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ABSTRACT: Epigenetic mechanisms are emerging as one of the major factors of the dynamics of gene expression in the human malaria parasite, . To elucidate the role of chromatin remodeling in transcriptional regulation associated with the progression of the intraerythrocytic development cycle (IDC), we mapped the temporal pattern of chromosomal association with histone H3 and H4 modifications using ChIP-on-chip. Here, we have generated a broad integrative epigenomic map of twelve histone modifications during the IDC including H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K9ac, H3K14ac, H3K56ac, H4K20me1, H4K20me3, H3K4me3, H3K79me3 and H4R3me2. While some modifications were found to be associated with the vast majority of the genome and their occupancy was constant, others showed more specific and highly dynamic distribution. Importantly, eight modifications displaying tight correlations with transcript levels showed differential affinity to distinct genomic regions with H4K8ac occupying predominantly promoter regions while others occurred at the 5' ends of coding sequences. The promoter occupancy of H4K8ac remained unchanged when ectopically inserted at a different locus, indicating the presence of specific DNA elements that recruit histone modifying enzymes regardless of their broad chromatin environment. In addition, we showed the presence of multivalent domains on the genome carrying more than one histone mark, highlighting the importance of combinatorial effects on transcription. Overall, our work portrays a substantial association between chromosomal locations of various epigenetic markers, transcriptional activity and global stage-specific transitions in the epigenome.
PLoS Pathogens 02/2013; 9(2):e1003170. · 9.13 Impact Factor
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ABSTRACT: DNA microarray is presently one of the most powerful and fastest growing technologies for genomic research of infectious diseases. Accordingly, DNA microarray-based global analyses of Plasmodium parasites provided many insights into the general biology of malaria infection. From the parasite perspective, it was shown that the complex Plasmodium life cycle is characterized by a high level of coordination in gene expression but at the same time parasites have a considerable capacity to alter their transcriptional profile as a response to external stimuli and/or adaptation to varying growth conditions in their host. In addition to transcriptional profiling, DNA microarrays were shown to be useful for quantitative analyses of Plasmodium genomic DNA including characterizations of sequence polymorphisms and copy number variants (CNV) as well as genomic loci associated with different chromatin factors (e.g., immunoprecipitated material (ChIP-on-chip)). Here, we present protocols for transcriptional profiling, comparative genomic hybridization (CGH), and ChIP-on-chip analyses that have been developed for the use of low-density long oligonucleotide DNA microarrays of Plasmodium species. Many of the presented procedures including RNA purification, DNA amplification, and chromatin immunoprecipitation are likely to be transferable to other genomic platforms such as other microarray technologies and new generation sequencing.
Methods in molecular biology (Clifton, N.J.) 01/2013; 923:189-211.
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Antoine Claessens,
Yvonne Adams,
Ashfaq Ghumra,
Gabriella Lindergard,
Caitlin C Buchan,
Cheryl Andisi,
Peter C Bull,
Sachel Mok, Archna P Gupta,
Christian W Wang,
Louise Turner,
Mònica Arman,
Ahmed Raza,
Zbynek Bozdech,
J Alexandra Rowe
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ABSTRACT: Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected and unselected parasites was analyzed using a variant surface antigen-supplemented microarray chip. After selection, the most highly and consistently up-regulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7, and ITvar19) that showed 11- to >100-fold increased transcription levels. These var genes encode P. falciparum erythrocyte membrane protein (PfEMP)1 variants with distinct N-terminal domain types (domain cassette 8 or domain cassette 13). Antibodies to HB3var3 and PFD0020c recognized the surface of live IEs and blocked binding to HBEC-5i, thereby confirming the adhesive function of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann-Whitney test, P = 0.029) but not by antibodies from controls with uncomplicated malaria (Mann-Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria.
