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

Area of Science:

• Molecular Biology
• Genetics
• Parasitology

Background:

• Transcription in trypanosomatids, like Trypanosoma brucei, is predominantly polycistronic, involving RNA polymerase II.
• RNA polymerase II promoters in T. brucei feature chromatin enriched with histones exhibiting specific post-translational modifications on their N-terminal tails.

Purpose of the Study:

• To investigate the role of histone H4 N-terminal tail residues in controlling gene expression in T. brucei.
• To engineer T. brucei strains expressing exclusively mutant histones to study gene expression regulation.

Main Methods:

• Utilized an inducible CRISPR-Cas9 system to delete endogenous histone H4 genes and complement with a single ectopic H4 gene.
• Performed saturation mutagenesis on six histone H4 N-terminal tail lysine residues, creating 384 distinct mutants.
• Conducted whole-genome sequencing, transcriptome analysis, and proteomic analysis to characterize mutant strains.

Main Results:

• Engineered "histone H4" strains exclusively expressing mutant histones, validated by sequencing and transcriptome analysis.
• Identified nineteen viable mutant strains expressing distinct H4 lysine 4 or lysine 14 mutants; H4 lysine 10 mutations were lethal.
• H4 lysine 4 glutamine mutants showed significantly reduced expression of genes near RNA polymerase II promoters, mimicking elevated acetylation.

Conclusions:

• Demonstrated direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.
• Specific histone modifications, particularly at H4 lysine 4, play a crucial role in regulating gene expression in T. brucei.
• Findings provide insights into the unconventional gene regulation mechanisms in parasitic protozoa.