Deciphering mechanisms of epigenetic regulation in Trypanosoma brucei, an evolutionarily distinct human pathogen.

Context:
Organisms like yeasts, plants and animals keep their DNA within a nucleus, a structure that allowed evolution of complex lifestyles. This promoted the emergence of plants and animals with distinct cells and tissues specialised for specific tasks. Similarly, single-celled organisms, like yeasts and protozoa, undergo changes in cell shape and physiology to adapt to different environments by changing which genes they express. In parasites, these changes in cell structure and biology are important for surviving in different hosts such as vector insects and mammals.
To adapt to different challenges cells need to turn on specific groups of genes in a particular setting and turn them off when not needed. The DNA thread is organised around specialised protein spools known as nucleosomes, which block gene activity. Specific chemical changes ('flags') on nucleosomes can profoundly affect the activity of genes allowing them to be turned on or off. The regulation of genes by such chemical flags in generally known as epigenetic regulation.

Aims:
We want to understand how such epigenetic regulation works in an evolutionary distinct organism. Much is known about epigenetic regulation in mammals and yeasts but these do not represent the full diversity of eukaryotic life. We have very little idea how these epigenetic processes work in divergent organisms, such as the trypanosome, Trypanosoma brucei, a parasite that causes disease in humans and cattle in sub-Saharan Africa and is related to other human parasites that cause Chagas disease and Leishmaniasis. In evolutionary terms, human and yeast cells are much more closely related to each other than they are to trypanosomes. Current evidence suggests that while the spooling of DNA around nucleosomes does also play an important role in trypanosomes, the components that make up its regulatory systems are distinct from those that have been studied in yeasts and humans. Key proteins involved are the 'writers', 'readers', and 'erasers' of information carried as chemical flags on nucleosomes. To characterise these differences in how these proteins act we have selected several interesting proteins for which we will determine their protein partners, the chemical marks that they add to nucleosomes and the protein modules that bind these chemical marks. This will be achieved by making large amounts of the individual component molecules, including nucleosomes and testing what flags they add to nucleosomes and how they alter and interact with each other.

Impact:
The protein writers, readers and erasers of mammalian nucleosome marks are targets for many drugs developed against diseases like cancer, where normal gene expression programs are disrupted. Once we have obtained sufficient data for components of the trypanosome regulatory machinery, it is likely that some of these will be sufficiently different from any human or mammalian component to allow them to be eventually be targeted for the development of anti-parasite drugs.

Molecular understanding of the role of distinct chromatin types in gene regulation, & the activities involved, are most advanced in mainstream eukaryotes (ie animals, yeasts) that represent only one eukaryotic super-group. However, distinct early-branching lineages have highly divergent histones & chromatin modifiers - histone post-translational modification writers, readers & erasers. An excellent example are trypanosomes, human parasites which use distinct machinery to interpret their genome. Currently, our understanding of trypanosome chromatin modifications & modifiers is rudimentary - few have been characterised.
Our preliminary systematic analysis of putative trypanosome chromatin modifiers showed that several are enriched at RNAPII promoters including a putative histone methyltransferase Set27 and Chromo-domain partner protein. Surprisingly, none of the many chromatin modifiers we examined coat silent gene regions or repetitive elements that were expected to assemble distinct types of chromatin. This included the silent telomeric variant surface glycoprotein (VSG) genes that allow trypanosomes to evade human host immunity. However, the expression of synthetic TALE proteins designed to bind telomeric repeats identified two zinc-finger RNA binding proteins enriched in telomeric chromatin.
This proposal addresses three questions concerning epigenetic regulation in trypanosomes:
1. How do putative histone writers and readers congregate at RNAPII promoters?
2. What is the role of zinc-finger RNA binding proteins in gene silencing at telomeres?
3. What is the composition and function of chromatin formed over silent VSG genes & repetitive elements?
These studies will provide understanding of the mechanisms by which specific chromatin modifiers influence gene expression & heterochromatin formation in Trypanosoma brucei & related human pathogens. Key divergent chromatin modifiers have the potential to be exploited as novel parasite specific therapeutic targets.

Our research will:
- reveal conserved and adaptive features of epigenetic control in a divergent human eukaryotic pathogen, providing an important evolutionary comparator for conventional eukaryotic models.
- characterise chromatin modulation complexes including novel components of previously unknown function.
- adapt and optimize tools to dissect epigenetic mechanisms in trypanosomes.
- illuminate the roles of chromatin-associated complexes in forming 'heterochromatin' to silence expression from specialized regions of the trypanosome genome.
- identify pathogen-specific chromatin modulators that may have longer-term potential for therapeutic development.

Our specific aims employ a battery of assays to dissect fundamental molecular mechanisms of chromatin-based epigenetic regulation in trypanosomes. Improved technologies and experimental tools will enhance our ability to determine how the chromatin-associated machinery functions and these tools, resources, datasets and procedures will be made available for use by a broad community of researchers. Our multipronged approach and application of knowledge of epigenetic mechanisms, gained in other systems, to trypanosomes could significantly advance our broad understanding of such processes in these divergent eukaryotes. For example, understanding mechanisms that control gene expression in African trypanosomes may uncover fundamental aspects of immune evasion which have direct bearing on parasite virulence and spread, as well as being relevant for a broad range of related pathogens. These include Trypanosoma cruzi and Leishmania parasites, both of which affect millions of people worldwide and particularly those highlighted as the most vulnerable or resident in the least developed countries, as defined by the DAC list of the OECD. For Trypanosoma cruzi, this burden affects 6-7 million in 21 endemic countries in latin America and, for Leishmania, there are up to 1 million cases annually, with 26,000-65,000 deaths. African trypanosome infections in humans are currently few, but diagnosis is poor and up to 65 million remain at risk. Further, livestock infections restrict economic development by 1 billion USD annually and sustain the potential for zoonotic transmission to humans.

Our highly mechanistic studies of trypanosome chromatin modifiers are likely to pinpoint differences between mammals and kinetoplastids that could be exploited, either as targets against parasites or to enhance host defence. As evolutionarily divergent eukaryotes, a large proportion of trypanosome proteins have no detectable similarity to other molecules and no known function. Identifying these novel components of the chromatin machinery therefore provides significant enhancement to our understanding of trypanosome biology and also the general biology of chromatin regulation across all nucleated organisms. In the longer term, such findings could be transformative from a treatment perspective. Inhibitors of bromodomain proteins and histone deacetylases are already marketed for cancer and immunosuppression, showing that chromatin modifiers make excellent drug targets. While commercialisation is not a major immediate objective, our proposal has long-term potential to uncover novel molecular mechanisms central to human and animal diseases and potential zoonoses. The application of comparative genomics through collaboration may identify novel pathways in kinetoplastid parasites, absent from their mammalian hosts. Opportunities to exploit such results with bespoke screens will be pursued through discussion with the Edinburgh technology transfer office (Edinburgh Innovation) and the University of Edinburgh Drug Discovery facility, as well as with collaborators at the University of Dundee Drug Discovery Unit.