Authors

M Novotna¹; D Horn¹;  ¹ University of Dundee, UK

Discussion

Trypanosomatid genomes lack sequences readily identifiable as promoters and replication origins and it remains unclear to what extent transcription, DNA replication and DNA repair rely upon chromatin-based controls. The N-terminal histone tails, and tail modifications, such as acetylation, play key roles in these processes in other eukaryotes. Genetic manipulation and subsequent study of histone functions have proven particularly challenging, however, because core histone genes are typically present as many copies of each gene. In trypanosomatids, these genes are present in long polycistronic transcription units containing approx. 40 copies. The N-terminal tails are also highly divergent relative to the usual model eukaryotes. I have used an inducible CRISPR/Cas9 system in Trypanosoma brucei to delete all native gene copies of histone H4, complementing the defect with a single, recoded and highly expressed ectopic copy. CRISPR/Cas9 was then used for site saturation mutagenesis of an N-terminal tail lysine residue in the ectopic gene (H4K14 initially). Current results suggest that amplicon-seq can be used to monitor the relative fitness of these mutants. I am also using these strains to determine whether the N-terminal tail of histone H4 can be removed altogether, which was surprisingly possible in budding yeast.