Abstract
Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
Highlights
Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts
Genome sequences of several pathogens have revealed a partitioning of chromosomes, with housekeeping genes often being located in the central core and antigen genes being located in subtelomeric regions[5,6]
This study aimed to identify the process that restricts antigen expression
Summary
No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment. In situ Hi-C was performed based on previously published protocols[35,36] and adapted to T. brucei: 2 × 108 cells (wild type, single marker, ΔH3.V, ΔH4.V and ΔH3.VΔH4.V) were collected and resuspended in 40 ml of 1× trypanosome dilution buffer All Hi-C reads were combined, mapped onto the error-corrected HGAPv3 contigs and a balanced 10-kb bin heat map matrix of DNA–DNA interactions was generated as previously described. For the immunoprecipitation of cell lines with Ty1-tagged proteins (Ty1-H3.V/ΔH3.V, Ty1-H4.V ΔH4.V/ΔH4.V and Ty1-SCC1/ΔSCC1 cells), 50 μl of Dynabeads protein G (ThermoFisher) was separated on a magnet, the supernatant was removed and the beads were resuspended in 200 μl of PBS-Tween (0.02%) containing 10 μg of BB2 antibody[49], and incubated with slow rotation at 4 °C overnight.
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