The role of nuclear architecture in transcriptional regulation in the African Trypanosome, Trypanosoma brucei

Trypanosoma brucei (T. brucei) is a unicellular protozoan parasite that can cause fatal infections in several
mammalian hosts including humans and livestock (Matthews, 2005). In a mammalian host, T. brucei resides
extracellularly in the bloodstream, where it is continuously exposed to the immune system. To evade
clearance by the immune system, T. brucei has evolved an elegant system of antigenic variation of its
protein coat - variant surface glycoprotein (VSG) (Pays, 2004).
There are over 1000 VSG genes and pseudogenes in the genome, with most being located within sub-
telomeric arrays. Crucial to the process of antigenic variation, only one VSG is expressed at any one time,
despite the large repertoire of variants available in the trypanosome genome. In bloodstream form
trypanosomes, the single active VSG is always expressed from one of multiple telomeric expression sites
(ESs), (Taylor, 2006).
An unusual feature of T. brucei is that VSG genes are transcribed by RNA Polymerase I (Pol I), a
polymerase usually associated with transcribing ribosomal RNA transcription within the nucleolus in
eukaryotes (Daneils, 2012). The active VSG ES has been shown to co-localise with an extra-nucleolar Pol I
containing body, which has been termed the ES body (ESB) (Navarro and Gull, 2001). There is evidence
that telomeres, and thereby active and inactive ESs, are distributed throughout the nucleus (Horn, 2014).
This is an unusual feature, and in many other eukaryotes telomeres are commonly sequestered at the
nuclear periphery. The positioning of telomeres and VSG ESs within the nucleus of T. brucei is very
interesting as the location of genes within the nucleus has been shown to be an important aspect of
regulation in other organisms (Lemaitre, 2015).
Despite the strict monoallelic expression of VSG in T. brucei, it has been possible to force two VSG ESs to
be simultaneously active using drug selection. These so called 'double-expressors' have a protein coat
made up of two VSG variants, but these cells are generated at a very low frequency. Much remains
unknown about what allows these two ESs to be simultaneously active in T. brucei. These double
expressor cells can be used to test the hypothesis that sub-nuclear localisation is involved in the stringent
transcriptional control of VSG, and that active ESs in double-expressors may share the same sub-nuclear
location within the cell.
The 3D nuclear organisation of VSG loci could provide some explanation as to how silent VSGs are
repressed. However, VSG gene arrangement within the nucleus must be dynamic to allow VSG switching
to occur, either by in situ switching or recombination. During this project the organisation and dynamics
VSG expressions sites within the nucleus will be investigated in T. brucei cell lines which have been
selected for 2 active VSG expressions sites. This should give insight into the dynamics of the T. brucei
nucleus, and how this is utilised by the parasite to repress silent VSGs and to enable VSG switching to
occur. Questions that will be addressed during the course of this project include:
Do double-expressor cells have two fully functional ESBs, or do the two active ESs share factors
required for transcription?
If two ESBs can not be detected, is there evidence for the physical proximity of the two active ESs
in a double-expressor, suggesting they are sharing an ESB?
Does the nuclear architecture and sub-nuclear localisation of the VSG ESs play a role in
transcriptional regulation and the stringent mono-allelic expression of VSG
What are the dynamics of ES positioning within the nucleus and how does this affect transcriptional
regulation?