Periodic Reporting for period 3 - 3D_Tryps (The role of three-dimensional genome architecture in antigenic variation)
Okres sprawozdawczy: 2020-04-01 do 2021-09-30
With the recent development of tools that allow the elucidation of the 3D genome architecture at unprecedented resolution (Hi-C technology) it is increasingly becoming clear that the folding of chromosomes plays a critical role in the regulation of gene expression and the frequency of translocation 1,2. Both the tight regulation of antigen expression and the translocation of genes coding for antigens are important processes required for antigenic variation in many pathogens.
Thus, the overall goal of the proposed work is to perform the first systematic analysis of the role of genome architecture in the mutually exclusive and hierarchical expression of antigens. To this end we will take advantage of CRISPR-Cas9 genome-editing approaches, genome-wide chromosome conformation capture (Hi-C), and different high-throughput sequencing-based technologies established in my laboratory to study the links between genome architecture and antigen expression.
However, factors linking 3D genome architecture, local chromatin conformation and antigenic variation have not been identified in any organism. One of the major obstacles in studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen arrays, which has precluded complete genome assembly in many pathogens.
Since the start of the action 2.5 years ago, we were able to generate a haplotype-specific genome assembly and performed scaffolding of the highly repetitive antigen arrays of the model protozoan parasite Trypanosoma brucei. In addition, employing genome-wide chromosome conformation capture (Hi-C) we observe a distinct partitioning of the genome with subtelomeric regions, which encode long antigen arrays, being folded into distinct, highly compacted compartments. Next, using a combination of Hi-C, FISH, ATAC-seq and single-cell RNA-seq analyses we were able to show that deletion of the histone variants H3.V and H4.V increases the clustering of antigen- coding genes, 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.
These findings have been published open access last year (L. S. M. Müller et al., Nature 563, 121, 2018).