Abstract LB-342: Targeted gene control with bifunctional molecules to harness endogenous chromatin-modifying activity

Abstract

Abstract Epigenetic pathways are commonly found disrupted in many human diseases including cancer, yet much remains unknown about the mechanisms by which dysregulation of chromatin regulatory pathways leads to disease pathology. To investigate and control these complex interactions in live cells, we developed a novel system that will unveil the underlying mechanisms of proteins responsible for modulating chromatin states using a gene-specific manipulation strategy. To explore how dynamic changes in acetylation levels impact chromatin structure we generated an endogenous reporter system at the murine Oct4 locus with a Gal4 DNA binding array upstream of an EGFP reporter allele. In our system, a FKBP protein fused to a Gal4 DNA-binding-domain serves as a protein anchor to the DNA binding array. Next, we synthesized novel cell-permeable, bifunctional molecules that include FK506 (which binds to FKBP) linked to a component that selectively binds to a histone tail chromatin-modifying enzyme. We characterized the effects of the Chemical Epigenetic Modifiers (CEM)s using flow cytometry, high-content microscopy and chromatin immunoprecipitation. We show that our lead repressive CEM can repress GFP expression by 50%. Next, we have made our system more modular by creating a deactivated Cas9 (dCas9)-FKBP fusion protein. The FKBP portion allows us to recruit our CEMs to any region of the genome to which we design a guide RNA. To study the addition of histone acetylation we utilized our new activating, HAT-binding CEMs and designed our dCas9-FKBP to target low-expression reporters. Our lead activating CEM increases expression over 20-fold. We are now using our activating CEM system to target tumor suppressor genes in hard to treat cancers. To show proof-of-principle, we have targeted the tumor suppressor p53 in a colon cancer cell line. We hypothesize that our activating CEM platform will activate the repressed P53 allele leading to slower tumor cell proliferation and apoptosis. This work represents a novel therapeutic strategy that is capable of modulating gene expression in a reversible and dose-dependent manner. Citation Format: Anna M. Chiarella, Kyle V. Butler, Jian Jin, Nathaniel A. Hathaway. Targeted gene control with bifunctional molecules to harness endogenous chromatin-modifying activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-342.