Authors

J Budzak¹; L E Kerry¹; C Davies¹; A Aristodemou¹; B Wickstead²; K Witmer¹; B Hall¹; M Kushwaha¹; M Povelones¹; G Rudenko¹;  ¹ Imperial College London, UK;  ² Queen's Medical Centre, University of Nottingham, Nottingham, UK

Discussion

The African trypanosome Trypanosoma brucei is coated with a dense layer of antigenically variable Variant Surface Glycoprotein (VSG) when in the bloodstream of the host.  Although a single trypanosome has thousands of VSG genes, only one is expressed at a time in a stringently monoallelic fashion from one of about 15 telomeric expression sites (ES)s. The active ES is transcribed by RNA polymerase I (RNA pol I), which normally exclusively transcribes rDNA.  ES transcription occurs in an extra-nucleolar body called the Expression Site Body (ESB).  In order to investigate the restriction operating on monoallelic exclusion, we have generated cell lines in which the VSG221 and VSGV02 ESs were simultaneously selected for using drug selection markers.  In addition, there is an eGFP gene in the VSG221 ES, and an mCherry gene in the VSGV02 ES, allowing ES activity to be monitored using flow cytometry.  These ‘double expresser’ (DE) cell lines appear to continuously switch back and forth between the two ESs.  We next introduced an RNAi construct into the DE KW01 cell line, allowing inducible knock-down of eGFP and mCherry.  This allows us to transiently ‘defluoresce’ the cells and further analyse ES expression using an epitope tagged Pol I subunit or by DNA or RNA FISH experiments. 

Interestingly, mNeonGreen tagged RNA pol I showed that around 64% of the DE KW01 trypanosomes contain only one ESB, 29% have no ESB, and 7% have two. DNA-FISH experiments showed that in 57% of all cells, both ESs either co-localise or are within 250 nm of each other. In contrast, in the parental ‘single expresser’ trypanosomes, only 3% of the two ESs are this close to each other. In order to monitor transcriptional activity in these DE lines, we used Stellaris RNA-FISH to detect nascent transcripts from both dynamically active ESs.  We find that trypanosomes predominantly transcribe either one or the other ES.  However strikingly, in cells where transcripts from both ESs are detected (about 20% of total cells), nascent transcripts from both ESs localise in close proximity in the nucleus. These data therefore show that ES double expression results in two active ESs sharing the same sub-nuclear location, presumably allowing the two ESs to occupy the same ESB. Although simultaneous transcription of two ESs appears possible, transcriptional activation of only a single ES is highly favoured. This argues that monoallelic exclusion places a physical restriction on the number of ESs that can be simultaneously activated at any one time in the nucleus.