Precision-editing used to probe unusual gene activity controls in deadly parasites

Scientists in the School of Life Sciences have used CRISPR-Cas9 based precision editing to demonstrate how parasitic trypanosomes use abundant DNA-packaging proteins, known as histones, to control gene expression. Trypanosomes, including parasites that cause a range of neglected tropical and veterinary diseases, are highly unusual among nucleated cells in that transcription of messenger RNA is minimally regulated;they transcribe almost ‘everything everywhere all at once’. This is because trypanosome genes are organised into long ‘polycistronic’ arrays that are transcribed from a common promoter, such that individual genes lack individual controls. Indeed, the African trypanosome uses only 150 promoters to express 8000 genes.

The promoters themselves lack obvious sequence features but are packaged in modified chromatin, comprising histones with specific chemical modifications, that may serve to control gene activity. Direct tests or demonstrations that histones control gene activity have been lacking, however, since precise histone manipulation has remained challenging, in nucleated cells generally. This is because most cells have many copies of each histone gene, >40 in trypanosomes.

The current paper is published in Nature Communications. David Horn, Professor of Parasite Molecular Biology in the School of Life Sciences, said “Advances in gene editing technology have finally allowed us to directly address this long-standing question in gene expression control in trypanosomes. It’s quite remarkable that cells exclusively expressing mutant histones display these very localised impacts that only affect genes immediately adjacent to promoters”. David also commented on the research carried out by Markéta Novotná during her PhD studies, saying “Many doubted whether we’d be able to completely replace native histones, and given the challenges, Markéta’s perseverance and technical skill were indeed essential to deliver such a successful outcome”.

Markéta Novotná, first author on the paper, said “We’ve been developing scalable precision gene editing and saturation mutagenesis approaches in the lab for some time, and these approaches will, I’m sure, now prove to be very versatile”.

David concluded “The current results indicate that histones coordinate the recruitment and action of RNA polymerase at specific sites in the genome. The platform Markéta developed now allows us to manipulate proteins encoded by other multi-copy genes in trypanosomes, of which there are many. The precision editing technologies we’re developing are also helping us to understand drug action and resistance in these deadly parasites”.

The work was supported by Wellcome, and was greatly facilitated by support from the Fingerprints Proteomics Facility at the University of Dundee. The paper can be found here.