Cohesin and chromosome translocation

这一补充是为了支持杰夫·舍恩的培训，他是一名来自贫困家庭的拉丁裔学生 背景，他在兰金实验室攻读细胞生物学博士学位。家长赠款包括 粘附素复合体如何通过修饰来调节细胞周期进程的研究 通过ESCO乙酰转移酶。兰金实验室长期以来一直对粘附素的调控感兴趣， 特别是在脊椎动物特有的凝聚力控制的阐述中，以及在 对DNA损伤的反应。 这个补充项目的目标是确定粘附素如何防止易位。易位 当DNA双链断裂被不适当地修复为无关的DNA片段时形成，结果 在整个基因组重排中。易位本质上是突变的，因此与 基因失调和某些疾病，如癌症。我们将使用以下内容定义基本机制 以下是具体的实验。 目的1.确定粘附素如何影响DNA双链断裂的流动性。模型细胞系 已经开发出可以通过实时监测邻近DNA双链断裂的位置的方法 和固定细胞显微镜。利用抑制剂和RNAi的组合，我们将开发这个系统来 了解粘附素的影响如何以及是否会破坏移动性。 目的2.检测粘附素及其调节因子对易位频率的影响。CRISPR- Cas9对将被用来诱导不同染色体上的双链断裂和易位 频率将通过定量聚合酶链式反应进行测量。我们将使用此系统了解哪些凝聚 调节因子和染色质环境影响易位频率。 目的3.测量粘附素调节剂对DNA断裂时粘附素载量的影响。 通过使用抑制剂和耗尽剂，我们将开发一种具有大量可诱导DNA双链的细胞系 中断以确定在DNA中断时粘附素稳定的关键上游调节因子。 本补充建议中描述的项目与父建议中的项目很好地结合在一起。这里, 这名学生将进行一项研究，研究如何调节粘附素以应对DNA损伤，重点是 尤其是关于粘附素上游的信号和机制。

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.