Chromatin associated proteins and histone post-translational modification dynamics in the development and immune evasion of the sleeping sickness para

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

The kinetoplastid parasites are major pathogens responsible for several important tropical diseases including Chagas disease, Leishmaniasis and African trypanosomiasis. Trypanosome parasites cause lethal disease in humans (sleeping sickness) and cattle (Nagana) across sub-Saharan Africa with devastating social and economic consequences. As trypanosomes multiply in the host bloodstream (slender forms) a proportion differentiate into quiescent stumpy forms. Stumpy forms subsequently survive uptake by feeding tsetse flies where they initially populate the mid-gut (procyclic forms) prior to migration and maturation in the salivary gland, finally infecting a new host when the fly feeds again.
The nucleus of trypanosomes contains a large amount of inert chromatin that cytologically appears dense and, as in most eukaryotes, is referred to as heterochromatin. One form of trypanosome heterochromatin mediates the repression of the many unexpressed copies of Variable Surface Genes (VSG) required to evade host immune systems. However, a molecular understanding of trypanosome heterochromatin and its contribution to developmental and VSG gene regulation, the control of transposable elements, centromere repeat sequence function and general nuclear architecture remains in its infancy. This is partly due to the extreme evolutionary divergence of trypanosomes and their histone proteins from the main eukaryotic paradigm.
Histones are subject to a plethora of post-translational modifications (PTMs; acetylation, methylation etc), particularly on the lysine residues, within the unstructured N-terminal tails that protrude from each nucleosome. Collectively such modifications are referred to as 'epigenetic marks' as they can confer properties independent from the underlying DNA sequences. Such marks regulate chromatin compaction and access to the enclosed DNA for the processes of transcription, replication, repair and recombination. In most conventional eukaryotes methylation of lysine 9 or 27 of histone H3 (K3K9, H3K27) forms repressive heterochromatin whereas H3K4 and H3K36 methylation assist transcription. Histone lysine acetylation also promotes transcription. Thus, the pattern of PTMs present on the eight histone subunits comprising each nucleosome supervise the release of information from the enclosed DNA. In kinetoplastids such as Trypanosoma brucei the conserved core histones are particularly divergent from the apparent eukaryotic norm; the equivalent conserved residues that are usually subject to PTMs in mainstream eukaryotes are either absent (e.g. H3K9) or the surrounding context is so distinct that alignments are tentative. Consequently few studies have been performed on the function of specific histone PTMs in trypanosomes, or the proteins that they attract, because the epitopes recognised by commercial antibodies raised against histone PTMs, and conserved in most eukaryotes, do not cross-react with these highly divergent trypanosome histones.

Aims
1. Implement cutting edge mass spectrometry methods to identify and quantify chromatin associated proteins and histone PTMs in the three tractable developmental forms of T. brucei (Bloodstream: slender and stumpy; Insect: procyclic).

2. Target CRISPR/Cas9-GFP to several heterochromatin regions via guide RNAs and utilize these to enrich specific loci by affinity selection. Quantify proteins and histone PTMs enriched in repeat element heterochromatin and silent VSG gene chromatin.

3. Characterize the role of specific PTMs and proteins identified in heterochromatin function and Trypanosoma brucei differentiation and development.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M010996/1 01/10/2015 30/09/2023
1940752 Studentship BB/M010996/1 01/10/2017 30/09/2021 Tadhg Devilin