Genome organization and DNA accessibility control antigenic variation in trypanosomes

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.

SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes

“…Some, like VSG427‐15 (BES10) and VSG‐14 (BES8), but not all were upregulated, while the active BES1 telomeric VSG 221 gene was consistently downregulated, suggesting that where SNF2PH was depleted, random derepression of inactive VSG‐ES promoters occurred. Thus, SNF2PH depletion induced derepression of a cluster of silent BES but not all, similar to recently reported for H3V‐H4V KO . As the PH domain is known to bind Histone 3 tails, the H3V KO and SNF2PH depletion phenotypes may be related.…”

“…Thus, SNF2PH depletion induced derepression of a cluster of silent BES but not all, similar to recently reported for H3V‐H4V KO . As the PH domain is known to bind Histone 3 tails, the H3V KO and SNF2PH depletion phenotypes may be related.…”

“…Interestingly, proteins regulating nuclear architecture also alter monoallelic VSG expression, including the lamina proteins NUP‐1 and cohesin, where silencing leads to a ~10‐fold increase in VSG switch frequency . Deletion of histone variants H3.V and H4.V highly increases VSG switch frequency via homologous recombination, and the telomeric VSG‐ES loci are confined to distinct compartments within the nucleus . Altogether, these data support a model where epigenetically marked VSG‐ES chromatin occupies a unique subnuclear compartment, facilitating monoallelic expression .…”

“…This global analysis confirmed previous ChIP data locating SNF2PH on developmentally regulated genes (EP and GPEET), RNA pol I driven rDNA (ribRNAs) and the SL cluster of small RNAs that are trans‐spliced in every mRNA. Interestingly, beside those essential genes for cell growth, SNF2PH occupies few other coding genes; noteworthy is H3V protein recently linked to the regulation of VSG monoallelic expression . In addition, SNF2PH was consistently enriched at tRNA gene clusters in 7 chromosomes.…”

“…As demonstrated by quantitative ChIP, the site of enrichment corresponded to developmentally regulated loci EP and GPEET2 procyclin, and 18S ribosomal DNA and SL‐RNA‐related sequences. Interestingly, SNF2PH localizes at H3.V, a histone variant recently associated with VSG monoallelic expression and its own coding sequence. SNF2PH was also significantly enriched at a substantial number of tRNAs gene arrays located in chromosomes XI, X, VIII, VII, V, IV, and III (Fig C); interestingly, tRNA clusters are known to function as a chromatin insulators in eukaryotes from yeast to human, reviewed in Ref.…”