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

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Trypanosoma brucei (T. brucei) is a unicellular protozoan parasite that can cause fatal infections in severalmammalian hosts including humans and livestock (Matthews, 2005). In a mammalian host, T. brucei residesextracellularly in the bloodstream, where it is continuously exposed to the immune system. To evadeclearance by the immune system, T. brucei has evolved an elegant system of antigenic variation of itsprotein 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 formtrypanosomes, 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), apolymerase usually associated with transcribing ribosomal RNA transcription within the nucleolus ineukaryotes (Daneils, 2012). The active VSG ES has been shown to co-localise with an extra-nucleolar Pol Icontaining body, which has been termed the ES body (ESB) (Navarro and Gull, 2001). There is evidencethat 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 thenuclear periphery. The positioning of telomeres and VSG ESs within the nucleus of T. brucei is veryinteresting as the location of genes within the nucleus has been shown to be an important aspect ofregulation in other organisms (Lemaitre, 2015).Despite the strict monoallelic expression of VSG in T. brucei, it has been possible to force two VSG ESs tobe simultaneously active using drug selection. These so called 'double-expressors' have a protein coatmade up of two VSG variants, but these cells are generated at a very low frequency. Much remainsunknown about what allows these two ESs to be simultaneously active in T. brucei. These doubleexpressor cells can be used to test the hypothesis that sub-nuclear localisation is involved in the stringenttranscriptional control of VSG, and that active ESs in double-expressors may share the same sub-nuclearlocation within the cell.The 3D nuclear organisation of VSG loci could provide some explanation as to how silent VSGs arerepressed. However, VSG gene arrangement within the nucleus must be dynamic to allow VSG switchingto occur, either by in situ switching or recombination. During this project the organisation and dynamicsVSG expressions sites within the nucleus will be investigated in T. brucei cell lines which have beenselected for 2 active VSG expressions sites. This should give insight into the dynamics of the T. bruceinucleus, and how this is utilised by the parasite to repress silent VSGs and to enable VSG switching tooccur. 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 factorsrequired for transcription?If two ESBs can not be detected, is there evidence for the physical proximity of the two active ESsin a double-expressor, suggesting they are sharing an ESB?Does the nuclear architecture and sub-nuclear localisation of the VSG ESs play a role intranscriptional regulation and the stringent mono-allelic expression of VSGWhat are the dynamics of ES positioning within the nucleus and how does this affect transcriptionalregulation?