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.
The cohesin protein complex is able to minimize the gross chromosomal rearrangements called translocations, but the underlying mechanisms are not known. Here we will define how cohesin is regulated to minimize the frequency of translocation. Translocations are implicated in genetic dysregulation and tumorigenesis; understanding their origins is critical to our understanding of how genome integrity is maintained when challenged by DNA damage.
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|Alomer, Reem M; da Silva, Eulália M L; Chen, Jingrong et al. (2017) Esco1 and Esco2 regulate distinct cohesin functions during cell cycle progression. Proc Natl Acad Sci U S A 114:9906-9911|
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|Sivakumar, Sushama; Daum, John R; Tipton, Aaron R et al. (2014) The spindle and kinetochore-associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit. Mol Biol Cell 25:594-605|