Please join the Department of Brain Sciences for a seminar from Dr Mahmoudreza Rafiee (Marie Curie & EMBO postdoc fellow at The Francis Crick Institute), hosted by the Division of Neuroscience.
“Exploring chromatin-RNA-binding proteins in pluripotency and ALS model system”
Chromatin functions are regulated by organizing the assembly of specialized machinery at specific loci. Phase separation is thought to play a key role in organizing chromatin, protein- protein and protein-RNA interactions. In particular, RNA-binding proteins (RBPs) contribute to the phase separations by their intrinsically disordered regions (IDRs). Although DNA-protein and RNA-protein interactions have been studied extensively, reliable quantification of chromatin- associated RBPs is necessary to understand how they are involved in transcriptional regulations and chromatin activities. Here, we present SPACE (Silica Particle Assisted Chromatin Enrichment), a sensitive yet stringent chromatin-purification method that allows identification of chromatin-binding sites of the RBPs. Our results in mouse embryonic stem cells reveal more than 600 RBPs that bind to chromatin most frequently via their IDRs. Furthermore, we assessed the capacity of SPACE to be used with limited input material, which demonstrated reproducible enrichment of 1700 proteins from 100,000 cells using a single injection to mass spectrometer. Additionally, we applied SPACE to neural precursors containing VCP mutations. As a result, we discovered reduced chromatin-binding of mutant VCP, which itself causes reduced chromatin- binding of other DNA-damage components such as P53BP1. Thus motor neurons with mutant VCP are more vulnerable to DNA damage. These results demonstrate that high sensitivity and specificity of SPACE can lead to new insights into disease-causing mechanisms, indicating that SPACE will be particularly valuable for studies that are limited by input material.
Meeting ID: 7305133865 Passcode: uMUZD5