Mutant/mutation
The mutant lacks expression of PFG and expresses RFP and GFP in female and male gametocytes, respectively.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2023.04.10.536204

Protein (function)
AP2-FG is a member of the Plasmodium AP2 family of transcription factors (TFs) . AP2-FG is a female-specific TF and plays an essential role in the development of female gametocytes. In P. berghei, AP2-FG is expressed from early gametocytes to mature females, and by disruption of AP2-FG, the development of the female gametocytes is impaired, showing immature morphologies and resulting in complete loss of capability to mediate parasite transmission to mosquitoes. This TF binds to a ten-base female-specific cis-acting element and regulates variety of genes include those for fertilization, meiosis, and the development of ookinetes. This broad repertoire of target genes and their essential role in female development suggest that this TF is a master regulator of female development. However, AP2-FG-disrupted parasites can produce ookinetes with decreased numbers and abnormal morphologies, suggesting that additional mechanisms for activating female-specific genes are still functional upon the disruption of AP2-FG.

From the paper: 'we screened for novel sequence-specific TFs among target genes that were functionally unannotated in PlasmoDB using highly conserved amino acid sequences among Plasmodium species as a criterion. We hypothesized that the amino acid sequences of DNA-binding domains of sequence-specific TFs would have been difficult to change during evolution because even a small change in the sequence-specificity of TFs could cause  catastrophic effects by global changes in gene expression. Through this screening, a gene encoding a 2709 amino acid protein with two regions highly conserved among Plasmodium was identified (PBANKA0902300, designated PFG. The two conserved regions comprised 108- and 139-amino acids, respectively, and each region showed 94% and 91% conservation between P. berghei and P. falciparum. Blastp search using these regions revealed that genes homologous to PFG exist broadly among apicomplexan parasites and also in alveolates closely related to Apicomplexa, such as Vitrella brassicaformis, whereas a large inter-region between these two regions present in all Plasmodium homologs was not observed in these proteins. Based on domain prediction using the SMART database, no functionally characterized domains were identified to be significantly similar to these two regions.

Phenotype
Normal growth/multiplication of asexual blood stages and normal numbers of mature female and male gametocytes. In ookinete cultures, only approximately 20% of the female gametocytes were converted into zygotes, and no banana-shaped or retort-form ookinetes were generated. No oocyst formation. 
Female gametocytes with mature morphology were observed on Giemsa-stained blood smears. However, no RFP-positive parasites were detected in the PFG(-) parasites. In contrast, GFP-positive parasites were observed. These results indicated that the promoter activity of the female-specific CCP2 gene used for RFP expression in parasites was severely reduced by the disruption of PFG.

Additional information
From the paper: 'To investigate the expression of PFG, we generated parasites expressing GFP-fused PFG (PFG::GFP parasites; RMgm-5362). Fluorescence analysis showed that this gene is expressed only in female gametocytes and is localized in the nucleus, suggesting its involvement in the transcriptional regulation of female gametocytes. According to sex-specific transcriptome data, the pfg gene is specifically transcribed in female gametocytes. We examined the target genes of the female-specific transcriptional activator AP2-FG and found that it had been predicted as a target of AP2-FG and harbored AP2-FG peaks with binding motifs in the upstream region. These results suggest that PFG was activated in two steps, that is, by AP2-G and then by AP2-FG, during female development. Temporal profiling of the expression of PFG using PFG::GFP parasites showed that the expression became visible from 20 hpi (hours post-erythrocyte infection). The timing of the expression was approximately four hours later than that of AP2-FG.

In the cross-fertilization experiment with PFG(-) parasites, ookinetes were formed when crossing with normal females (P48/45(-)) but not when crossing with normal males (P47(-)), confirming that the phenotype of lack of production of mature ookinetes  observed in PFG(-) was derived from female gametocytes

 In the paper evidence is presented for the following:

- Female-specific genes are globally downregulated in PFG(-) parasites
- PFG is co-localized with AP2-FG on ten-base motif
To examine whether PFG targeted these female-specific genes, ChIP-seq analysis was performed using PFG::GFP parasites and anti-GFP antibodies. In two biologically independent experiments, 1073 and 1,204 peaks were identified in the genome, respectively. These peaks were observed upstream of the genes significantly downregulated in PFG(-) parasites, suggesting that PFG is directly involved in the transcriptional activation of these genes. Statistical analysis of genomic sequences around the summits of peaks showed that binding of PFG to the genome was associated with ten-base motif  sequences, TGTRNNYACA, the female-specific cis-acting element identified in the ChIP-seq of AP2-FG. In comparison with the graphical views, the peaks identified in the ChIP-seq of PFG were co-localized with those of AP2-FG. An heat map positioned at the peak summit of PFG in the center showed that the position of the peaks was consistent with the ChIP-seq peaks of AP2-FG throughout the genome. These results strongly suggest that these two TFs form a complex on the ten-base motif and co-operatively activate their targets. On the other hand, graphic images also showed that some of the AP2-FG peaks lacked the corresponding ChIP-seq peaks of PFG.
- PFG is essential for AP2-FG binding to ten-base motif.
- Ten-base motif is essential for binding of PFG to the genome.
To demonstrate that the ten-base motif is essential for binding PFG to the genome, we performed a ChIP-qPCR assay using transgenic parasites in which the motif upstream of a target was mutated. A CPW-WPC family protein gene (PBANKA_1346300) harbors a 23 ChIP-seq peak for PFG and a ten-base motif under the summit of the peak. Three-point mutations were introduced into the motif using the CRISPR/Cas9 system, and GFP was fused to the PFG gene in these parasites. Introducing these mutations reduced the input value to background levels, demonstrating that the motif is essential for PFG binding to the genome.
- AP2-FG binds to the five-base motifs directly with its AP2 domain
- Five-base motif acts as a cis-activating element on the promoter

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