Loss of an ectopic VSG is dependent on active transcription and does not disrupt the number or variety of templates used to repair a BES DSB

In the mammalian host, Trypanosoma brucei is coated in a single variant surface glycoprotein (VSG) species. Stochastic switching of the expressed VSG allows the parasite to escape detection by the host immune system. DNA double strand breaks (DSB) trigger VSG switching, and repair via gene conversion results in an antigenically distinct VSG being expressed from the single active bloodstream form expression site (BES). By stably expressing a second telomeric VSG from a ribosomal locus, we were able to disrupt monoallelic VSG expression. We found that cells expressing two VSGs contained one VEX2 focus that was significantly larger in size, as compared to the wild-type cells. VEX2 marks the single active BES and this therefore suggests the ectopic VSG is expressed from the same nuclear position as the active BES. In the double VSG expressing cells, the DNA sequence of the ectopic copy is lost following a DSB in the active BES, despite it being spatially separated in the genome. The loss of the ectopic VSG is dependent on active transcription and does not disrupt the number or variety of templates used to repair a BES DSB. We propose that there are stringent mechanisms within the cell to reinforce monoallelic expression during antigenic variation.