ChIP-seq on PfAP2-G2 in trophozoite and gametocyte stages of parasites.

Differentiation from asexual blood stages to sexual gametocytes is required for transmission of malaria parasites from the human host to mosquitos, where sexual fertilization occurs to complete the lifecycle. Although preventing gametocyte development would block parasite transmission, the molecular mechanisms underlying sexual commitment and gametocyte maturation are still relatively unknown. Previous studies identified an ApiAP2 protein, AP2-G2, which plays a critical role in gametocyte maturation in rodent malaria parasite Plasmodium berghei by acting as the repressor of asexual stage genes in gametocytes. In this work we characterize the P. falciparum orthologue (PF3D7_1408200) of PbAP2-G2 and report that it plays a critical role in maturation of gametocytes. Disruption of pfap2-g2 did not obstruct commitment to sexual development but the majority of parasites were unable to develop normally beyond stage III gametocytes. In asexual stages PfAP2-G2 binds to the promoters of wide variety of genes expressed in diverse range of parasite's life cycle stages including gametocytes, mosquito lifecycle stages early ring stage genes and genes encoding for proteins involved in egress and invasion. Surprisingly, we also identify binding of PfAP2-G2 in the gene body of almost 3000 genes. We could also show that PfAP2-G2 interacts with chromatin remodeling proteins and another ApiAP2 protein (PF3D7_1139300) whose motif is also overrepresented in the ChIP-seq data. Overall this work suggests that PfAP2-G2 is a transcriptional regulator that regulates genes possibly by recruiting additional transcription factors and chromatin remodeling machinery and plays a critical role in the development of malaria parasites as they transition from the asexual stage to gametocytes. Overall design: Three replicates of ChIP-seq were performed during the trophozoite stages of asexual development using GFP taged PfAP2-G2. PfAP2-G2-GFP parasites were highly synchronized for that stage, followed by crosslinking and isolation of the chromatin. Chromatin bound by PfAP2-G2 GFP was isolated via anti-GFP and input material was used as a negative control for each replicate. We also performed two replicates of ChIP-seq using GFP-tagged PfAP2-G2 parasites on gametocyte stage III to define the target genes during sexual development using purified population of gametocytes.