Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes [RNA-seq]

Male and female gametocytes are sexual precursor cells essential for transmission of malaria parasite in the mosquitoes. Differentiation of gametocytes to fertile gametes (gametogenesis) relies on the gender-specific transcriptome. However, how the parasites establish distinct repertoire of gene transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 (AP2) family transcription factor (TF) AP2-O3 operates as a transcription repressor regulating female gametocyte transcriptome. AP2-O3 is specifically localized in the nucleus of the female gametocytes. AP2-O3-deficient parasites produce apparently normal female gametocytes, which fail to differentiate to fully fertile female gametes, leading to developmental arrest in fertilization and early development post-fertilization. AP2-O3 disruption causes massive up-regulation of transcriptionally dormant male genes and simultaneously down-regulation of highly transcribed female genes in female gametocytes. ChIP-seq and EMSA analysis establish AP2-O3 as a transcription repressor that targets a significant proportion of the upregulated male genes by recognizing an eight-base DNA motif in the promoters. In addition, the maternal AP2-O3 is removed after fertilization, which is required for the zygote to ookinete development. These results demonstrate that global transcriptional repression of male genes in the female gametocytes is required for safeguarding female-specific transcriptome and essential for the mosquito transmission of Plasmodium. Overall design: We performed RNA-Seq to gain insights into the genome-wide gene expression changes due to the loss of AP2-O3 in female gametocytes. To obtain female gametocytes of high purity, we used a double fluorescence reporters strain P. yoelii DFsc7, in which GFP and mCherry are specifically expressed in the male and female gametocytes respectively. We deleted the endogenous ap2-o3 in the DFsc7 background and generated the mutant DFsc7;?ap2-o3.Using fluorescence-activated cell sorting (FACS), the mCherry+ female gametocytes from both DFsc7 and DFsc7;?ap2-o3 strains were collected with more than 99% purity for RNA-seq.