Epigenetic regulation through post-translational histone modifications plays crucial roles in various cellular processes. Whilst efforts have been focused on understanding the function of chromatin states from different histone modifications, their regulators are still largely unknown, partly due to limitations with current methods. Moreover, the regulators of chromatin states often differ in different cellular contexts. Therefore, new methods of detecting individual and combinatorial histone marks are crucial. By implementing the split-Venus bimolecular fluorescence complementation system, we have developed a novel doxycycline-inducible biosensor to detect individual and combinatorial histone marks in live single cells. We have developed biosensors that are able to detect individual H3K4me3 and H3K27me3, as well as bivalent chromatin, which is the co-occurrence of H3K4me3 and H3K27me3, known to mark promoters of poised genes that are lowly expressed. We have established the biosensors in the SUM159 triple negative breast cancer cell line as it is well characterised, and dysregulation of histone modifications has been shown facilitate cellular adaptation in this cell line.
Sensitivity of the biosensors were validated by flow cytometry and immunofluorescence experiments following drug treatments that disrupt global levels of different histone modifications. Chromatin immunoprecipitation of the biosensors, using an antibody that recognises the reconstituted Venus protein, also revealed correct localisation across the genome. Pilot CRISPR-Cas9 screens on the H3K27me3 biosensor revealed known components of the H3K27me3 writer complex as top screen hits, validating the ability to use these novel biosensors to uncover upstream regulators. Ongoing high throughput CRISPR-Cas9 screens on the other biosensors are using a custom nuclear-proteome boutique library developed by the lab to uncover factors that regulate global levels of different chromatin states. This project developed a novel method to measure and detect individual and combinatorial histone marks in live single cells, enabling the identification of regulators that establish or maintain unique chromatin states.