Transcriptional regulation of VSG monoallelic expression in the African trypanosome - Trypanosoma brucei

T. brucei is a unicellular protozoan parasite that is best known for its sophisticated strategy of antigenic variation of the surface coat protein, VSG, to evade host immune responses. There are over one thousand VSG genes in the T. brucei genome and ~20 telomeric expression sites (ESs) from where VSG may be expressed. All but one of these ESs is strictly silenced, which is crucial for antigenic variation to be successful, and a single ES is actively transcribed in a manner known as monoallelic expression. VSG ESs are transcribed using RNA Polymerase I from within a unique sub-nuclear structure called the Expression Site Body (ESB). The observation that an ESB is not detected in all cells within a given population suggests that the active ES is dynamically regulated. In this research project I adapted a fluorescent timer reporter called FastFT for use in T. brucei, to investigate this phenomenon. I established that the kinetics of the FastFT fluorophore maturation in T. brucei make this a promising tool for studying dynamic transcriptional states, and potentially other aspects of trypanosome biology. I also used this novel tool to test the model that the ESB is a limiting structure contributing to monoallelic expression of VSG, and showed that transcriptional dynamics are altered when monoallelic expression is disrupted. I also sought to identify factors that suppress transcription from silent ESs, of which there are two types – bloodstream form and metacyclic form. I analysed data from three genome-wide functional screens designed to identify repressors of metacyclic form expression site transcription and validated several of the candidate genes. I further characterised the function of one of these genes, TbSAP, which is a predicted DNA binding protein. I showed that perturbation of TbSAP led to up-regulation of both bloodstream and metacyclic ES transcription. I also discovered that TbSAP is a nuclear protein that appears to be enriched at the nuclear periphery. Chromatin immunoprecipitation experiments indicated that TbSAP interacts with ES promoters, and may mediate transcriptional repression through either regulation of ES localisation or ES chromatin structure. In this research project I have attempted to further our understanding of the positive and negative regulators of ES transcription, which underpin the paradigmatic system of monoallelic expression in this divergent eukaryote.