Investigating the role of potential PRDM9 interactors FUS and DEK in recombination hotspot activation.
In: Student Reports, Summer 2022, The Jackson Laboratory
Anna Struba and Aditya Mahadevan Iyer, Ph.D.
FUS and DEK were identified as potential PRDM9 interactors through multiple proteomic assays in the Baker laboratory. To study the role of FUS and DEK in PRDM9-associated functions, we generated stable FUS, DEK, and HELLS (known interactor of PRDM9) knockdowns in the HEK-P9 model cell system (dox-inducible PRDM9 overexpression system in HEK293). We used CRISPRoff to selectively target our genes of interest for silencing through methylation and heterochromatinization.. Transfected cells were treated with doxycycline for 24 hours to induce Prdm9 expression, and then harvested to study the effects of their absence on PRDM9 binding and methyltransferase activity. We assayed Prdm9 activity by looking at enrichment of H3K4me3 marks at known Prdm9 recombination hotspots using chromatin immunoprecipitation (ChIP). Compared to untransfected controls, , we found that H3K4me3 in our FUS and DEK knockdown cell lines showed no difference. Conversely, the HELLS knockdown cell line had decreased levels of H3K4me3 compared to the control, as expected. Thus, the PRDM9 protein complex does not require the presence of FUS or DEK to deposit the H3K4me3 marks at recombination hotspots.
Misherghi, Adel, "Investigating the role of potential PRDM9 interactors FUS and DEK in recombination hotspot activation." (2022). Summer and Academic Year Student Reports. 2708.