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Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite

ABSTRACT: The var multigene family encodes clonally variant surface antigens that are key to immune evasion and pathogenesis in the human malaria parasite, Plasmodium falciparum. Epigenetics and nuclear organization regulate the var gene family in a system of mutually exclusive expression; however, few factors have been shown to play a direct role in these processes. Thus, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for identification of novel factors associated with var genes in their natural chromatin context. A tagged, catalytically inactive Cas9 (“dCas9”) was targeted to the promoters or introns of a subset of var genes and subjected to immunoprecipitation followed by label-free LC-MS/MS. A non-targeted dCas9 served as a control.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Plasmodium Falciparum (isolate 3d7)

TISSUE(S): Erythrocyte

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Valentin SABATET

LAB HEAD: Damarys Loew

PROVIDER: PXD013131 | Pride | 2020-07-15

REPOSITORIES: Pride

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Discovery of RUF6 ncRNA interacting proteins involved in P. falciparum immune evasion

Project description:RUF6 is a ncRNA gene family that is transcribed by RNA Polymerase III but actively regulates the Pol II-transcribed var virulence gene family. Understanding how RUF6 ncRNA connect to downstream effectors is lacking. We developed an RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA protein interactions. The RUF6 ncRNA interactome was purified with biotinylated antisense oligonucleotides. Quantitative label-free mass spectrometry identified several unique proteins linked to gene transcription including. Affinity purification of Pf-DDX5 identified proteins originally found by our RUF6-ChIRP protocol, validating the technique’s robustness for identifying ncRNA interactomes in P. falciparum. Our work identifies a RUF6 ncRNA protein complex that interacts with RNA Pol II to sustain var gene expression.

2022-11-15 | PXD036801 | Pride

Exploring the var gene interactome with CRISPR/dCas9

Project description:Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.

2020-05-01 | GSE129397 | GEO

Mutually Exclusive Transcription of Subtelomeric Gene Families in Plasmodium falciparum is Restricted to var Genes

Project description:The P. falciparum genome is equipped with several subtelomeric gene families that are implicated in parasite virulence and immune evasion. The members of these gene families are uniformly positioned within heterochromatic domains of the genome and are thus subject to variegated expression. The best-studied example is that of the var gene family encoding the major parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). Transcriptional regulation of other subtelomeric gene families and their role in parasite biology is much less understood. Here, we investigated the mode of transcriptional control of var, rif, stevor, phist and pfmc-2tm families by comparative genome-wide transcriptional profiling of transgenic parasite lines. Our results establish a clear functional distinction between var and non-var transcriptional control mechanisms. Unlike var promoters, we find that promoters of non-var families are not silenced by default. Moreover, we show that mutually exclusive transcription is unique to the var gene family. 3D7 wild-type parasites were transfected with constructs carrying eight different promoters that drive expression of the drug-selectable marker hdhfr-gfp. Thereof seven promoters are members of the multigene families upsA var, upsB var, upsC var, rif, stevor, phistb and pfmc-2tm. The cam promoter was used as transfection-based control and also a wild-type 3D7 cell line was included as control. These nine cell lines were subjected to genome-wide transcriptional profiling. Parasites were synchronized to obtain an 8 hour growth window and were harvested at four consecutive timepoints (TP): TP1 (6-14 hours post-invasion (hpi)); TP2 (14-22 hpi); TP3 (22-30 hpi); TP4 (30-38 hpi) to monitor intra- and inter-family specific linkage of multigene family expression.

2011-09-01 | E-GEOD-31829 | biostudies-arrayexpress

Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe [ChIP-Seq data]

Project description:Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway leading to the expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive (pho7+) and negative (csk1+) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response for the ScPHO5 (pho1+), ScPHO84 (spbc8e4.01c+), and ScGIT1 (spbc1271.09+) orthologs. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. We provide a global analysis of the PHO pathway in S. pombe. Our results elucidate the conserved core regulon required for responding to phosphate starvation between distantly related ascomycetes and a better understanding of flexibility in environmental stress response networks. ChIP Sequencing of the Schizosaccharomyces pombe 972h- transcription factor Pho7-TAP in high-Pi, no-Pi, and csk1M-NM-^T conditions

2013-01-02 | E-GEOD-39498 | biostudies-arrayexpress

Comparative genomic hybridization by array of Arabidopsis thaliana, Arabidopsis suecica, Arabidopsis arenosa and frankenfurter shows homeolog-specific retention in allotetraploid Arabidopsis suecica

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2011-01-01 | E-MEXP-2969 | biostudies-arrayexpress

Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

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