Using locus-specific proteomics to investigate heterochromatin in Trypanosoma brucei

Tadhg Devlin

Research output: ThesisDoctoral Thesis

Abstract / Description of output

The eukaryotic nucleus is classically divided into two broad categories: gene-poor heterochromatin and gene-rich euchromatin. In most model eukaryotes, heterochromatin is epigenetically defined by histone H3 lysine 9 methylation (H3K9me), and is rich in repetitive DNA sequences. Trypanosoma brucei is a kinetoplastid parasite that branched early in eukaryotic evolution, and is the causative agent of African sleeping sickness. Trypanosome histone proteins are divergent, and classical heterochromatin histone post-translational modifications (PTMs) such as H3K9me are absent. As a result, the proteins and histone PTMs that define heterochromatin in T. brucei are unknown. Using transcription activator-like effector (TALE) DNA-binding proteins, we have developed a system to purify proteins associated with repetitive DNA sequences, which are candidate heterochromatin regions. We designed TALEs to bind telomeres, centromeres, transposable elements, and the approximately 100 transcriptionally silent minichromosomes, as these repetitive sequences are likely to form heterochromatin. In combination with label-free quantitative mass spectrometry, we hoped this approach would identify previously unknown heterochromatin-associated proteins. Results obtained with telomeres provided proof-of-principle that this was a viable strategy for purifying chromatin-associated proteins in T. brucei, identifying both known and novel telomereinteracting proteins. This included the discovery of a potential role for two zinc-finger proteins at telomeres, in addition to their previously characterised role in post-transcriptional gene regulation. Expansion of the technology to minichromosomes identified that these chromosomes may assemble a kinetochore, in contrast to the prevailing model in the literature. Overall, this work shows that locus-specific proteomics can be a useful tool for investigating trypanosomatid chromatin biology, and could be applied to other genomic loci of interest in future.
Original languageEnglish
  • Auchynnikava, Tatsiana, Supervisor, External person
  • Staneva, Desislava, Supervisor, External person
Publication statusPublished - 23 Jun 2022


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