Abstract
Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR‐based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 (“dCas9”) was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl‐CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene‐associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin‐associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.
Highlights
In the malaria parasite Plasmodium falciparum, antigenic variation is key to evasion of the immune system and persistence of infection in the human host
We show specific deficient Cas9 (dCas9) enrichment at targeted var gene DNA regulatory elements with chromatin immunoprecipitation followed by sequencing (ChIP-seq). dCas9 immunoprecipitation followed by liquid chromatography-mass spectrometry (IP LC-MS/ MS) confirmed previously identified var gene interactors and revealed enrichment of several novel chromatin-associated factors possibly involved in transcriptional control and/or chromatin organization of var genes
To identify factors associated with putative var gene DNA regulatory elements in an unbiased manner, we adapted the CRISPR/dCas9based engineered DNA-binding molecule-mediated chromatin immunoprecipitation method developed in Fujita and Fujii (2013) to P. falciparum
Summary
In the malaria parasite Plasmodium falciparum, antigenic variation is key to evasion of the immune system and persistence of infection in the human host. Functional studies of orthologous histone writers, readers, and erasers have implicated several chromatin-associated proteins in mutually exclusive var gene transcription including heterochromatin protein 1 (HP1), the histone deacetylases HDA2 and silent information regulator 2 (SIR2a and b), and the histone methyltransferases SET2 and SET10 (Freitas-Junior et al, 2005; Flueck et al, 2009; Perez-Toledo et al, 2009; Tonkin et al, 2009; Volz et al, 2012; Jiang et al, 2013; Coleman et al, 2014; Ukaegbu et al, 2014). The histone modifications H3K9ac and H3K4me2/3 and the histone variants H2A.Z and H2B.Z were shown to be enriched at the active var gene promoter (Lopez-Rubio et al, 2007; Petter et al, 2013), but the molecular machinery involved in var gene activation, such as histone-modifying enzymes or nucleosome remodelers, has yet to be elucidated
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