Functional genomics has helped to identify global regulators of sexual differentiation in the malaria parasite.
Sexual differentiation of malaria parasites (Plasmodium spp.) occurs in the mammalian host, when the parasite exits the vegetative asexual cycle and morphs into male and female gametocytes, which mate in the gut of the mosquito vector. At a molecular level, little is known about the regulation of this sexual differentiation; however, in 2014, a transcription factor, AP2-G, was identified as a master regulator of sexual commitment in Plasmodium berghei1 and Plasmodium falciparum2. Now, Yuda et al.3 describe a second transcriptional regulator of sexual differentiation, AP2-G2, which they further characterize in P. berghei using functional genomics.
These regulators belong to a family of 27 genes homologous to plant APETALA2/ethylene response factor (AP2/ERF) DNA-binding proteins and encode the first bona fide transcription factors to be identified in malaria parasites. As ap2-g2 deletion had previously been shown to result in the near-complete loss of mature gametocytes1, Yuda et al. used P. berghei ap2-g2 deletion mutants to investigate whether this AP2 family member has a role in the transcriptional regulation of gametocytogenesis3. They found that AP2-G2 is indispensable for transmission to the mosquito vector. Furthermore, the mutants were competent for sexual commitment but the maturation of gametocytes was halted, suggesting that AP2-G2 regulates progression of gametocytogenesis after sexual determination.
To explore the molecular effects of the loss of AP2-G2 in P. berghei, the authors used two functional genomics approaches. First, changes in the transcriptome were assessed through genome-wide microarray analysis. Deletion of ap2-g2 resulted in the downregulation of 354 genes, including all of the genes known to be expressed by gametocytes. Second, to identify AP2-G2 genomic binding sites, the authors generated a transgenic parasite that expressed GFP-tagged AP2-G2, which they profiled with chromatin immunoprecipitation followed by sequencing (ChIP−seq) using the ABI SOLiD 5500 sequencing platform. Mapping the ChIP−seq reads from two independent experiments onto the genome of the parasite revealed 3,584 putative AP2-G2 binding sites common to both datasets and a 5 bp AP2-G2 binding motif, which was confirmed by an electrophoretic mobility shift assay.
The combination of ChIP−seq and microarray analyses identified 1,505 genes that are potentially regulated by AP2-G2. Strikingly, genes with gametocyte-specific expression were not present in this dataset. Instead, predicted target genes included those expressed during the asexual proliferation stage in erythrocytes, during the liver stage, and during mosquito stages of the P. berghei life cycle. These results led the authors to postulate that AP2-G2 is a repressive factor that, during gametocytogenesis, silences genes that are dispensable for gametocyte formation. Indeed, the presence of a putative AP2-G2 motif within a 1.2 kb region upstream of the start codon in more than 30% of P. berghei genes suggests that AP2-G2 is responsible for genome-scale repression. As the repression of these genes seems necessary for the reprogramming of asexual parasites into mature gametocytes, their mistimed expression may be responsible for halted gametocyte maturation in ap2-g2 deletion mutants.
Yuda et al. noted that global transcriptional repression also occurs in the early stages of germline formation in metazoans, in which asexual primordial germ cells are generated prior to sexual commitment. This strategy may enable germline cells to maintain their potential to differentiate into any cell type or, in Plasmodium spp., into different life cycle stages. Transcriptional repression therefore seems to be an essential step in sexual cell fate determination in organisms as divergent as malaria parasites and higher eukaryotes, although whether this has arisen from ancient conserved mechanisms or convergent evolution is unknown.
The study of both AP2-G and AP2-G2 has begun to clarify the pathways that govern sexual commitment and maturation in Plasmodium spp., by identifying target genes and by establishing that the expression of AP2-G precedes the expression of AP2-G2. However, the nature of the genetic and environmental interactions of these factors remains unknown. Indeed, the interplay of these factors with one another and with additional regulatory mechanisms (including epigenetics), as well as the identification of downstream sex-specific regulatory networks, provide some of the most interesting questions in malaria biology.
Sinha, A. et al. A cascade of DNA binding proteins for sexual commitment and development in Plasmodium. Nature 507, 253–257 (2014).
Kafsack, B. F. C. et al. A transcriptional switch underlies commitment to sexual development in human malaria parasites. Nature 507, 248–252 (2014).
Yuda, M. et al. Global transcriptional repression: an initial and essential step for Plasmodium sexual development. Proc. Natl Acad. Sci. USA 112, 12824–12829 (2015).
The authors declare no competing financial interests.
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Rita Gomes, A., Talman, A. Commitment issues in Plasmodium. Nat Rev Microbiol 14, 4 (2016). https://doi.org/10.1038/nrmicro.2015.9