Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes

Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei , approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. RNA polymerase II promoters are unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their highly divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei , we engineered strains exclusively expressing novel mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 native copies of histone H4 , complementing the tandem arrays with a single ectopic H4 gene. The resulting ‘hist ^one H4’ strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine (K) residues and used multiplex amplicon-seq to profile the relative fitness of 384 distinct precision edited mutants. H4 ^K10 mutations were not tolerated, but we could derive a panel of nineteen strains exclusively expressing novel H4 ^K4 or H4 ^K14 mutants. Both proteomic and transcriptomic analysis of H4 ^K4Q mutants revealed significantly reduced expression of genes adjacent to RNA polymerase II promoters, where the glutamine (Q) mutation mimics an abnormally high level of acetylation. Thus, we present direct evidence for polycistronic expression control by histone H4 N-terminal tails in trypanosomes.