Our group is interested in functional genomics. Using the protozoan parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, we study some of the very fundamental and evolutionarily conserved mechanisms of genome organization and chromatin formation. At the same time we try to understand how changes in chromatin structure can help the parasite to evade the host immune response via antigenic variation.
Key to our work is the ability generate high quality genome assemblies and to study their organization on a genome-wide scale. To this end we have established numerous system-wide approaches in T. brucei, including ribosome-profiling, high-resolution ChIP-seq and Hi-C assays as well as a mass spectrometry-based approach to quantify levels of histone modifications. Using these techniques, we are able to generate genome assemblies and to investigate how different chromatin structures are established at specific loci along the genome, how they are formed across the nucleus in 3-dimensions and how they affect gene expression. Currently, we are adopting several of these techniques to the single cell level.
Key questions of our research are:
- How are genomes organized?
- What ensures partitioning of genomes into ‘stable’ regions coding for housekeeping genes and in ‘unstable’ regions allowing the formation of new antigen-encoding genes?
- Are genomes compartmentalized?
- If yes, how is this compartmentalization established and maintained?
- What ensures that only one antigen is expressed at a time?
- How does the chromatin structure differ between active and inactive antigen-coding genes
- Do changes in 3D genome architecture affect antigen expression
- How is the T. brucei genome organized in 3-dimensions?
- What ensures the hierarchical activation of antigens?
- Does spatial proximity in 3D affect the order of antigen activation?
Why study antigenic variation?
It is very important!
Viruses, bacteria, and protozoan parasites all face similar challenges when infecting a susceptible host, including the evasion of the host immune response. This common challenge has led to the evolution of remarkably similar survival strategies even among evolutionarily distant pathogens. One of these strategies is antigenic variation. Antigenic variation refers to the capacity of an infecting organism to systematically alter the identity of proteins displayed to the host immune system making it difficult or impossible for the host to eliminate the pathogen. This leads to prolonged infections and enhanced transmission to a new host (1). As a consequence, antigenic variation represents a key challenge in the fight against infectious diseases and the ability to interfere with the process would represent a major breakthrough, as it would help hosts to clear the infection and facilitate the development of vaccines.
The general requirements for antigenic variation are very similar among pathogens and include a) mechanisms to evolve large gene families coding for different antigen isoforms, b) the mutually exclusive expression of one or a few antigens from large multigene families and c) the periodic, nonrandom switching between the expression of different antigens. It is important that antigens are not activated at random during the course of an infection as this would lead to heterogeneous pathogen populations in the host and to a rapid exhaustion of available antigens. Despite extensive research and significant findings in different organisms, the underlying mechanisms of antigenic variation are not fully understood in any organism (2).
Why study antigenic variation now?
The recent, groundbreaking development of high-throughput sequencing technologies and CRISPR-Cas9-based genome editing tools has created unprecedented opportunities to study antigenic variation.
For example, it is now possible to probe chromosome organization by measuring the frequency of physical interactions or proximity of any two genomic loci by combining high-throughput sequencing technologies with chromosome conformation capture assays, an approach referred to as Hi-C. Hi-C analyses have revealed the three-dimensional (3D) organization of chromosomes at unparalleled resolution (3) and shown that the spatial organization and compartmentalization of DNA inside the nucleus play critical roles in the regulation of gene expression and the frequency of translocation (4,5). In addition, CRISPR-Cas9 technology has increased the efficiency of genome editing such that it can be performed at nucleotide-resolution without the need to insert selectable markers (6). Thus, CRISPR-Cas9 technology will enable individual antigen genes to be moved to spatially distinct locations in the genome providing a means to elucidate the role of genome architecture in the hierarchical switching of antigen expression.
Why study antigenic variation in Trypanosoma brucei?
T. brucei contains the largest family of mutually exclusively expressed surface antigens described to date and possesses many attributes that have made it one of the most important models to study antigenic variation.
For example, trypanosomes can be easily cultured in liquid media, genetic manipulation is straightforward and RNA-interference (RNAi) can be used to efficiently deplete essential proteins or to perform genome-scale forward genetic screens (7,8).
The major T. brucei surface antigen is the variant surface glycoprotein (VSG) (9) and the parasite’s genome codes for ~ 2,000 VSGs (10). While most of the VSG genes are found in long, subtelomeric arrays (11), a small subset of VSGs is located in distinct telomere-proximal polycistronic transcription units, called ‘expression sites’ (12). VSGs are transcribed by RNA polymerase I (RNA pol I) and can only be transcribed when located within an expression site. However, at any one time only one of ~15 expression sites is transcribed while the others are transcriptionally repressed (9). Switching of VSG expression occurs by two mechanisms: a) by switching transcription from one expression site to another (transcriptional switch, epigenetic) or b) by a recombination-based event that leads to the replacement of the previously active (old) VSG with a new VSG from elsewhere in the genome (recombinational switch, genetic) (13).
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2. Turner, C. M. A perspective on clonal phenotypic (antigenic) variation in protozoan parasites. Parasitology 125 Suppl, S17-23 (2002).
3. Hakim, O. & Misteli, T. SnapShot: Chromosome Confirmation Capture. Cell 148, 1068-1068.e2 (2012).
4. Hager, G. L., McNally, J. G. & Misteli, T. Transcription dynamics. Mol Cell 35, 741-753 (2009).
5. Misteli, T. & Soutoglou, E. The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol 10, 243-254 (2009).
6. Hsu, P. D., Lander, E. S. & Zhang, F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157, 1262-1278 (2014).
7. Ngo, H., Tschudi, C., Gull, K. & Ullu, E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 95, 14687-14692 (1998).
8. Clayton, C. E. Genetic manipulation of kinetoplastida. Parasitol Today 15, 372-378 (1999).
9. Horn, D. Antigenic variation in African trypanosomes. Mol Biochem Parasitol 195, 123-129 (2014).
10. Cross, G. A., Kim, H. S. & Wickstead, B. Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol Biochem Parasitol 195, 59-73 (2014).
11. Berriman, M. et al. The genome of the African trypanosome Trypanosoma brucei. Science 309, 416-422 (2005).
12. Hertz-Fowler, C. et al. Telomeric expression sites are highly conserved in Trypanosoma brucei. PLoS ONE 3, e3527 (2008).
13. Taylor, J. E. & Rudenko, G. Switching trypanosome coats: what’s in the wardrobe? Trends Genet 22, 614-620 (2006).