Control of gene expression in trypanosomes: Defining the nuclear lamina

Parasitic protozoa are important agents of disease, and afflict a major proportion of the world's population. In evolutionary terms parasites are very different to their hosts, and this is frequently reflected in the presence of a great many unique or unusual mechanisms that underpin their biology. This aspect presents multiple opportunities for the understanding of pathogenesis and also the possibility of identifying therapeutic targets.
In trypanosomes, which as a group cause Sleeping sickness, Chagas' disease and Kala Azar (visceral leishmaniasis) and a great many other diseases, immune evasion is vital for survival and transmission. African trypanosomes are highly reliant on antigenic variation for their longterm survival - the process is so successful that trypanosomes can infect their host for many months and in some cases years. The primary mechanism of immune evasion is antigenic variation, the periodic switching of the parasite surface through sequential expression of immunologically distinct forms of the variant surface glycoprotein (VSG). Understanding VSG expression is central to unravelling the mechanisms of trypanosome virulence and is a longstanding goal in the field.
At the rim of the nucleus DNA is compacted into heterochromatin, which is transcriptionally silent and acts as a mechanism for the inactivation of genes placed there. To ensure that only one VSG is expressed at a time trypanosomes use heterochromatin as a store for all potentially expressed VSG genes except one. Recently we have uncovered a system of proteins that are localised to the rim of the trypanosome nucleus, and which regulate heterochromatin, nuclear structure and other functions. Most remarkably these proteins are distinct from those that make up the equivalent system in the host. This application proposes to interrogate in detail the manner in which the proteins comprising this system are organised, how they function and ultimately how they regulate VSG gene expression.

Trypanosomatids evade the host immune response by multiple mechanisms, but in all species the parasite surface is central to virulence. In Leishmania and the American trypanosome, mechanisms are focused on invasion and survival within host cells. For African trypanosomes, where the parasite resides in the host bloodstream, the major mechanism is antigenic variation. All of these parasites rely on specific expression of highly complex gene cohorts encoding surface molecules, and significantly many of these molecules are expressed from genes that are at sub-telemetric loci. Our recent identification of the protein NUP-1, that is an analog to the lamins from mammalian cells, and which has a role in maintaining heterochromatin, provides a major insight into mechanisms of gene regulation. Given that NUP-1 is present across the trypanosomatids, but is restricted to them, it is likely that common mechanisms pertain to this entire group of pathogens.

In African trypanosomes, antigenic variation facilitates long term infection, and is predicated upon the monoallelic expression of a single variant surface glycoprotein (VSG) gene, and which involves inactivation within heterochromatin of other VSG genes. In most eukaryotes heterochromatin is maintained by the lamina, a proteinaceous network subtending the nuclear envelope. In mammals the lamina is comprised of 60kDa lamin proteins, but these are absent from trypanosomes and replaced by NUP-1. Proteomics has identified additional components of the trypanosome lamina, including proteins we have designated as NUP-2 and NUP-3, together with connections to the nuclear pore complex.

We propose to gain insights into composition, location and interactions between NUP-1 and 2 and other nuclear structures including DNA by a range of state of the art techniques, and specifically super-resolution microscopy, proteomics and next generation sequencing, applied to both transcriptional analysis and chromatin IP.

As a basic biology application, clearly most of the impact here is to individuals in directly related fields, and impact to the more general public is limited and rather non-specific by nature. However, clearly the advancement of understanding of immune evasion and potential host interactions via control of gene expression is of significant interests to a broad audience.
The RA will certainly benefit hugely. The application is highly cross discipline in nature, and will expose the RA to a great range of methods and collaborative opportunities. Specifically, analysis using imaging, knockdown, ChIP and biochemical approaches will provide opportunities to learn specific and advanced analytical methodology. Imaging and ultrastructural work will facilitate exposure to these methods. As the project will encompass expertise from several laboratories, this will also provide great networking potential. The work will be presented at several international meetings, encompassing nuclear transport, gene regulation and parasitology, again providing opportunity for networking, advancement and considerable broadening of expertise.
Most directly, research communities working with trypanosomes and their close relatives will see a major empowerment, and which includes the direct experimental community, parasitologists/trypanosomatid cell biologists working at the fundamental biology level, plus those working on direct impact of these organisms on health and agriculture. These individuals and their research goals will be aided in formulating improved hypotheses specifically targeting essential systems for study or therapeutic potential.
Next are researchers with interests in wider aspects of biology. Specifically here we consider the general protist and evolutionary biology communities. The analysis of divergent mechanisms for gene expression and nuclear organisation in trypanosomes is clearly of general impact, and the detailed mechanistic and novel methodological aspects of the work we seek to perform will broaden this further. As the UK has a particularly large parasitology and trypanosome research community this is therefore of specific benefit to the domestic scientific community.
Due to the broad geographical range of trypanosomes, beneficiaries will also include those with interests in ecology, environmental monitoring and human impact plus potentially climate change and agricultural management.
Further, individuals seeking to develop therapeutics will benefit from this work in the medium term - it is hoped that we will continue to refine our understanding of how the trypanosome organises chromatin and to fully assess opportunities for the design, and possible execution of small molecule screens arising from the interactions that will be identified.
Finally, in public understanding and education: Trypanosomes and tropical diseases are organisms most school children and the general public encounter at some level, either more formally or in the media. Advances in understanding the biology of these organisms may increase the appeal and enthusiasm for basic and applied bioscience to the next generation and general public.