Cohesin and chromosome translocation

这个补充是为了支持杰夫舍恩，一个来自弱势群体的拉丁裔学生的培训。 他在兰金实验室攻读细胞生物学博士学位。父母补助金包括 研究如何通过修饰调节粘附素复合物对细胞周期进程的反应 乙酰基转移酶的作用。兰金实验室一直对粘着蛋白的调节感兴趣， 特别是在脊椎动物特定的阐述衔接控制，以及如何衔接修改， DNA损伤的反应。 这个补充项目的目标是确定粘附蛋白如何防止易位。易位 当DNA双链断裂被不适当地修复为不相关的DNA片段时， 基因组重排的研究易位本质上是致突变的，因此与 基因失调和某些疾病如癌症。我们将定义底层机制， 以下具体实验。 目标1。确定粘附素如何影响DNA双链断裂的迁移率。模型细胞系 其中邻近DNA双链断裂的位点可以通过实时监测。 和固定细胞显微镜。使用抑制剂和RNAi的组合，我们将利用这个系统， 了解内聚蛋白的影响如何以及是否会破坏流动性。 目标2.测量粘附素及其调节因子对易位频率的影响。CRISPR- Cas9对将用于诱导不同染色体上的双链断裂和易位 将通过定量PCR测量频率。我们将用这个系统来了解哪些内聚 调节子和染色质环境影响易位频率。 目标3。测量粘附素调节剂对DNA断裂时粘附素负载的影响。 使用抑制剂和耗竭，我们将利用具有大量可诱导DNA双链的细胞系 断裂，以确定关键的上游调节剂的凝聚稳定在DNA断裂。 本补充提案中描述的项目与主提案中的项目融合良好。在这里， 学生们将进行一项研究，研究粘蛋白是如何对DNA损伤做出反应的， 特别是关于粘附素本身上游的信号传导和机制。

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.