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

TY - JOUR

T1 - Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes

AU - Novotna, Marketa

AU - Tinti, Michele

AU - Correia Faria, Joana

AU - Horn, David

N1 - © The Author(s) 2025

PY - 2025/7/4

Y1 - 2025/7/4

N2 - Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, for example, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. The RNA pol-II promoters are also unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 histone H4 genes, complementing the defect with a single ectopic H4 gene. The resulting “histoneH4” strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine residues, that are either acetylated or methylated, and profiled relative fitness of 384 distinct precision-edited mutants. H4lys10 mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4lys4 or H4lys14 mutants. Proteomic and transcriptomic analysis of H4lys4 glutamine mutants revealed significantly reduced expression of genes adjacent to RNA pol-II promoters, where glutamine mimics abnormally elevated acetylation. Thus, we present direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.

AB - Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, for example, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. The RNA pol-II promoters are also unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 histone H4 genes, complementing the defect with a single ectopic H4 gene. The resulting “histoneH4” strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine residues, that are either acetylated or methylated, and profiled relative fitness of 384 distinct precision-edited mutants. H4lys10 mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4lys4 or H4lys14 mutants. Proteomic and transcriptomic analysis of H4lys4 glutamine mutants revealed significantly reduced expression of genes adjacent to RNA pol-II promoters, where glutamine mimics abnormally elevated acetylation. Thus, we present direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.

U2 - 10.1038/s41467-025-61480-z

DO - 10.1038/s41467-025-61480-z

M3 - Article

C2 - 40615444

SN - 2041-1723

VL - 16

JO - Nature Communications

JF - Nature Communications

M1 - 6194

ER -