Genome-wide investigation of transcriptional regulation in malaria sporozoite [ChIP-Seq]

Sporozoite is the stage in which malaria parasites initially infect the vertebrate host. Elucidation of gene regulation in this stage will promote the investigation of mechanisms of liver infection by this parasite and contribute to development of strategies for preventing the malaria transmission. AP2-Sp is a transcription factor essential for formation of sporozoites or sporogony, which take place in oocysts on the midgut of infected mosquitoes. To understand the role of this transcription factor in the transcriptional regulatory system of this stage we performed ChIP-seq analysis using whole mosquito midguts containing late oocysts as start materials and explore its target genes genome-widely. Target genes were composed of 640 genes, which encompassed various functional categories and were contained genes involved in distinct processes parasites pass through in this stage, from sporogony to development into the liver stage. Furthermore, RNA-seq analysis showed that these genes constituted majority of the genes highly expressed in in this stage. These results suggested that this TF determines basal pattern of gene expression of this stage by targeting a broad range of genes directly. Overall design: Mosquito midgut was dissected and fixed immediately in 1% paraformaldehyde solution and kept in it until dissection of all mosquitoes were completed. ChIP-seq was performed in the same procedure as reported previously. Briefly, fixed midguts were sonicated with Bioruptor (Tosho Denki, Yokohama, Japan) in 0.25 ml of lysis buffer. After centrifugation at 4?, supernatant was subjected to ChIP with anti-GFP antibodies, and harvested DNA fragments were subjected to sequencing. Input DNAs were obtained from the chromatin without IP by the same procedure. Sequencing was performed with SOLID 5500 system (Life Technologies) in experiment 1 and illumine NextSeq system in experiment 2.