Abstract
Antigenic variation in African trypanosomes requires monoallelic transcription and switching of variant surface glycoprotein (VSG) genes. The transcribed VSG, always flanked by ‘70 bp’-repeats and telomeric-repeats, is either replaced through DNA double-strand break (DSB) repair or transcriptionally inactivated. However, little is known about the subtelomeric DSBs that naturally trigger antigenic variation in Trypanosoma brucei, the subsequent DNA damage responses, or how these responses determine the mechanism of VSG switching. We found that DSBs naturally accumulate close to both transcribed and non-transcribed telomeres. We then induced high-efficiency meganuclease-mediated DSBs and monitored DSB-responses and DSB-survivors. By inducing breaks at distinct sites within both transcribed and silent VSG transcription units and assessing local DNA resection, histone modification, G2/M-checkpoint activation, and both RAD51-dependent and independent repair, we reveal how breaks at different sites trigger distinct responses and, in ‘active-site’ survivors, different switching mechanisms. At the active site, we find that promoter-adjacent breaks typically failed to trigger switching, 70 bp-repeat-adjacent breaks almost always triggered switching through 70 bp-repeat recombination (∼60% RAD51-dependent), and telomere-repeat-adjacent breaks triggered switching through loss of the VSG expression site (25% of survivors). Expression site loss was associated with G2/M-checkpoint bypass, while 70 bp-repeat-recombination was associated with DNA-resection, γH2A-focus assembly and a G2/M-checkpoint. Thus, the probability and mechanism of antigenic switching are highly dependent upon the location of the break. We conclude that 70 bp-repeat-adjacent and telomere-repeat-adjacent breaks trigger distinct checkpoint responses and VSG switching pathways. Our results show how subtelomere fragility can generate the triggers for the major antigenic variation mechanisms in the African trypanosome.
Highlights
Several important parasites, including those that cause malaria and Human African Trypanosomiasis (HAT), achieve antigenic variation and evasion of the host adaptive immune response through monoallelic expression and clonal phenotypic variation of surface proteins [1,2]
ligationmediated PCR (LM-PCR) assays revealed double-strand break (DSB) in all three subtelomeric regions and, in contrast to a previous report [10], transcription status had little impact on the number of DSBs, which were detected at a similar frequency regardless of whether the variant surface glycoprotein (VSG) was transcribed or silent (FIG. 1B)
We suggest that breaks within the 70-bp repeats, or between two blocks of 70-bp repeats [10], would generate effective substrates for single-strand annealing [42], a recombination pathway which would generate a ‘repeat’ deletion, rather than lead to VSG replacement
Summary
Several important parasites, including those that cause malaria and Human African Trypanosomiasis (HAT), achieve antigenic variation and evasion of the host adaptive immune response through monoallelic expression and clonal phenotypic variation of surface proteins [1,2]. The African trypanosomes are flagellated parasitic protists of major medical and veterinary importance. They are the causative agents of HAT, and Nagana in cattle, and they proliferate in the mammalian host bloodstream. In Trypanosoma brucei, antigenic variation requires mono-telomeric expression and switching of variant surface glycoprotein genes (VSGs). It is this continuous process of allelic exclusion, transcription of only one telomeric VSG at a time in each cell, which is essential for the persistence of a chronic infection. T. brucei has long been a paradigm for antigenic variation but the molecular triggers and the mechanisms mediating VSG recombination and switching are not fully understood
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