Periodic Reporting for period 4 - 3D_Tryps (The role of three-dimensional genome architecture in antigenic variation)
Okres sprawozdawczy: 2021-10-01 do 2023-03-31
Antigenic variation is a widely used strategy for evading the host immune response. Even among evolutionarily divergent pathogens, the functional requirements for antigenic variation are similar and include 1) the mutually exclusive expression of one or a few antigens from large multigene families and 2) the periodic, non-random switch in 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 a rapid exhaustion of available antigens. Despite decades of research and significant findings made in different organisms, the mechanisms of antigenic variation are not fully understood in any organism.
With the recent development of tools that allow the elucidation of the 3D genome architecture at unprecedented resolution (Hi-C technology), it is becoming increasingly 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 was to provide the first systematic analysis of the role of genome architecture in the mutually exclusive and hierarchical expression of antigens. To this end, we took advantage of CRISPR-Cas9 genome-editing approaches, genome-wide chromosome conformation capture (Hi-C), and various high-throughput sequencing-based technologies established in my laboratory to study the links between genome architecture and antigen expression.
With the recent development of tools that allow the elucidation of the 3D genome architecture at unprecedented resolution (Hi-C technology), it is becoming increasingly 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 was to provide the first systematic analysis of the role of genome architecture in the mutually exclusive and hierarchical expression of antigens. To this end, we took advantage of CRISPR-Cas9 genome-editing approaches, genome-wide chromosome conformation capture (Hi-C), and various high-throughput sequencing-based technologies established in my laboratory to study the links between genome architecture and antigen expression.
The common need to evade the host immune response has led to the evolution of remarkably similar survival strategies even among evolutionarily distant organisms such as fungi, bacteria and protozoa. One of these strategies is antigenic variation, where an infecting organism systematically alters the identity of proteins that are visible to the host immune system to prevent elimination by the host. Antigenic variation involves mechanisms that allow homologous recombination to generate large reservoirs of immunologically diverse antigens and to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by 3D genome architecture and local DNA accessibility.
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 6 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.
In addition, using the newly assembled genome and data from our newly established genome-wide chromosome conformation assays, we have discovered that distinct DNA-DNA interactions correlate with activation of the dominant surface antigen, presumably by enhancing splicing of the corresponding transcript. Finally, we have established scRNA-seq approaches that will allow us and others in the field to disentangle the factors that influence the mutually exclusive expression of VSG and the switching of VSG isoform 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 6 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.
In addition, using the newly assembled genome and data from our newly established genome-wide chromosome conformation assays, we have discovered that distinct DNA-DNA interactions correlate with activation of the dominant surface antigen, presumably by enhancing splicing of the corresponding transcript. Finally, we have established scRNA-seq approaches that will allow us and others in the field to disentangle the factors that influence the mutually exclusive expression of VSG and the switching of VSG isoform expression.
As part of the ERC-funded research, we have established genome-wide chromosome conformation capture (Hi-C) and single-cell RNA-sequencing in T. brucei. Taking advantage of these technologies, we hope to identify the factors determining the hierarchy of antigen activation.