Dynamic Epigenetic Regulation of Gene Expression during the Life Cycle of Malaria Parasite Plasmodium falciparum
Figure 5
Histone modification patterns on ectopically integrated promoters.
(A) Cloning strategy for ectopic integration of promoter regions. Four promoter regions (1.5–2 kb upstream of the ATG) corresponding to upstream regions of MAL13P1.122, PF14_0705, PFD0240c and PFC0210c were cloned upstream of the luciferase reporter gene pLN-luc (see Materials and Methods). P. falciparum strain Dd2attB was transfected with the above vectors to achieve integration at the cg6 locus and the transgenic cell lines were selected on blasticidin. Primer pair P2/P4 was used to confirm integration (data not shown). (B) H4K8ac occupancy at the four ectopically integrated promoter regions. The graphs represent real time PCR results carried out on H4K8ac-immunoprecipitated DNA from rings (R), trophozoites (T) and schizonts (S). In order to distinguish between the endogenous and luciferase tagged promoter, specific primers were designed to amplify regions spanning the 3′ end of the promoter and either the start of the endogenous gene or the start of the luciferase gene. The positions of forward (F) and reverse (R) primers are shown in panel A. Grey lines refer to the ChIP enrichment of the native cg6 locus in the untransfected parasites. Orange and green lines represent the ChIP enrichment of native promoters and integrated promoters, respectively, in the transfectants. The error bars give the standard deviation from triplicate experiments.