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.
Project description:Antigenic variation in Plasmodium falciparum is achieved by transcriptional switching amongst polymorphic var genes, enforced by epigenetic modification of chromatin. Histone-modifying ‘sirtuin’ enzymes PfSir2a and PfSir2b have been implicated in this process. We examined the effect of genetic disruption of sirtuins on var gene expression. Comparative Genomic Hybridization profile indicate that loss of PfSir2a in 3D7 resulted in strikingly rearranged chromosomes.
Project description:The parasite Plasmodium falciparum is responsible for severe malaria, which remains a major cause of death, particularly in sub-Saharan Africa. The reference strain NF54 (or its subclone 3D7) is commonly used for controlled human malaria infection (CHMI), but recently strains with a different geographic and genomic background have become available for CHMI, including 7G8, which was subcloned from the Brazilian isolate IMTM22 in 1984 (Burkot TR et al. 1984. Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Trans R Soc Trop Med Hyg 78 (3):339-41. doi: 10.1016/0035-9203(84)90114-7). In contrast to NF54, in which var gene expression resets after mosquito transmission, 7G8 shows a partial reset with retention of the C-type var gene PF7G8_040025600 in the human host. The three subclones A1G9 (almost exclusive var2csa expression, control), A2E10, and A2G2 (both predominantly expressing var gene PF7G8_040025600) recently obtained by limited dilution from the Sanaria 7G8 parasite working cell bank (Lot: SAN03-021214 dated 20. February 2014) were selected for gDNA sequencing to test whether parasite subclones expressing PF7G8_040025600 differ in their genomic background. 150 mL of P. falciparum cell culture with >10% parasitemia was harvested and gDNA isolation was performed using the MasterPure™ Complete DNA Purification Kit (Lucigen). The gDNA samples were tested for degradation and RNA contamination on an agarose gel and quantified using the Qubit™ dsDNA BR Assay Kit (ThermoFischer). DNA-seq was performed at BGI Genomics (Shenzhen, China) on the DNBseq platform to generate 150 bp paired-end sequencing reads.
Project description:Analyses of new genomic, transcriptomic or proteomic data commonly result in trashing many unidentified data escaping the ‘canonical’ DNA-RNA-protein scheme. Testing systematic exchanges of nucleotides over long stretches produces inversed RNA pieces (here named “swinger” RNA) differing from their template DNA. These may explain some trashed data. Here analyses of genomic, transcriptomic and proteomic data of the pathogenic Tropheryma whipplei according to canonical genomic, transcriptomic and translational 'rules' resulted in trashing 58.9% of DNA, 37.7% RNA and about 85% of mass spectra (corresponding to peptides). In the trash, we found numerous DNA/RNA fragments compatible with “swinger” polymerization. Genomic sequences covered by «swinger» DNA and RNA are 3X more frequent than expected by chance and explained 12.4 and 20.8% of the rejected DNA and RNA sequences, respectively. As for peptides, several match with “swinger” RNAs, including some chimera, translated from both regular, and «swinger» transcripts, notably for ribosomal RNAs. Congruence of DNA, RNA and peptides resulting from the same swinging process suggest that systematic nucleotide exchanges increase coding potential, and may add to evolutionary diversification of bacterial populations.