Authors

C Davies¹; E Lockyear¹; G Sioutas¹; H Sidhu¹; S Alsford²; B Wickstead³; B S Hall¹; G Rudenko¹;  ¹ Imperial College London, UK;  ² London School of Hygiene and Tropical Medicine, UK;  ³ University of Nottingham, UK

Discussion

Trypanosoma brucei relies on an elegant system of antigenic variation to survive extracellularly in the bloodstream of its mammalian host. Strict mono-allelic expression of the protective variant surface glycoprotein (VSG) is crucial to this process. VSG is always expressed from a telomeric expression site, of which there are two types:   bloodstream form (BES) and metacyclic form (MES), which are each utilised at different life cycle stages.

We would like to identify factors that play a role in keeping the MESs silent in bloodstream form trypanosomes. Do the same key players regulate both types of expression sites or do MESs have their own distinct set of regulatory machinery? To tackle this unanswered question, we have carried out whole genome RNAi library screens. These were performed using a trypanosome line containing a puromycin resistance gene integrated behind a silent MES promoter. RNAi mediated knock down of genes involved in silencing MESs would lead to increased expression of puromycin resistance protein.  We successfully performed three whole genome RNAi screens at three different concentrations of puromycin.

High throughput RITseq analysis allowed us to identify a number of candidate genes, many of which were picked up in multiple screens. These candidates have been validated as having a role in ES regulation using a cell line with a reporter GFP gene in a silent MES.  This allowed us to measure fluorescence intensity following RNAi knockdown. These analyses have resulted in us identifying an MES silencing candidate which we call SAP, due to the presence of a predicted SAP DNA binding domain (Tb927.10.4440).  SAP domain containing proteins are typically chromatin associated and have been implicated in the organisation of chromatin domains within chromosomes.  Knock-down of SAP results in derepression of multiple MESs as monitored by qPCR.  We have also shown that SAP is essential for normal growth in bloodstream form trypanosomes. SAP localises in the nucleus, with enrichment at the nuclear periphery.

Further characterisation of these candidate genes will provide more insight into the transcriptional regulation occurring at MESs, and potentially all telomeric VSG expression sites. Understanding the monoallelic expression in this organism is crucial to understanding its success as a parasite.