Showing papers by "Karine G. Le Roch published in 2019"

Comparative 3D genome organization in apicomplexan parasites.

Abstract: The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum, the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum, the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei, and absent in Plasmodium yoelii In Plasmodium knowlesi, telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi, the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.

Kinesin-8B controls basal body function and flagellum formation and is key to malaria transmission.

Abstract: Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

Plasmodium kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission.

Abstract: Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Δkinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host.

Predicting gene expression in the human malaria parasite Plasmodium falciparum using histone modification, nucleosome positioning, and 3D localization features.

Abstract: Empirical evidence suggests that the malaria parasite Plasmodium falciparum employs a broad range of mechanisms to regulate gene transcription throughout the organism's complex life cycle. To better understand this regulatory machinery, we assembled a rich collection of genomic and epigenomic data sets, including information about transcription factor (TF) binding motifs, patterns of covalent histone modifications, nucleosome occupancy, GC content, and global 3D genome architecture. We used these data to train machine learning models to discriminate between high-expression and low-expression genes, focusing on three distinct stages of the red blood cell phase of the Plasmodium life cycle. Our results highlight the importance of histone modifications and 3D chromatin architecture in Plasmodium transcriptional regulation and suggest that AP2 transcription factors may play a limited regulatory role, perhaps operating in conjunction with epigenetic factors.

The role of epigenetics and chromatin structure in transcriptional regulation in malaria parasites.

Abstract: Due to the unique selective pressures and extreme changes faced by the human malaria parasite Plasmodium falciparum throughout its life cycle, the parasite has evolved distinct features to alter its gene expression patterns. Along with classical gene regulation by transcription factors (TFs), of which only one family, the AP2 TFs, has been described in the parasite genome, a large body of evidence points toward chromatin structure and epigenetic factors mediating the changes in gene expression associated with parasite life cycle stages. These attributes may be critically important for immune evasion, host cell invasion and development of the parasite in its two hosts, the human and the Anopheles vector. Thus, the factors involved in the maintenance and regulation of chromatin and epigenetic features represent potential targets for antimalarial drugs. In this review, we discuss the mechanisms in P. falciparum that regulate chromatin structure, nucleosome landscape, the 3-dimensional structure of the genome and additional distinctive features created by parasite-specific genes and gene families. We review conserved traits of chromatin in eukaryotes in order to highlight what is unique in the parasite.

The Arabidopsis RRM domain protein EDM3 mediates race-specific disease resistance by controlling H3K9me2-dependent alternative polyadenylation of RPP7 immune receptor transcripts.

Abstract: The NLR-receptor RPP7 mediates race-specific immunity in Arabidopsis. Previous screens for enhanced downy mildew (edm) mutants identified the co-chaperone SGT1b (EDM1) and the PHD-finger protein EDM2 as critical regulators of RPP7. Here, we describe a third edm mutant compromised in RPP7 immunity, edm3. EDM3 encodes a nuclear-localized protein featuring an RNA-recognition motif. Like EDM2, EDM3 promotes histone H3 lysine 9 dimethylation (H3K9me2) at RPP7. Global profiling of H3K9me2 showed EDM3 to affect this silencing mark at a large set of loci. Importantly, both EDM3 and EDM2 co-associate in vivo with H3K9me2-marked chromatin and transcripts at a critical proximal polyadenylation site of RPP7, where they suppress proximal transcript polyadeylation/termination. Our results highlight the complexity of plant NLR gene regulation, and establish a functional and physical link between a histone mark and NLR-transcript processing.

Concise Synthesis of the Antiplasmodial Isocyanoterpene 7,20‑Diisocyanoadociane

Abstract: The flagship member of the antiplasmodial isocyanoterpenes, 7,20-diisocyanoadociane (DICA), was synthesized from dehydrocryptone in 10 steps, and in 13 steps from commercially available material. Our previous formal synthesis was reengineered, leveraging only productive transformations to deliver DICA in fewer than half the number of steps of our original effort. Important contributions, in addition to the particularly concise strategy, include a solution to the problem of axial nucleophilic methylation of a late-stage cyclohexanone, and the first selective synthesis and antiplasmodial evaluation of the DICA stereoisomer with both isonitriles equatorial.

