Mutant/mutation
The mutant lacks expression of gSNF2 and expresses RFP and GFP in female and male gametocytes, respectively.

Protein (function)
Multiple AP2 family transcription factors (TFs) are involved in these steps. In the initial step of the gametocytogenesis, AP2-G plays essential roles. AP2-G is responsible for the onset of the gametocytogenesis as a master TF; upon disruption of this, TF parasites lose the ability to produce gametocytes, and by conditional overexpression of AP2-G gametocytes can be induced in asexual erythrocytic stage parasites. Our recent study on Plasmodium berghei demonstrated that this TF induces multiple TFs essential for gametocyte development and generates a driving force for gametocytogenesis. AP2- 2 is one of the target genes of AP2-G and supports transition of asexual stage parasites to early gametocytes as a repressor. In the second step of differentiation of early gametocytes into female gametocytes, at least two targets, AP2-FG and AP2-O3, play a critical role. AP2-FG plays essential roles in female maturation. It binds the ten-base motif TGYAYRTRCA, on the promoter of target genes. AP2-O3 plays a role in development as a transcriptional repressor, repressing several male-specific genes, and supports female differentiation. Differentiation of males, which starts at the same time as that of females, also contains two steps. The first step is common between the two sexes because disruption of AP2-G2 results in developmental arrest of both sexes.  Male-specific development begins in the second step and male-specific repertoire of gene expression is established during this period. 

In eukaryotes, gene regulation by a sequence-specific TF involves several steps. The series of events require a nucleosome-free region (NFR) which allows TFs, coregulators, and basic TFs to access genomic DNA. Thus, the regulatory region sometimes has to be altered to NFR beforehand by removing or repositioning nucleosomes. Mechanisms involving ATP-dependent chromatin remodeling complexes (remodelers) play important roles in this process; they slide or evict nucleosomes in ATP-dependent manner and make the regulatory DNA region accessible. Genome-wide change of chromatin structures by a remodeler can govern gene expression repertoire and contribute to establishment of a cell-type-specific gene expression program. ATPase subunit is a core subunit of the remodeler, and it constitutes the complex with multiple subunits associated with it. Chromatin remodelers are classified into four major families based on the domain structure of the ATPase subunit: SWI/SNF, SWI, INO80, and CHD families. These families are involved in various cellular processes dependent on chromatin remodeling, such as transcription and genome replication and repair. SWI/ SNF plays an important role in transcriptional regulation. In the budding yeast Saccharomyces cerevisiae, one of the member RSC (essential remodeling complex) is recruited to the promoter of the target gene, slides nucleosomes, and creates NFR limited by stably positioned +1 and −1 nucleosomes, initiating transcription. We analyzed the target genes of AP2-G and found a gene encoding an ATPase subunit of the SWI/SNF family (SNF2 ATPase) in the targets (Gene ID: PBANKA_0312700). Based on its specific expression in gametocytes, we designated this gene as gSNF2.  

From the paper:  'The gSNF2 gene is one of the targets of AP2-G, harboring a ChIP-seq peak of AP2-G approximately 0.8 kbp upstream. This gene is included in 30 genes that are up-regulated by the conditional expression of AP2-G in P. berghei in the earliest period (within 6 h after the induction of AP2-G), which is consistent with our ChIP-seq result showing that the gSNF2 gene is a direct target of AP2-G.
he protein is classified as a DEAD-box RNA helicase, because it possess DEXDc and HELICc domains. However, phylogenetic analyses using its helicase-related region found these to be more similar to ATPase subunit of remodelers, particularly to the ATPase subunit of the SWI/SNF family (SNF2 ATPase). Analysis with SMART and Pfam domain databases using amino acid regions conserved in Plasmodium orthologues, identified two putative domains, a HSA (helicase/SANT-associated) and SnAC (SNF2 ATPase-coupling). HSA domains are involved in binding to actin-related proteins. SnAC domains are specific to the ATPase subunit of the SWI/SNF2 family chromatin remodeler and participates in attaching to the nucleosome. On the other hand, bromodomains, which have the capacity to bind acetylated lysine residues of proteins and are located typically in the C-terminal portion of the SNF2 ATPase of these model organisms including S. cerevisiae, were not identified. These results strongly suggested that this gene is an ATPase subunit of the SWI/SNF family that possess a unique domain structure'.

Phenotype
Normal growth/multiplication of asexual blood stages and normal numbers of mature female and male gametocytes. Absence of male gamete formation (exflagellation). no oocyst formation. 

To examine whether the development of both female and male gametocytes was impaired, parasites were subjected to cross-fertilization experiments. Ookinetes were produced by fertilization between their females and normal males, but no ookinetes were generated by fertilization between their male and normal females, demonstrating that only male gametocytes were functionally impaired by the disruption.

FACS analysis of the mutant knock-out parasites showed that green fluorescence in male gametocytes was significantly weaker than that in the parent parasite line (approximately fivefold), suggesting that males were produced but their gene expression was affected by the disruption.

The phenotype described here was different from that reported by Kent et al. (RMgm-4523), which reported that parasites with this gene disruption lacked the capacity to produce male gametocytes.

Additional information
To study the expression profile of gSNF2, parasites were generated that express it as a protein fused with mNeonGreen (RMgm-5364; gSNF2::mNeon parasites). 
Clear GFP signals were observed in the nucleus of both male and female gametocytes but not in the asexual stages. Time-course study showed that expression of the gene starts at 20 hpi (hours post erythrocyte infection), a few hours before manifestation of sexual dimorphism in the early gametocytes, and continued until they developed into mature gametocytes. The expression started a few hours after the expression of AP2-G, consistent with being a target of AP2-G.

In the paper evidence is provide for the following:
- Expression of Male-Specific Genes Decreased in gSNF2-Disrupted Parasites 
- gSNF2 Is Recruited Upstream of Female- and Male-Specific Genes 
The results suggested that gSNF2 molecules are recruited to ten- and five-base motifs, which are present upstream of genes expressed in females and males, respectively.
- Five and Ten-Base Motif Sequences Are Essential for the Recruitment of gSNF2. 
The results demonstrated that the recruitment was dependent solely on the motifs in the promoter and that independent sequence-specific TFs, that is, AP2-FG in females and a sequence-specific TF binding to the five-base motif in male, determined genomic regions of gSNF2 recruitment.
- Five-Base Motif Acts as a Male-Specific Cis-Acting Element
- gSNF2 Broadly Targets Male-Enriched Genes
- gSNF2 Disruption Reduced Nucleosome-Free Region (NFR) Formation Upstream of Target Genes.
The results indicate that gSNF2 contributes to transcriptional activation of male genes through NFR formation.

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