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Whole genome expression profiling across the asexual life cycle in PfBDP1 knockdown malaria parasites

ABSTRACT: To study the bromodomain protein PfBDP1 in the malaria parasite P. falciparum, a transgenic parasite line was generated (PfBDP1DD) in which PfBDP1 was tagged with a haemagglutinin tag and a ligand regulatable FKBP destabilization domain that allowed conditional knockdown of PfBDP1 by removal of the stabilizing ligand Shld1 from cultures. Shld1 was removed 30 hours post invasion (hpi) and the cells allowed to reinvade fresh red blood cells. The effects of PfBDP1 knockdown on gene expression were assessed by transcriptional profiling on microarrays over 6 consecutive time points (8 hour intervals) in the intraerythrocytic developmental cycle (IDC).

ORGANISM(S): Plasmodium falciparum Plasmodium falciparum 3D7

PROVIDER: GSE64688 | GEO | 2015/01/07

SECONDARY ACCESSION(S): PRJNA271650

REPOSITORIES: GEO

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Whole genome expression profiling across the asexual life cycle in PfBDP1 knockdown malaria parasites

Project description:To study the bromodomain protein PfBDP1 in the malaria parasite P. falciparum, a transgenic parasite line was generated (PfBDP1DD) in which PfBDP1 was tagged with a haemagglutinin tag and a ligand regulatable FKBP destabilization domain that allowed conditional knockdown of PfBDP1 by removal of the stabilizing ligand Shld1 from cultures. Shld1 was removed 30 hours post invasion (hpi) and the cells allowed to reinvade fresh red blood cells. The effects of PfBDP1 knockdown on gene expression were assessed by transcriptional profiling on microarrays over 6 consecutive time points (8 hour intervals) in the intraerythrocytic developmental cycle (IDC). Two condition matched time course experiment, PfBDP1DD ON versus OFF Shld1. Transgenic P. falciparum 3D7 parasites (PfBDP1DD) expressing an endogeneous PfBDP1-HA-DD fusion protein were grown in the presence of 2.5 nM WR99210/500 nM Shld1 (PfBDP1DD_ON) or 2.5 nM WR99210 (PfBDP1DD_OFF). After invasion, RNA was extracted at 6 consectutive time points in 8 hour intervals during the IDC, starting at 4 hpi, and was processed for microarray analysis. Three matched biological replicates were performed for both conditions, independently grown and harvested. Each array represents one RNA sample.

2015-01-07 | E-GEOD-64688 | biostudies-arrayexpress

Genome-wide localisation of the bromodomain protein PfBDP1 in the malaria parasite P. falciparum

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2016-03-22 | E-GEOD-79135 | biostudies-arrayexpress

Genome-wide localisation of the bromodomain protein PfBDP1 in the malaria parasite P. falciparum

2016-03-22 | GSE79135 | GEO

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Whole genome expression profiling across the life cycle of PfBDP1 overexpressing P. falciparum malaria parasites

Project description:Erythrocyte invasion is an essential step in the life cycle of the malaria parasite Plasmodium falciparum that involves specific interactions between host cell receptors and parasite ligands. How the parasite regulates the expression of invasion-related genes is to date largely unknown. Here we show that a novel, parasite-specific bromodomain protein (PfBDP1) binds to chromatin at the transcriptional start site of invasion-related genes and directly controls their expression. Conditional PfBDP1 knockdown causes a dramatic defect in parasite invasion and growth and results in transcriptional down-regulation of multiple invasion-related genes at a time point critical for invasion. This is the first report of a histone binding protein that activates genes in P. falciparum and our data place PfBDP1 in a central position for controlling the coordinated expression of invasion genes. Bromodomains are emerging therapeutic targets and drugs that specifically inhibit PfBDP1 could be an invaluable tool in the effort to eradicate malaria. P. falciparum 3D7 parasites over-expressing PfBDP1-3xHA (3D7/PfBDP1 OE) [PMID: 23181666] were grown in presence of 5Î¼g/ml BSD-S-HCl. The parasite line 3D7/cam [PMID: 22435676.] grown under the same conditions and expressing hDHFR instead of PfBDP1-3xHA from the same promoter was used as control. RNA extracted from these samples at four consecutive time points each was processed for microarray analysis.

2015-01-07 | E-GEOD-64690 | biostudies-arrayexpress

Whole genome expression profiling across the life cycle of PfBDP1 overexpressing P. falciparum malaria parasites

2015-01-07 | GSE64690 | GEO

ChIPseq of the P. falciparum bromodomain proteins PfBDP7 and PfBDP1 in early and mature schizonts

Project description:Epigenetic regulation is recognized as a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite P. falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the bromodomain proteins PfBDP1 and PfBDP2 (BDP1/BDP2 core complex) was shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7 (P. falciparum bromodomain protein 7), because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, Stevor, and PfMC2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and identified PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

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Plasmodium falciparum Kelch13, Eps15 and Clathrin HC interactomes obtained using a dimerisation-induced quantitative BioID (DiQ-BioID)

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2021-12-02 | PXD008219 | Pride

Ligand-induced disorder-to-order transitions characterized by structural proteomics and molecular dynamics simulations

Project description:We have studied both the Spy and the FKBP protein-ligand systems by structural proteomics and molecular -dynamics simulations. We have used hydrogen/deuterium exchange, differential crosslinking, and surface modification to characterize the conformational changes that occur upon both peptide binding (Im7 with Spy) and small molecule binding (rapamycin with FKBP) to the protein. We have shown that, in both cases, the proteins are considerably disordered but their structures are different from the unfolded structure obtained with 8M urea in solution, and both proteins undergo a dramatic increase in secondary structure content upon ligand binding.

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