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

Abstract

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 "hist^oneH4" 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. H4^lys10 mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4^lys4 or H4^lys14 mutants. Proteomic and transcriptomic analysis of H4^lys4 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.

Fig. 1. Complementation of >40 T. brucei histone H4 genes with a recoded H4 gene.

a The schematic shows hist^oneH4 strain construction. The 2T1^T7-Cas9 strain was transfected with a cassette containing a recoded, and ectopic histone H4 gene (H4^ECT, synonymous changes highlighted) and a single guide RNA (sgRNA) targeting H4^NAT genes, both under the control of a T7 RNA polymerase promoter (T7). Cas9 was induced for 24 h in the resulting cell line, which was then transfected with an NPT (neomycin phosphotransferase) cassette to delete the native histone H4 (H4^NAT) tandem arrays. Created in BioRender. Novotna, M. (2025) https://BioRender.com/f0do2k7. b The Southern blots were probed for the 5′ end of the native H4 genes (left), which detected the expected 736 bp band (approx. 43 copies), and 1505 bp band (1 copy from each of 2 alleles) only in the parental cell line. The blot was stripped and re-probed for the ectopic H4 gene (right) revealing the expected 4664 bp and 13803 bp bands in both independent hist^oneH4 strains, but not in the parent. Some residual signal, or cross-reactivity, can be seen from the 736 bp band; the ectopic H4 probe shares 84% sequence identity with native H4 genes. M, digoxigenin (DIG)-labelled DNA ladder. c The circular plot shows whole-genome sequencing data for the hist^oneH4 strains compared to the parental strain, and the parental strain plus the H4^ECT gene. The zoom at top shows precise deletion of the native H4 arrays; clone 1, blue; clone 2, magenta. d RNA-seq analysis. A strain expressing the H4^ECT gene was compared to the parent strain in the upper panels. Both hist^oneH4 strains were compared to the strain expressing the H4^ECT gene in the lower panels. The H4^NAT and H4^ECT transcripts (orange) and the super-abundant VSG transcript are highlighted in the left-hand panels, while five genes immediately downstream of the H4 array are highlighted (blue) in the right-hand panels. n = 8934.

Fig. 2. H4 tail lysine saturation mutagenesis and multiplex fitness-profiling.

a The alignment shows the N-terminal tail sequence of T. brucei histone H4, compared to the equivalent sequences from yeast and human. Lysine residues are highlighted in the T. brucei sequence. b Schematic of the saturation mutagenesis and amplicon-sequencing strategy. An sgRNA cassette targeting the 5′ end of the H4^ECT gene was introduced into an hist^oneH4 strain. Cas9 was induced, and 24 h later the cells were transfected with single-stranded oligodeoxynucleotide (ssODN) editing templates. DNA was extracted at different timepoints, the edited region of the H4^ECT gene was PCR-amplified, and the amplicons were deep-sequenced. Created in BioRender. Novotna, M. (2025) https://BioRender.com/21f7d0g. c The radial plots show the abundance of sequence-reads representing each codon at each edited position during the time course. Data for six edited N-terminal tail lysine’s are shown. Values represent the averages of duplicate samples and are relative to codon scores obtained at the 12 h timepoint. D2, day-2; D4, day-4; D6, day-6. *, stop codons. Source data are provided as a Source Data file. d The logo shows relative overrepresentation or underrepresentation at day-6 for those cognate amino acids that were either tolerated or not tolerated following editing at each of the six lysine positions targeted.

Fig. 3. A panel of strains exclusively expressing histone H4 mutants.

a The plots show the fitness of a panel of K4 and K14 mutants relative to H4^K4K or H4^K14K strains, respectively. Cell density was recorded every 24 h for 4–5 days (left to right) and for two technical replicates. Sanger sequencing traces show the edited codon for each mutant. b Protein blotting analysis of selected K4 mutants, showing H4^K4 acetylation, non-acetylated H4^K4, and H4^K10 acetylation. EF-1α (elongation factor 1 α; red) served as a loading control. Similar results were obtained from at least two independent experiments. Source data are provided as a Source Data file.

Fig. 4. Proteomic analysis of histone H4^K4Q and H4^K14Q mutants.

a Proteomics analysis. Three strains expressing the H4^K4Q mutant were compared to an H4^K4K control in the upper panels. A strain expressing the H4^K41Q mutant was compared to an H4^K14K control in the lower panels. The core and variant histones are highlighted. n = 5776. b The boxplot shows log₂ fold-change for the two comparisons in (a), and for either genes within 10 kbp of transcription start-sites (n = 468) or >10 kbp distal from those sites (n = 5308). Boxes indicate the interquartile range (IQR), the whiskers show the range of values within 1.5 × IQR and a horizontal line indicates the median. The notches represent the 95% confidence interval for each median. p-values were calculated using two-sided t-tests.

Fig. 5. Expression of promoter-adjacent genes is disrupted in H4^K4Q mutants.

a The schematic illustrates a canonical RNA polymerase II transcribed polycistronic transcription unit in T. brucei, showing the distribution of genes closest to the start-site (orange), closest to a termination-site (blue) and all other genes (grey). The boxplot shows log₂ fold-change in RNA-seq data when we compared three strains expressing the H4^K4Q mutant to an H4^K4K control. Genes within 10 kbp of a transcription start-sites (orange, n = 736), genes within 10 kbp of a termination-site (blue, n = 576), all other genes (grey, n = 8755). Boxes indicate the interquartile range (IQR), the whiskers show the range of values within 1.5 × IQR and a horizontal line indicates the median. The notches represent the 95% confidence interval for each median. p-values calculated using two-sided t-tests. b RNA-seq analysis focussing on genes adjacent to divergent start-sites (top left), genes adjacent to convergent termination-sites (top right), genes adjacent to start-sites where another polycistron ends (bottom left), and genes adjacent to termination-sites where another polycistron starts (bottom right). Distances from the start or end are shown on the x-axis. Numbers of transcripts within 10 kbp of a start- or termination-site that were significantly (FDR < 0.01) increased or decreased in abundance in the H4^K4Q mutant relative to the H4^K4K control are indicated. These numbers were used to calculate p-values using χ² tests. FDR, False Discovery Rate. c The circular plot shows the full RNA-seq dataset mapped to the T. brucei chromosome cores (data in grey). Transcripts from genes that are within 10 kbp of a promoter (black bars, as defined by the upstream borders of H4^K10 acetylation footprints), and that are significantly (FDR < 0.01) increased or reduced in abundance in the H4^K4Q mutant relative to the H4^K4K control, are highlighted.