Histone variant H3.3 demarcates GC-rich coding and subtelomeric sequences and serves as a potential memory mark for virulence gene expression in P. falciparum

ChIP-seq experiments were performed to profile PfH3.3 (PF3D7_0617900) in the malaria parasite Plasmodium falciparum. Sequencing of ChIP samples showed enrichment of PfH3.3 at GC-rich coding sequences and subtelomeric repetitive regions throughout the intraerythrocytic life cycle and additionally in intergenic regions during trophozoite stages. Also the promoter and the coding sequence of the active and poised var2CSA gene were marked (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) Overall design: DCJ P. falciparum parasites (Dzikowski et al., 2006) were transfected with a pHH1-plasmid (Reed et al., 2000) encoding a C-terminally Ty1-tagged version of PfH3.3 that contained a CTG instead of the start codon ATG and resulted in endogenous expression of PfH3.3-Ty1. These endogenously PfH3.3-Ty1 expressing DCJ P. falciparum parasites were selected for var2CSA expression by affinity purification as in (Noviyanti et al., 2001). Afterwards, parasite cultures were used to isolate nuclei from formaldehyde cross-linked early ring (10hpi) stages, late ring stages (20hpi), trophozoite stages (30hpi) or schizont stages (40hpi). Subsequently chromatin was prepared and used for chromatin immunoprecipitations using monoclonal anti-Ty1 antibody (BB2) (Brookman et al., 1995) or anti-histone H3 (recognizes PfH3 and PfH3.3, Abcam Ab1791). Total RNA was isolated from the same starting material (10hpi, 20hpi, 30hpi or 40hpi), PolyA-selected and fragmented. Subsequent cDNA synthesis was modified to maintain directional information for strand-specific RNA-Seq as described in (Kensche et al., 2015). ChIP-seq and directional RNA-seq libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification. Obtained data were normalized to the amount of mapped reads per million (RPM). For log2 ratio tracks PfH3.3 values were divided through the respective PfH3core values and log2 was calculated.