Chromatin organisation and transcription regulation in Trypanosoma brucei

Abstract

In eukaryotic nuclei, DNA is packaged into chromatin by association with histone proteins. Specific histone post-translational modifications result in the formation of transcriptionally active and silent chromatin domains. The aim of this project was to characterise chromatin organisation and assess the role of transcription regulation in Trypanosoma brucei, the causative agent of sleeping sickness in Africa. The approach taken was endogenous fluorescent tagging of putative readers, writers and erasers of histone modifications in T. brucei and subsequent use of the tags to localise the candidate proteins in the cell and to identify their genomic associations and protein interaction networks. Chromatin immunoprecipitation followed by sequencing showed that many of the nuclear proteins associate with RNAPII transcription start regions (TSRs). Whereas most proteins were enriched broadly over those regions, Chromo1 and SET27 displayed sharp overlapping peak profiles. Moreover, co-immunoprecipitation and mass spectrometry revealed that Chromo1 and SET27 are likely part of the same complex together with four uncharacterised proteins and JBP2, an enzyme involved in the synthesis of the DNA modification base J. Overall, this work provides the basis for investigating the role of chromatin factors in trypanosome transcription regulation.