In its bloodstream form (BF), Trypanosoma brucei escapes elimination by its host's immune response by periodically switching its variant surface glycoprotein (VSG). Although there are many VSG genes, only the VSG positioned at an active expression site (ES) is transcribed. When T. brucei returns to its tsetse vector, it differentiates into a non-infective, procyclic form (PF), and all ESs are inactivated. The mechanisms of ES inactivation in BF and PF are not understood but appear to be distinct. Studies from a variety of organisms indicate that core and variant histones often play important roles in the control of transcription and development. Recently, two genes from T. brucei that encode distinct histone variants were identified and the experiments outlined in this proposal explore how they may affect the transcriptional control of ESs. Upon establishing that both variants are likely to exist in nucleosomes in vivo, mutant alleles of each will be introduced into both BF and PF cells and tested for their ability to perturb the silencing of VSG and reporter genes at inactive ESs. Additionally, to determine if either variant is present at unique sites within the nucleus, immunofluorescence microscopy and chromatin immunoprecipitations (ChrIP) will be performed. Finally, to examine if the chemical modifications of histones differ between active and inactive ESs, chromatin purified from both BF and PF cells will be analyzed by mass spectroscopy and by ChrIP.
