Experiments, drug-treated cells were pelleted down and washed with RPMI at least twice before culturing in fresh media without drug for specified time intervals and subsequently harvested for protein extraction, RNA extraction or chromatin immunoprecipitation. All experiments were done in either duplicates or triplicates.Plasmid construction and transfectionHistone H4 gene was mutated such that the targeted lysine (K) residues (K5, K8, K12, K16) were replaced with arginine (R). These amino acid exchanges were previously shown to preserve the positive charge but prevent acetylation [59, 60]. For this, plasmid pBCamR-3HA [35] was modified to create HA-tagged version of wild-type or mutated histone H4 gene in all constructs. Mutations in H4 gene were introduced by amplifying complete H4 gene of P. falciparum using forward primer carrying the respective change in LIMKI 3 chemical information nucleotide sequence (see Additional file 1). Unmodified H4 or H4 carrying mutations were cloned upstream of 3HA at Bam H1/Nhe 1 sites of pBcamR-3HA to make a fusion with the 3HA tag epitope and transfected into P. falciparum using the blasticidindriven regulatable transgene expression systems [61]. Plasmodium falciparum strain 3D7 was transfected as described [62] to carry episomal copies of the plasmids in presence of selection marker blasticidin. Once the transfectants were stably maintained, the concentration of blasticidin was increased from 2.5 to 10 /ml in a dominant-negative selection system, which allowed the dominant expression of HA-tagged H4 protein.Protein extraction and immunodetectionincubated at 100 for 10 min and centrifuged at maximum speed for 10 min to recover the supernatant. Nuclear fractionation was done as described [35]. Briefly, saponin-lysed parasite pellets were incubated in cell lysis buffer CLB (20 mM HEPES (pH7.9), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.65 NP40, 1 mM DTT, protease inhibitors (Complete TM, Roche Diagnostics)) for 5 min PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29045898 on ice. Nuclei were pelleted at 5000 rpm, washed twice with CLB and digested with 300U MNase (Fermentas) in digestion buffer DB (20 mM TrisHCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 5 mM MnCl2, 300 mM sucrose, 0.4 NP40, 1 mM DTT, protease inhibitors) for 20 min at 37 . Soluble low salt nuclear fractions were recovered by centrifugation for 10 min at 13,000 rpm. Remaining nuclear debris was washed twice in DB and resuspended in high salt buffer HSB (20 mM HEPES (pH 7.9), 800 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitors) by vortexing for 20 min at 4 . High salt nuclear fraction was recovered; after centrifugation for 5 min at 13,000 rpm, the high salt nuclear fraction was saved. The insoluble pellet was solubilized in SDS extraction buffer [2 SDS, 10 mM TrisHCl (pH 7.5)] by vortexing for 20 min at room temperature. Acid extraction of histones was modified from the original protocol [63]. Briefly, the insoluble nuclear pellet containing DNA and histones was treated overnight with 0.25 M HCl at 4 . The acid-extractable protein was precipitated with trichloroacetic acid (TCA), washed in icecold acetone, and resuspended in Laemmli sample buffer. For immunoblotting, identical amount of total protein lysates separated by 12 SDS-PAGE were transferred onto nitrocellulose membrane. Western blot analyses were carried out using primary antibodies probed against the core histone modifications (or HA from Sigma) obtained from Millipore, Upstate and horseradish peroxidase-conjugated.