Discussion

Antigenic variation in Trypanosoma brucei is mediated by transcription of Variant Surface Glycoprotein (VSG) genes located in telomeric VSG expression sites (ES). Only one VSG is expressed at a time from 1 of 15 ESs. Despite the fact that monoallelic exclusion of VSG is critical for continued survival of the parasite, little is known about how this process is regulated mechanistically. To gain more insight into this, we generated three �double-expressing� (DE) T. brucei lines with the VSG221 ES (marked with eGFP) and the VSGV02 ES (marked with mCherry) both simultaneously active. Generation of DE lines is rare (10 7 and they are highly unstable without selection. Flow cytometry quantitating expression of mCherry or eGFP has allowed us to characterise dynamics of ES expression in these three independently generated DE lines. These T. brucei DE lines show different degrees of stability of the DE phenotype, which is reflected in differences revealed through RNA-seq analysis. This indicates that different sets of events can occur to maintain a DE state. However some genes are universally up or downregulated in all three DE lines. These include the telomere binding protein TRF, which has earlier been shown to be key for ES control. We are currently analysing these differentially expressed candidate genes through knock-down experiments for their role in mono-allelic control of ESs