Research Initiation Award: Deciphering the Mechanistic Pathways of the Novel Epigenetic Modulator EBdCas9

The Historically Black Colleges and Universities Undergraduate Program (HBCU-UP) through Research Initiation Awards (RIA) provide support for junior and mid-career faculty at Historically Black Colleges and Universities who are building new research programs or redirecting and rebuilding existing research programs. It is expected that the award will help to further the faculty member's research capability and effectiveness and improve research and teaching at the home institution. Recent literature has demonstrated the role of a computationally designed protein EB-dead-Cas 9 (EBdCas9) in modulating gene expression, particularly as a transcriptional activator resulting in down-stream cellular phenotypic changes. In this project, researchers seek to investigate the molecular mechanisms of cellular differentiation by elucidating the protein-protein iterations that facilitate and/or regulate EBdCas9 activity and their intracellular localization. It is anticipated that this project will lay the groundwork for future research to understand how EBdCas9 functions in living cells, and how EBdCas9 may be a potential therapeutic target in various disease states. The project also will engage undergraduate and graduate students in laboratory research and mentoring to prepare them for future study and careers in biology-related careers. The epigenome consists of chemical modifications to DNA, RNA and proteins that modulate gene expression and control cellular identity. Proper epigenetic regulation is critical for healthy development while dysfunctional or aberrant epigenetic regulation is associated with disease. Molecular tools that modulate the epigenome give us the ability to study the mechanism of epigenetic processes and how they define cellular state for diseases. One such tool is EBdCas9, a synthetic embryonic ectoderm development (EED) binder that inhibits activity of the polycomb repressive complex 2 (PRC2) and causes the reduction of the histone modification H3K27me3, in a locus specific manner. While the mode of action of EBdCas9 is mostly understood, its binding partners in its DNA-bound and unbound states have not been fully investigated. This project will test the hypothesis that EBdCas9 does not globally disrupt all PRC2 protein-protein interactions but rather only disrupts direct interactions between EED and other PRC2 core proteins when bound to DNA. EBdCas9 and guide RNAs will be expressed in the SKOV3 ovarian cancer cell line and targeted to PRC2 repressed ABC transporter genes. Experimental validation of protein interactions will be conducted using a series of immunoprecipitations and co-immunoprecipitation on the nuclear and cytoplasmic cellular fractions of EBdCas9 transfected cells with and without the presence of guide RNAs. It is anticipated that EBdCas9 will only bind with EED in the presence of guide RNAs in the nuclear fraction, and that global levels of other PRC2 proteins will not be affected. These studies will reveal how EBdCas9 disrupts epigenetic repression while preserving broader PRC2 complex integrity. Defining the molecular interaction network of EBdCas9 will provide critical mechanistic insight into a new epigenetic editing technology. These findings will also facilitate the broader application of EBdCas9 in studies of cell state and cancer biology. The project also will increase awareness of and engagement undergraduate and graduate students in the research and build capacity for epigenetics research at Bowie State University. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.