is intrinsically mutagenic, and therefore correlated with gene dysregulation and certain diseases such as cancer. We will define the underlying mechanisms with the following specific experiments. Aim 1. Determine how cohesin affects mobility of DNA double strand breaks. Model cell lines have been developed in which sites adjacent to DNA double strand breaks can be monitored by live and fixed cell microscopy. Using combination of inhibitors and RNAi we will exploit this system to understand how and if cohesin impacts break mobility. Aim 2. Measure the impact of cohesin and its regulators on translocation frequency. CRISPR- Cas9 pairs will be used to induce double strand breaks on different chromosomes and translocation frequency will be measured by quantitative PCR. We will use this system to understand which cohesin regulators and chromatin environments affect translocation frequency. Aim 3. Measure the impact of cohesin regulators on cohesin loading at DNA breaks. Using inhibitors and depletions we will exploit a cell line with numerous inducible DNA double strand breaks to identify critical upstream regulators of cohesin stabilization at DNA breaks. The project described in this supplement proposal integrates well with that in the parent proposal. Here, the student will undertake a study of how cohesin is regulated in response to DNA damage, focusing particularly on the signaling and mechanisms upstream of cohesin itself.

这种补充是为了支持杰夫·舒恩（Jeff Schoen）的培训，这是一名不利的拉丁裔学生 背景，他在兰金实验室攻读细胞生物学博士学位时。父母赠款包括 研究如何通过修饰对细胞周期进程调节粘蛋白复合物的研究 通过ESCO乙酰转移酶。 Rankin实验室对粘蛋白调节有很长的兴趣， 特别是在脊椎动物特异性的内聚力控制中，以及如何修改内聚力 对DNA损伤的反应。 该补充项目的目的是确定粘蛋白如何防止易位。易位 当DNA双链断裂不当修复到无关的DNA片段时形式，导致 在总体基因组重排中。易位是固有的诱变，因此与 基因失调和某些疾病，例如癌症。我们将使用 以下特定实验。 目标1。确定粘着蛋白如何影响DNA双链断裂的迁移率。模型单元线 已经开发了与DNA双链断裂相邻的地点可以通过现场监测 和固定细胞显微镜。使用抑制剂和RNAi的组合，我们将利用该系统 了解粘着蛋白如何以及是否影响破裂的活动能力。 AIM 2。测量粘着蛋白及其调节剂对易位频率的影响。 crispr- Cas9对将用于诱导不同染色体和易位的双链断裂 频率将通过定量PCR测量。我们将使用该系统了解哪种粘蛋白 调节器和染色质环境会影响易位频率。 目标3。测量粘着蛋白调节剂对DNA断裂处粘蛋白负荷的影响。 使用抑制剂和耗竭，我们将利用具有许多诱导DNA双链的细胞系 断裂以识别DNA断裂处粘蛋白稳定的关键上游调节剂。 该补充提案中描述的项目与父母提案中的计划很好地整合了。这里， 学生将研究如何根据DNA损伤调节粘蛋白，集中精力 特别是在粘蛋白本身上游的信号传导和机制上。