Kinesin-8B controls basal body function and flagellum formation and is key to malaria parasite transmission

Abstract: Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not colocalise with kinetochores in the nucleus but instead revealed dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We thus uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Abstract: Kinesin-8 proteins are microtubule motors that are often involved in the regulation of mitotic spindle length and chromosome alignment. They move towards the ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics are not well understood in the different proliferative stages. Genome-wide homology analysis of Plasmodium spp. revealed the presence of two Kinesin-8 motor proteins (Kinesin-8X and Kinesin-8B). Here we have studied the biochemical properties of Kinesin-8X and its role in parasite proliferation. In vitro, Kinesin-8X showed motile and depolymerization activities like other Kinesin-8 motors. To understand its role in cell division, we have used protein tagging and live cell imaging to define the location of Plasmodium Kinesin-8X during all proliferative stages of the parasite life cycle. Furthermore, we have used gene targeting to analyse the function of Kinesin-8X. The results reveal a spatiotemporal involvement of Kinesin-8X in spindle dynamics and its association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the Kinesin-8X gene showed that this protein is required for endomitotic division during oocyst development and is, therefore, necessary for parasite replication within the mosquito gut, and for transmission to the vertebrate host. Consistently, transcriptome analysis of Δkinesin-8X parasites reveals modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression confirming its role in cell division.

Three-Dimensional Genome Organization and Virulence in Apicomplexan Parasites.

Abstract: Mounting evidence supports the idea that epigenetic, and the overall 3-dimensional (3D) architecture of the genome, plays an important role in gene expression for eukaryotic organisms. We recently used Hi-C methodologies to generate and compare the 3D genome of 7 different apicomplexan parasites, including several pathogenic and less pathogenic malaria parasites as well as related human parasites Babesia microti and Toxoplasma gondii. Our goal was to understand the possible relationship between genome organization, gene expression, and pathogenicity of these infectious agents. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes that are unique in their effect on chromosome folding, indicating a link between genome organization and gene expression in more virulent pathogens.

Genomics and precision medicine for malaria: A dream come true?

Abstract: The fight against malaria has led to various strategies to understand the biological mechanisms of the parasite, and its interactions with both the human and mosquito hosts. The malaria parasite has evolved diverse tactics to evade host detection. Recent advances in genomics and genome editing such as development of the CRISPR-Cas9 system in malaria parasites as well as next generation sequencing technologies, proteomics and metabolomics have allowed us to better understand the parasite biology and survey genes involved in drug resistance the parasites have acquired over the years. Most importantly these new technologies are allowing us to identify new drug targets and design improved vaccine strategies. In mosquito vector control, revolutionary approaches using synthetic biology are being designed to develop gene drive systems that can reduce vector population. This new concept could have a wide impact on most vector-borne diseases. These combined innovative approaches to malaria research are intended to reduce infection rates with the ultimate goal to eliminate one of the most prevalent and deadly infectious Plasmodium species in the world.

Plasmodium condensin core subunits (SMC2/SMC4) mediate atypical mitosis and are essential for parasite proliferation and transmission

Abstract: Condensin is a multi-subunit protein complex that regulates chromosome organization, segregation and condensation during cell division in eukaryotes. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin during endomitosis in schizogony and endoreduplication in male gametogenesis. SMC2 and SMC4 localize at discrete foci during schizogony, and with a diffuse nuclear distribution during male gametogenesis. ChIP-seq analyses suggest a centromeric location of SMC2/SMC4 only during schizogony. Co-immunoprecipitation data reveal the presence of both condensin complex I and II during male gametogenesis, but only the SMC2/SMC4 heterodimer during schizogony. Finally, knockdown of smc2 and smc4 gene expression revealed their essential roles in parasite proliferation and transmission. This study shows that condensin core subunits (SMC2/SMC4) have differential complex and distinct functions at different stages of the parasite life cycle.

Measuring significant changes in chromatin conformation with ACCOST

Abstract: Chromatin conformation assays such as Hi-C cannot directly measure differences in 3D architecture between cell types or cell states. For this purpose, two or more Hi-C experiments must be carried out, but direct comparison of the resulting Hi-C matrices is confounded by several features of Hi-C data. Most notably, the genomic distance effect, whereby contacts between pairs of genomic loci that are proximal along the chromosome exhibit many more Hi-C contacts that distal pairs of loci, dominates every Hi-C matrix. Furthermore, the form that this distance effect takes often varies between different Hi-C experiments, even between replicate experiments. Thus, a statistical confidence measure designed to identify differential Hi-C contacts must accurately account for the genomic distance effect or risk being misled by large-scale but artifactual differences. ACCOST (Altered Chromatin Conformation STatistics) accomplishes this goal by extending the statistical model employed by DEseq, re-purposing the “size factors,” which were originally developed to account for differences in read depth between samples, to instead model the genomic distance effect. We show via analysis of simulated and real data that ACCOST provides unbiased statistical confidence estimates that compare favorably with competing methods such as diffHiC, FIND, and HiCcompare. ACCOST is freely available with an Apache license at https://bitbucket.org/noblelab/accost.