One might describe Trypanosoma brucei as a moderately fashionable parasite, as it changes its outer coat faster than models can strut their stuff down the exclusive catwalks in Paris or Milan! Other than making this troublesome unicellular protozoan look very sharp, its ever-changing coat helps it to keep out of reach of the host immune system after infection, thereby promoting the development of sleeping sickness in humans.
The “fabric” of the T. brucei coat mainly comprises numerous variant surface glycoproteins (VSGs) that are coded for by immunologically diverse genes (more than 2, 500, generated by homologous recombination) and primarily present in large arrays near the telomeric ends of chromosomes. Importantly, VSG gene expression depends on their localization to distinct telomere-proximal polycistronic transcription units (expression sites), ensuring the presence of only a few VSG proteins on the parasite coat at any one time.
In the search for the snazzy mechanisms that control VSG gene expression and immune evasion in T. brucei, researchers from the laboratory of T. Nicolai Siegel (Ludwig-Maximilians-Universität München, Germany) examined 3D genome architecture and local chromatin conformation, following clues from previous studies they employed microscopy-based analyses of T. brucei and Plasmodium falciparum, another unicellular eukaryotic parasite (See Cell Host Microbe for the latest of these studies).
Müller and colleagues first required a more complete genome assembly of T. brucei; a task made difficult by the highly repetitive nature and heterozygosity of the VSG gene arrays. However, via PacBio single-molecule real-time (SMRT) sequencing technology and the application of known conserved features of chromosome folding, the authors reported the de novo haplotype-specific assembly and scaffolding of the telomeric VSG gene arrays. They then used these maps to assess the contribution of genome organization to VSG gene expression and immune avoidance by T. brucei.
Here are all the details for this hip and stylish, cutting-edge 3D genome organization study:
- Through genome-wide chromosome conformation capture (Hi-C) analysis, the authors discovered folding of VSG-encoding subtelomeric regions into distinct, highly compact compartments, while highly expressed housekeeping genes lie in a less compact genome core
- RNA sequencing (RNA-Seq) revealed partitioning of the genome into a transcribed homozygous core and non-transcribed heterozygous subtelomeric regions, where VSG genes lie
- Transcriptional data, therefore, mirrors the 3D organization of the genome
- The high degree of compaction in subtelomeric regions ensures the exclusive expression of single VSG genes (and the repression of the many thousand others) and promotes homologous recombination
- RNA sequencing (RNA-Seq) revealed partitioning of the genome into a transcribed homozygous core and non-transcribed heterozygous subtelomeric regions, where VSG genes lie
- Chromatin immunoprecipitation with sequencing (ChIP-seq) analyses of the major subunit of cohesin (SCC1), which aids the establishment and maintenance of higher-order genome structures, revealed binding to regions near the 3´ ends of VSG expression sites, suggesting the presence of a chromatin domain boundary
- The H3.V and H4.V histone variants display similar distributions to SCC1, suggesting that they may function in combination with SCC1 to shape genome organization and regulate VSG gene expression
- Single-cell RNA-seq (scRNA-seq) analysis demonstrated that the loss of both H3.V and H4.V leads to common switching in the expression of VSG genes via recombination events (usually occurring rarely and in response to an adapting host immune system) and the expression of multiple VSG genes
- Transposase-accessible chromatin using sequencing (ATAC-seq) analysis suggests that multiple VSG gene expression and recombination events occur thanks to increased inter-chromosomal interactions, interactions among repressed expression sites, and local DNA accessibility
Overall, the authors of this en mode study show that changes in global genome architecture and local chromatin configuration can induce switches in VSG gene expression and, therefore, play vital roles in the ability of T. brucei to avoid immune detection via the creation of a brand-new glycoprotein coat.
For more of the details on this eye-catching “little-black-dress” of a paper, see Nature, October 2018
If you’d like to read more about the ATAC-Seq method, please visit this great blog article from our friends at Active Motif – Complete Guide to Understanding and Using ATAC-Seq.