Coordinated regulation of gene expression in Plasmodium female gametocytes by two transcription factors

Reviewer #1 (Public Review):

Gametocytes are erythrocytic sexual stages of the malaria-causing parasite Plasmodium, and are essential for parasite transmission and reproduction in the mosquito vector. In this study, Murata et al investigated the mechanisms of gene regulation in female gametocytes in the rodent malaria model parasite Plasmodium berghei. According to current views, gene regulation in Plasmodium parasites is dominated by the family of AP2 transcription factors (TFs), such as the AP2-G TF, which drives sexual commitment. The same authors previously identified one AP2 TF, called AP2-FG, as an essential TF mediating differentiation of female gametocytes. Here, they identified a novel protein, called PFG (for partner of AP2-FG), which cooperates with AP2-FG to regulate a subset of female gametocyte genes.

PFG was identified among AP2-G targets, but possesses no known DNA binding or other characterized domain. The authors show that PFG-knockout P. berghei parasites can form male and female gametocytes yet cannot transmit to mosquitoes, due to a defect in female gametocyte development. Using RNA-seq, they show that many female-specific genes are down-regulated in PFG(-)parasites. Chromatin immunoprecipitation combined with DNA sequencing (ChIP-seq) revealed that PFG colocalizes with AP2-FG on a ten-base motif that is enriched upstream of female-specific genes. Importantly, the ChIP-seq profile of PFG is unchanged in the absence of AP2-FG, suggesting that PFG binds to DNA independently of AP2-FG. Mutation of the ten-base motif in one target gene using CRISPR-Cas9 demonstrates that this motif is required for PFG localization at the gene locus. The data also show that binding of AP2-FG is affected in the absence of PFG, with disruption of AP2-FG interaction with the ten-base motif, but conservation of AP2-FG binding to distinct five-base motifs. Using a recombinant AP2 domain from AP2-FG, the authors demonstrate that the AP2 domain of AP2-FG binds to the five-base motifs. Using CRISPR they show that disruption of the five-base motifs in the genome abrogates AP2-FG binding, and using a reporter expression system they confirm that these motifs act as a cis-activating promoter element.

Through the analysis of target genes based on the presence of the ten- versus five-base motifs, the authors propose a model where AP2-FG can function in two forms, associated or not with PFG, and acting on the ten- or five-base motifs, respectively, to regulate distinct gene subsets during development of female gametocyte development.

The paper is very well written, with a clear narrative, and the work is very well performed, relying on robust molecular approaches. Generally the conclusions and the model proposed by the authors are well supported by the data. Nevertheless, the study as it stands raises a number of questions. First, it is unclear how the authors selected PFG as a candidate protein as the protein lacks any known DNA binding or regulatory domain. Detailing the reasoning that led to the identification of PFG would make the entire study more appealing. While the data convincingly show that PFG and AP2-FG cooperate to regulate the expression of a subset of female-specific genes, the paper does not show whether the two proteins actually interact with each other to form a complex. Finally, how PFG binds to DNA and whether the protein has transactivating activity remains elusive, as the protein apparently possesses no known DNA-binding or activating domain. These points could be discussed in more detail in the manuscript and/or be the subject of follow up studies.

In summary, this work reveals the essential role of a Plasmodium protein with no known DNA binding or regulatory domain, illustrating that unknown facets remain to be uncovered in this fascinating pathogen.

Reviewer #2 (Public Review):

Murata et al have characterized a new transcription activator termed PFG, which regulates gene expression in female gametocytes. The authors show solid evidence that PFG is a partner of the previously described transcription factor AP2-FG and describe three sets of genes: genes activated by PFG or AP2-FG alone and genes activated by the complex. The authors also show differential binding to the target DNA sequences by AP2-FG to either a 10bp, if in a complex with PFG or a 5bp motif if alone. In all, this is a useful study which further elucidates the underlying regulatory network that drives development of sexual stages and ultimately transmission to mosquitoes. The data presented are clear and solid and the conclusions drawn are mostly supported by the results shown. However, in the absence of evidence of physical interaction, it remains unclear if AP2-FG and PFG actually interact directly or as part of the same complex.

Reviewer #3 (Public Review):

This study is well designed and executed and provides new and important insights into the role of two TFs during the maturation of female gametocytes and fertilization in the mosquito midgut. However, it would benefit from a more thorough characterization of the phenotype to understand at which step of development these factors are required.

The gene at the center of this study (PBANKA_0902300) was identified in an earlier genetic screen by Russell et al. as being a female specific gene with essential role in transmission and named Fd2 (for female-defective 2). Since this name entered the literature first and is equally descriptive, the Fd2 name should be used instead of PFG to maintain clarity and avoid unnecessary confusion.

This study is well designed and executed and provides new and important insights into the role of two TFs during the maturation of female gametocytes and fertilization in the mosquito midgut. However, it would benefit from a more thorough characterization of the phenotype to understand at which step of development these factors are required.

The gene at the center of this study (PBANKA_0902300) was identified in an earlier genetic screen by Russell et al. as being a female specific gene with essential role in transmission and named Fd2 (for female-defective 2). Since this name entered the literature first and is equally descriptive, the Fd2 name should be used instead of PFG to maintain clarity and avoid unnecessary confusion.