Antigenic variation is a common strategy utilized by intracellular pathogens to evade immune detection. Plasmodium falciparum, the causal agent of the most lethal form of malaria, modulates antigenic variation of the ~60 member 'var' virulence gene family by exhibiting mutually exclusive, monoallelic expression. This is achieved by spatially and epigenetically regulating var transcription such that silent var loci cluster at the nuclear periphery and are associated with nucleosomes modified by silencing epigenetic marks such as hypoacetylation and trimethylation of histone 3 lysine 9. However, the exact mechanism of clustering and placement of histone marks at var genes is poorly understood. The recent identification of the novel DNA- and RNA-binding protein family PfAlba in P. falciparum may provide new insights into these mechanisms. The PfAlbas bind to DNA and RNA in vitro, and localize to either the nuclear periphery or the parasite cytoplasm in a stage-dependent manner during intra-erythrocytic development. We therefore postulate a role for these proteins in var regulation and more generally, in chromatin biology and RNA metabolism. To determine this, we propose to: (1) Analyze the genome-wide localization of the PfAlbas using CHIP-seq and qPCR assays, (2) Identify PfAlba RNA substrates using in vitro and in vivo techniques such as SNAAP and iCLIP, and (3) Examine the effect of PfAlba knockdown on var regulation and RNA metabolism by qPCR assays and RNA-seq, respectively. The research will take place in Dr. Artur Scherf's laboratory at Insitut Pasteur, Paris. The host has the technological know-how to perform the assays described in this proposal while the fellow has the biochemical and genetic skills required to successfully achieve the goals. Overall, this study will result in the transfer of knowledge from the US to Europe and simultaneously train the fellow to become an independent researcher.
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