Kinetoplastid parasites are major pathogens responsible for important tropical diseases including Chagas disease, Leishmaniasis and African trypanosomiasis. Trypanosomes cause lethal disease in humans (sleeping sickness) and cattle (Nagana) across sub-Saharan Africa with devastating social and economic consequences.
The trypanosome nucleus contains a significant component of silent chromatin that cytologically appears dense and, as in most eukaryotes, is referred to as heterochromatin. One form of trypanosome heterochromatin mediates repression of the many unexpressed copies of Variable Surface Glycoprotein (VSG)
genes required to evade host immune systems. However, a molecular understanding of trypanosome heterochromatin and its contribution to developmental and VSG gene regulation, 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, their histone proteins and other components from the main eukaryotic paradigm.
A direct route to characterising the chromatin associated with specific chromosomal elements or regions is to design specific DNA binding proteins that allow their affinity selection. Enabled by Tadhg Devlin, together we have successfully developed a synthetic TALE-GFP fusion protein that binds terminal TTAGGG telomeric repeats and its affinity selection allowed the associated proteome to be defined by mass spectrometry. Initially designed as a proof-of-concept, Tadhg is now exploring the role of two unexpected proteins of interest that he has identified as enriched in telomeric chromatin.
The purpose of the proposed project is to capitalise on our ability to design and use synthetic TALE-GFP fusion proteins to target other chromosomal elements, affinity select associated chromatin and determine its composition using high resolution mass spectrometry.
CIR147 (147bp) repeat arrays of 20-100kb are found at trypanosome centromeres but essentially nothing is known about the associated chromatin. Likewise, we do not know if dispersed transposable elements or VSG genes themselves are coated with distinctive chromatin. Several TALE-GFP fusion proteins will be designed to bind different regions of the CIR147 repeat, the 79 bp region common to most retroelements and the 70bp repeat associated with VSG genes. ChIP-seq will be used to determine if the resulting TALE proteins are enriched over the sequences that they were designed to target. Affinity selection coupled with mass spectrometry will then be used to characterise the associated proteomes.
Because trypanosomes are so divergent from other eukaryotes (at least 500 million years) homologous proteins are frequently difficult to identify. Bespoke bioinformatics pipelines (collaboration with Field lab - UoDundee) will allow the student to gain expertise in comparative genomics, to analyse the structures, evolutionary distributions and origins of novel chromatin-associated proteins. This will also allow us to investigate the presence of such novel factors in related pathogenic protists as well as across the eukaryotic tree of life, providing perspective on the mechanisms behind adaptation in chromatin-regulatory mechanisms.
Lab Websites: https://www.wcb.ed.ac.uk/research/allshire https://www.allshirelab.com http://www.biology.ed.ac.uk/research/groups/kmatthews/index.htm
Institute Websites: https://www.wcb.ed.ac.uk https://www.ed.ac.uk/biology/cell-biology http://ciie.bio.ed.ac.uk/ http://www.ed.ac.uk/schools-departments/biology/immunology-infection
Developmental competence and antigen switch frequency can be uncoupled in Trypanosoma brucei. McWilliam K, Ivens A, Morrison L, Mugnier M, and Matthews KR, (2019). Under revision at Proceedings of the National Academy of Sciences, USA.
Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing.
Rojas F, Silvester E, Young J, Milne R, Tettey M, Houston DR, Walkinshaw MD, Pérez-Pi I, Auer M, Denton H, Smith TK, Thompson J, Matthews KR.
Cell. 2019 Jan 10;176(1-2):306-317.e16.
Interspecies conservation of organisation and function between nonhomologous regional centromeres.
Tong P, Pidoux AL, Toda NRT, Ard R, Berger H, Shukla M, Torres-Garcia J, Müller CA, Nieduszynski CA, Allshire RC.
Nat Commun. 2019 May 28;10(1):2343. doi: 10.1038/s41467-019-09824-4.