Precision mutagenesis of histone tail residues in trypanosomes

Abstract

Trypanosoma brucei is a divergent unicellular organism and the causative agent of sleeping sickness in people and Nagana in cattle. The incidence of the human disease has decreased significantly, but Nagana still causes devastating economical damage in Sub-Saharan Africa. These cells are covered in a variant surface glycoprotein (VSG) coat, which facilitates host immune evasion. Only a single VSG gene from a repertoire of >2500 is expressed at a time and it is periodically switched. Typically conserved histones are strikingly divergent in trypanosomes, especially in the tail regions, which harbour posttranslational modifications, e.g. lysine acetylation. Epigenetic modifications can impact transcription, DNA replication and repair, but the mechanisms are not fully characterized in T. brucei. Current insights mostly derive from investigating the histone modification enzymes: writers, erasers, and readers. However, interpretation of enzyme-defective phenotypes is complex, due to potential multiple histone and non-histone substrates. Manipulation of the histone genes themselves has proven to be challenging, as they are present as many identical arrayed copies.

In this work, I used an inducible CRISPR/Cas9 system to delete all copies of native histone H4 genes, as confirmed by Southern blotting and whole genome sequencing, and replaced them with a single ectopic copy, which was optimised for high expression. The resulting Hist^H4one strains maintained fitness and histone H4 protein expression comparable to the parental controls. Next, Cas9-driven saturation mutagenesis was performed on H4 N-tail residues (K4, K10, and K14). Multiplex amplicon-seq profiling was used to monitor relative fitness, revealing those tolerated H4K4 or H4K14 mutations; H4K10 mutations were not tolerated. Remarkably, viability was maintained even when the H4K4 or H4K14 residues were removed. Using these outputs, a panel of strains exclusively expressing novel histone H4 mutants, including arginine (R; non-acetylated mimic) or glutamine (Q; constitutively acetylated mimic) substitutions, was phenotypically profiled; using growth, proteomics, microscopy, protein blotting, flow cytometry and DNA-damage sensitivity assays. I detected derepression (up to 7.5x) of silent VSGs in the acetylation mimic H4K4Q mutants. These strains were also hyper-sensitive to a DNA damaging agent. Thus, I present the first direct evidence that histone tails and their modification impact these processes in trypanosomes.

Date of Award	2024
Original language	English
Awarding Institution	University of Dundee
Supervisor	David Horn (Supervisor) & Mark Field (Supervisor)