Functional characterization of the putative Plasmodium falciparum orthologue of the YEATS domain protein Yaf9

Background and aims: Malaria is a serious infectious disease that affects many people worldwide. Plasmodium falciparum, a protozoan parasite, causes the most severe form of malaria. Pregnant women and children in Southeast Asia and Central Africa are particularly vulnerable to this disease, which can be fatal. The pathogen's virulence is due, in part, to its ability to change the expression of surface antigens, like for example PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1). These antigens are encoded by the 60-member var gene family, of which only one gene is active at a time. The monoallelic expression of var genes is regulated by epigenetic mechanisms that control the accessibility of the var gene promoter for the transcriptional machinery. The exchange of canonical H2A/H2B dimers for the variants H2A.Z and H2B.Z is correlated with the activation of var genes. In other eukaryotic organisms, this process is known to be mediated by ATP-dependent chromatin remodeling complexes of the SWR1 (SWI2/SNF2-Related 1) family, but the mechanisms in P. falciparum have not been described. Previous research identified an orthologous complex to the yeast SWR1 protein complex in P. falciparum. The ATPase subunit PfSwr1 interacts with the YEATS (YAF9, ENL, AF9, TAF14, SAS5) domain protein PF3D7_0807000, which is structurally similar to GAS41 (Glioma amplified sequence 41) in humans and Yaf9 in yeast. It is hypothesized that the function of PfYaf9 is to recruit chromatin remodeling complexes to modified histone side chains to exchange H2A/H2B dimers with H2A.Z/H2B.Z dimers. Hence, the aim of this thesis was to functionally characterize the P. falciparum protein PfYaf9. Methods: To test if PfYaf9 has a similar function as its structural orthologues in binding histones, a Far Western Blot with recombinant GST-PfYaf9 was carried out. Western Blot and immunofluorescence analysis determined the expression of PfYaf9 across the life cycle and its subcellular localization, while knock down experiments combined with RNAseq analysis were used to get insights about PfYaf9’s essentiality and its role in gene regulation. Chromatin immunoprecipitation was conducted to map the genome wide binding sites of PfYaf9. Results: This study identified PfYaf9 as a nuclear protein and revealed that PfYaf9 predominantly binds to Plasmodium histone H3. PfYaf9 expression levels vary throughout the asexual life cycle in human erythrocytes and peak in the late stages. Although conditional knock down of PfYaf9 had only minor effects on parasite replication, many of the parasite's genes were deregulated, in particular surface antigens and invasion genes. Consistent with a regulatory function in gene expression, genome wide mapping of PfYaf9 binding sites indicated that the protein is enriched at invasion gene promoters. Moreover, PfYaf9 was found to be associated with the centromeres, suggesting it may have a function in chromosome segregation. Conclusion: With this research the YEATS domain protein PfYaf9 was identified as a new key player in Plasmodium gene regulation. With its involvement in the regulation of surface antigen expression and invasion genes, two essential processes in the Plasmodium life cycle, it can be considered as a possible new target for future malaria therapy. Further research will focus on elucidating its essentiality for parasite survival and its role in the stress response.