Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum

Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. In this project, histone peptide pull-downs and protein-protein interaction quantitative proteomics were used to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. One of the identified bromodomain proteins (BDP5, PF3D7_1234100) has previously been proposed to be the P. falciparum homologue of TBP-associated factor 1 (TAF1). As the bdp5/taf1 locus was refractory to disruption, knock-sideways strategy was employed to disrupt BDP5/TAF1 function by tethering it to the cell membrane (Birnbaum, 2017, Nature Methods). To gain insight in the transcriptional defects upon BDP5/TAF1 knock-sideways (KS), directional RNA seq libraries were generated at 2, 6 and 12 hours after knock-sideways induction. Overall design: Asexual blood stage bdp5/taf1-2xFKBP-GF-2xFKBP P. falciparum 3D7 parasites with the Lyn-FRB-T2A-mCherry mislocalizer plasmid were synchronized (~4hr) and split in two at 14 hours post red blood cell invasion. One half was kept in normal medium ('control' condition) while the second half was put on 250 nM rapalog to induce knock-sideways. RNA was collected at 2, 6 and 12 hours post knock-sideways induction and prepared to directional RNA-seq libraries. A culture of bdp5/taf1-2xFKBP-GF-2xFKBP parasites without the mislocalizer was treated in a similar fashion but collected 12 hours after rapalog addition to control for any effects of rapalog itself. RNA-seq libraries were sequenced paired-end for 42 bp on a NextSeq 500 system (Illumina).