P. falciparum-specific Genomic Context Protein-Binding Microarray
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ABSTRACT:
PROVIDER: PRJNA947215 | ENA |
REPOSITORIES: ENA
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Project description:Development of the human malaria parasite, Plasmodium falciparum is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites to regulate target genes is still largely unknown. To address TF target specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC and further characterized PfAP2-G and PfAP2-EXP which bind unique DNA motifs (GTAC and TGCATGCA). We interrogated the impact of DNA sequence context and the chromatin landscape on P. falciparum TF binding using high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We determined that DNA sequence context does not greatly impact binding site selection for CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization contribute to differential binding. In contrast, GTGCAC-binding TFs prefer different sequence contexts, DNA shape profiles, and chromatin dynamics. Finally, we find that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions in vivo due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms.
2023-12-31 | GSE227873 | GEO
Project description:Global transcription of the malaria parasite, Plasmodium falciparum, is finely regulated, yet the genome encodes for a limited number of sequence-specific transcription factors (TFs) to coordinate this pattern. A subset of these TFs bind overlapping DNA motifs (i.e., CACACA and GTGCAC); however, mechanisms of binding site selection and redundancy have not yet been investigated in P. falciparum. Therefore, we integrated a variety of approaches from new and published work such as high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post translational modification mapping, and genome-wide chromatin accessibility mapping to comprehensively interrogate the impact of DNA sequence context and the chromatin landscape on TF binding in P. falciparum. We show that sequence context of the CACACA-binding TFs does not greatly impact binding site selection, while chromatin accessibility, epigenetic patterns, and co-factor recruitment differentiates in vivo binding. In contrast, the GTGCAC-binding TFs do prefer significantly different sequence contexts and DNA shape profiles both in vitro and in vivo, in concert with chromatin dynamics. Our results demonstrate that TFs select binding sites based on unique combinations of sequence context, DNA topology, epigenetic patterns, and chromatin accessibility, thereby contributing to the complex gene regulatory mechanisms governing malaria pathogenesis.
2024-07-31 | GSE212052 | GEO
Project description:Passive transfer studies in humans clearly demonstrated the protective role of IgG antibodies against malaria. Identifying the precise parasite antigens that mediate immunity is essential for vaccine design, but has proved difficult. Completion of the Plasmodium falciparum genome revealed thousands of potential vaccine candidates, but a significant bottleneck remains in their validation and prioritization for further evaluation in clinical trials. Focusing initially on the Plasmodium falciparum merozoite proteome, we used peer-reviewed publications, multiple proteomic and bioinformatic approaches, to select and prioritize potential immune targets. We expressed 109 P. falciparum recombinant proteins, the majority of which were obtained using a mammalian expression system that has been shown to produce biologically functional extracellular proteins, and used them to create KILchip v1.0: a novel protein microarray to facilitate high throughput multiplexed antibody detection from individual samples. The microarray assay was highly specific; antibodies against P. falciparum proteins were detected exclusively in sera from malaria-exposed but not malaria-naïve individuals. The intensity of antibody reactivity varied as expected from strong to weak across well-studied antigens such as AMA1 and RH5 (Kruskal-Wallis H test for trend: p-value <0.0001). The inter-assay and intra-assay variability was minimal, with reproducible results obtained in re-assays using the same chip over duration of 3 months. Antibodies quantified using the multiplexed format in KILchip v1.0 were highly correlated with those measured in the gold-standard monoplex ELISA (median (range) Spearman’s R of 0.84 (0.65-0.95)). KILchip v1.0 is a robust, scalable and adaptable protein microarray that has broad applicability to studies of naturally acquired immunity against malaria by providing a standardized tool for the detection of antibody correlates of protection. It will facilitate rapid high-throughput validation and prioritization of potential Plasmodium falciparum merozoite-stage antigens paving the way for urgently needed clinical trials for the next-generation of malaria vaccines.
2021-02-12 | PXD011724 | Pride
Project description:To more specifically define the sequence content that favors direct binding of GAF, a genomic-context protein binding microarray (gcPBM) was used to detect GST-tagged GAF binding to double stranded DNA probes using a fluorescently conjugated anti-GST secondary antibody
2018-02-15 | GSE110653 | GEO
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Project description:In vitro, genomic context identification of transcription factor binding sites
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Project description:The human malaria parasite Plasmodium falciparum employs intricate post-transcriptional regulatory mechanisms in different stages of its life cycle. Despite the importance of post-transcriptional regulation, key elements of these processes, namely RNA binding proteins (RBPs), are poorly characterized. In this study, the RNA binding properties of P. falciparum proteins were characterized including two putative members of the Bruno/CELF family of RBPs (PfCELF1 and PfCELF2), dihydrofolate reductase-thymidylate synthase (PfDHFR-TS), and adenosine deaminase (PfAda).The mRNA targets of these P. falciparum proteins were investigated by ribonomics using DNA microarrays.
2014-10-23 | GSE56329 | GEO
Project description:ChIP-seq experiments were performed for the putative Telomere Repeat-binding Zinc finger protein (PfTRZ) in the malaria parasite Plasmodium falciparum strain 3D7. The gene encoding this factor (PF3D7_1209300) was endogenously tagged with either a GFP- or a 3xHA-tag and these transgenic parasite lines were used in ChIP-sequencing experiments. Sequencing of the ChIP and input libraries showed enrichment of PfTRZ at all telomere-repeat containing chromosome ends (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) as well as in all upsB var promoters.In addition,PfTRZ was enriched at seven additional, intra-chromosomal sites and called in the PfTRZ-HA ChIP-seq only.
2016-07-15 | GSE76537 | GEO
Project description:The human malaria parasite Plasmodium falciparum employs intricate post-transcriptional regulatory mechanisms in different stages of its life cycle. Despite the importance of post-transcriptional regulation, key elements of these processes, namely RNA binding proteins (RBPs), are poorly characterized. In this study, the RNA binding properties of P. falciparum proteins were characterized including two putative members of the Bruno/CELF family of RBPs (PfCELF1 and PfCELF2), dihydrofolate reductase-thymidylate synthase (PfDHFR-TS), and adenosine deaminase (PfAda).The mRNA targets of these P. falciparum proteins were investigated by ribonomics using DNA microarrays. Two-condition ribonomic experiment, RBP vs. Mock enrichment of mRNAs. Ribonomic experimental replicates: 4-7 for each RBP, 5 for Mock. One replicate per array.
2014-10-23 | E-GEOD-56329 | biostudies-arrayexpress
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