Project description:Lobosphaera incisa is an oleaginous green microalga and a target organism of high biotechnological interest because it is the richest known plant source of arachidonic acid (ARA), a pharmaceutically and nutraceutically valuable omega-6 long-chain polyunsaturated fatty acid. ARA is deposited in considerable amounts in storage lipid triacylglycerols, most profoundly when L. incisa is exposed to nitrogen starvation. To get insights into genome-wide gene expression in L. incisa under nitrogen starvation, the whole transcriptome was sequenced by RNA-Seq. Total RNA was isolated under four growth conditions: exponential growth (control) and growth in nitrogen-depleted medium for 12 or 72 hours at normal or high light intensities. Statistical and differential gene expression analyses were conducted by comparing the various samples. The results of this study were used to identify patterns of gene regulation with special emphasis on genes involved in fatty acid and lipid metabolism.
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.