Proceedings of the National Academy of Sciences 05/2012; 109(26):E1772-81. · 9.68 Impact Factor
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ABSTRACT: Malaria genetic variation has been extensively characterized, but the level of epigenetic plasticity remains largely unexplored. Here we provide a comprehensive characterization of transcriptional variation in the most lethal malaria parasite, Plasmodium falciparum, based on highly accurate transcriptional analysis of isogenic parasite lines grown under homogeneous conditions. This analysis revealed extensive transcriptional heterogeneity within genetically homogeneous clonal parasite populations. We show that clonally variant expression controlled at the epigenetic level is an intrinsic property of specific genes and gene families, the majority of which participate in host-parasite interactions. Intrinsic transcriptional variability is not restricted to genes involved in immune evasion, but also affects genes linked to lipid metabolism, protein folding, erythrocyte remodeling, or transcriptional regulation, among others, indicating that epigenetic variation results in both antigenic and functional variation. We observed a general association between heterochromatin marks and clonally variant expression, extending previous observations for specific genes to essentially all variantly expressed gene families. These results suggest that phenotypic variation of functionally unrelated P. falciparum gene families is mediated by a common mechanism based on reversible formation of H3K9me3-based heterochromatin. In changing environments, diversity confers fitness to a population. Our results support the idea that P. falciparum uses a bet-hedging strategy, as an alternative to directed transcriptional responses, to adapt to common fluctuations in its environment. Consistent with this idea, we found that transcriptionally different isogenic parasite lines markedly differed in their survival to heat-shock mimicking febrile episodes and adapted to periodic heat-shock with a pattern consistent with natural selection of pre-existing parasites.
Genome Research 03/2012; 22(5):925-38. · 13.61 Impact Factor
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ABSTRACT: The cytoadherence of Plasmodium falciparum is thought to be mediated by variant surface antigens (VSA), encoded by var, rif, stevor and pfmc-2tm genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different P. falciparum strains. However, many P. falciparum genomes have recently been sequenced, allowing the development of specific microarray probes for each VSA gene.
All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer software. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray.
From each parasite strain 50-56 var genes, 125-132 rif genes, 26-33 stevor genes and 3-8 pfmc-2tm genes were identified. Bioinformatic analysis of the new VSA sequences showed that 13 rif genes and five stevor genes were well-conserved across at least three strains (83-100% amino acid identity). The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using P. falciparum clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by ITvar9. Whole transcriptome analysis showed that the most highly up-regulated gene in rosetting parasites was ITvar9 (19 to 429-fold up-regulated over six time points). Only one rif gene (IT4rifA_042) was up-regulated by more than four fold (five fold at 12 hours post-invasion), and no stevor or pfmc-2tm genes were up-regulated by more than two fold. 377 non-VSA genes were differentially expressed by three fold or more in rosetting parasites, although none was as markedly or consistently up-regulated as ITvar9.
Probes for the VSA of newly sequenced P. falciparum strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of ITvar9 was confirmed, and the data did not support the involvement of other VSA families in rosette formation.
Malaria Journal 06/2011; 10:180. · 3.19 Impact Factor
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ABSTRACT: The apparent paucity of molecular factors of transcriptional control in the genomes of Plasmodium parasites raises many questions about the mechanisms of life cycle regulation in these malaria parasites. Epigenetic regulation has been suggested to play a major role in the stage specific gene expression during the Plasmodium life cycle. To address some of these questions, we analyzed global transcriptional responses of Plasmodium falciparum to a potent inhibitor of histone deacetylase activities (HDAC). The inhibitor apicidin induced profound transcriptional changes in multiple stages of the P. falciparum intraerythrocytic developmental cycle (IDC) that were characterized by rapid activation and repression of a large percentage of the genome. A major component of this response was induction of genes that are otherwise suppressed during that particular stage of the IDC or specific for the exo-erythrocytic stages. In the schizont stage, apicidin induced hyperacetylation of histone lysine residues H3K9, H4K8 and the tetra-acetyl H4 (H4Ac4) and demethylation of H3K4me3. Interestingly, we observed overlapping patterns of chromosomal distributions between H4K8Ac and H3K4me3 and between H3K9Ac and H4Ac4. There was a significant but partial association between the apicidin-induced gene expression and histone modifications, which included a number of stage specific transcription factors. Taken together, inhibition of HDAC activities leads to dramatic de-regulation of the IDC transcriptional cascade, which is a result of both disruption of histone modifications and up-regulation of stage specific transcription factors. These findings suggest an important role of histone modification and chromatin remodeling in transcriptional regulation of the Plasmodium life cycle. This also emphasizes the potential of P. falciparum HDACs as drug targets for malaria chemotherapy.
PLoS Pathogens 01/2010; 6(1):e1000737. · 9.13 Impact Factor
