Efficient assembly of Drosophila histone gene arrays and endogenous insertion via Recombinase-mediated cassette exchange (RMCE)

Precise regulation of the genome is essential for metazoan development and survival. To organize DNA, eukaryotes evolved histone proteins to package the genome into nucleosomes which form high-order structured chromatin. Post-translational modifications (PTMs) on histones exert another layer of regulatory control, impacting many genome-based processes including DNA replication, transcription, and chromatin accessibility. There are gaps in understanding how specific histone PTMs impact these and other processes. Leveraging a histone gene replacement system in Drosophila melanogaster enables us to directly investigate the role of histone PTMs in biological processes. We seek to improve and test the functionality of a recently developed histone gene replacement system. This new system is an engineered histone deletion locus called ΔHisCCadillac. We utilized Golden Gate Assembly and a newly designed vector to rapidly construct Histone Array multimers amenable for Recombinase-mediated cassette exchange (RMCE) at the modified endogenous histone locus. In previous systems, the mutant array was not inserted at the endogenous locus and thus may not fully recapitulate histone gene expression. Using these reagents, we isolated flies with 4x Histone Arrays integrated at the ΔHisCCadillac locus. We also sought to determine the factors preventing full 12x Array construction via Golden Gate Assembly. This led to optimization of the assembly process and resulted in several new reagents for the construction of histone array multimers. The optimization of the histone gene replacement system is useful in studying specific histone PTMs in the future and contributes to knowledge on proper DNA organization essential to cell processes.