DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice.

Alix Thivolle,Emilia Mclaughlin,Eliane Tihon,Lucy Glover,Annick Dujeancourt-Henry,Ann-Kathrin Mehnert,David Sacks

doi:10.1371/journal.ppat.1010038

Alix Thivolle, Emilia Mclaughlin + Show 5 more

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https://doi.org/10.1371/journal.ppat.1010038

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Abstract

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. This process is facilitated by the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.

Highlights

Trypanosoma brucei exists as an extracellular parasite in the mammalian host, colonizing the blood, adipose tissue and skin [1,2]
In addition to the 70intsce cell line we integrated an SceR between ESAG1 and the smaller block of 70-bp repeats (Fig 1A; ESAG1sce), and between two blocks of 70-bp repeats in the Pseudo gene (Fig 1A; Pseudosce), as has been previously described [13], in both cases using an RFP:PAC fusion cassette containing the I-SceI recognition site
From our data we conclude that an I-SceI induced double strand breaks (DSB) that is flanked by 70-bp repeats and distal to the variant surface glycoprotein (VSG) gene is not toxic to bloodstream form trypanosomes

Summary

Introduction

Trypanosoma brucei exists as an extracellular parasite in the mammalian host, colonizing the blood, adipose tissue and skin [1,2]. A large proportion of the trypanosome subtelomeric nuclear genome encodes for VSG genes and their associated sequences, providing a vast repertoire of potential surface antigens for immune evasion [4]. The VSG is expressed from a subtelomeric locus called a bloodstream form expression site (BES) by RNA Polymerase I at an extra nucleolar transcription factory termed the expression site body (ESB) [5,6] which recruits the VSG exclusion (VEX) complex [7,8,9]. BESs are polycistronic transcription units which contain several protein coding genes termed expression site associated genes (ESAGs) along with the VSG. There are approximately fifteen BESs in the nuclear genome [10], monoallelic expression of a single BES ensures that only one VSG is exposed to the immune system at a time

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