Reporter-gene-assisted small molecule inhibitor screening of heterochromatin boundary maintenance in Plasmodium falciparum
Phenotypic variation between malaria parasites is one of the major contributors to the pathogens success and is enabled by differences in chromatin organisation. Epigenetic changes, especially in chromatin structure, are largely responsible for differential expression of genes involved in antigenic variation as well as transmission between the human host and mosquito vector. Genes contributing to phenotypic variability are often located close to the boundaries between densely-packed heterochromatin and transcriptionally active, loosely packed euchromatin. Heterochromatin occupancy at these loci is variable and the primary source of heterogeneity between parasite strains/clones. Despite the central importance of heterochromatin in parasite biology, mechanisms on how heterochromatin domains are organised are not yet understood.In order to identify small molecular inhibitors influencing maintenance of heterochromatin boundaries, parasites were genetically modified by GFP tagging of a euchromatic gene close to a heterochromatin domain and a control gene within euchromatin was marked with tdTomato to ensure proper parasites staging. In this Heterochromatin Spreading Sensor (HSS) line heterochromatin spreading is expected to suppress reporter gene expression and would lead to the loss of the GFP signal while tdTomato signal remain unaltered. Using this system we tested the effects of two different small molecule inhibitor libraries (in total ~300 epigenetic and kinase inhibitors) on reporter gene expression utilising a high-content imaging system, and identified eleven compounds with an effect on GFP expression levels. Nine of these compounds are histone demethylase or methyltransferase inhibitors and their effects on heterochromatin occupancy levels were assessed by Cleavage under Target & Tagmentation (CUT&Tag). Overall design: Parasites were grown in the presence of inhibitor compounds identified by microscopy. Parasites were harvested and HP1-CUT&Tag was performed.