Genome organization and DNA accessibility control antigenic variation in trypanosomes
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 host 1 . 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 threedimensional genome architecture and local DNA accessibility 2,3 . 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 haplotypespecific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using longread sequencing technology and conserved features of chromosome folding 4 . 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 transposaseaccessible 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. Genome sequences of several pathogens have revealed a partitioning of chromosomes, with housekeeping genes often being located in the central core and antigen genes being located in subtelomeric regions 5,6 . These assemblies suggest that the linear organization of the genome may be important for restricting high levels of recombination to regions that code for antigens and for ensuring that all but one antigen is repressed. Recently, genome-wide Hi-C analyses have begun to uncover the 3D organization of chromosomes at high resolution 4 , which has highlighted the critical role of spatial organization and compartmentalization of DNA in the regulation of gene expression and recombination 2,3 . OPEN 1 N O V e M B e r 2 0 1 8 | V O L 5 6 3 | N A t U r e | 1 2 1 Fig. 1 | Long-read and Hi-C-based de novo assembly of the T. brucei Lister 427 genome. Only one of the two homologous chromosomes (chr.) is depicted for the homozygous chromosomal core regions (22.71 Mb). Both chromosomes are shown for the heterozygous subtelomeric regions (19.54 Mb). Relative transcript levels (window size, 5,001 bp; step size, 101 bp) are shown as a black line above each chromosome. BESs and MESs were assigned to the respective subtelomeric region if an unambiguous assignment based on DNA interaction data was possible (see Supplementary Information). Centromeres were assigned based on KKT2 ChIP-seq data 30 . 1 2 2 | N A t U r e | V O L 5 6 3 | 1 N O V e M B e r 2 0 1 8 . for critical reading of the manuscript. We thank T. Achmedov for scRNA-seq technical assistance, M. Berriman, G. Ramasamy, P. Myler and L. Barquist for assistance with the genome assembly, J. Dekker, M. Imakaev, J. M. Belton and B. R. Lajoie for advice on Hi-C experimental design and analysis, K. Ersfeld for advice on epitope tagging of SCC1 and FISH, S. Kirchner and A. R. Batista for suggestions on ATAC-seq, T. Straub and F. Goth for providing server space and all members of the Engstler, Janzen, Kramer, Morriswood and Ladurner laboratories for valuable discussions. We thank C. Clayton and L. Glover for reagents and M. Urbiniak for sharing unpublished SCC1 data.