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Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum

ABSTRACT: Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. In this project, histone peptide pull-downs and protein-protein interaction quantitative proteomics were used to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. One of the identified bromodomain proteins (BDP5, PF3D7_1234100) has previously been proposed to be the P. falciparum homologue of TBP-associated factor 1 (TAF1). As the bdp5/taf1 locus was refractory to disruption, knock-sideways strategy was employed to disrupt BDP5/TAF1 function by tethering it to the cell membrane (Birnbaum, 2017, Nature Methods). To gain insight in the transcriptional defects upon BDP5/TAF1 knock-sideways (KS), directional RNA seq libraries were generated at 2, 6 and 12 hours after knock-sideways induction.

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE138499 | GEO | 2019/10/15

REPOSITORIES: GEO

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Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells. Using homologous recombination, we generated ES cells and mice harbouring a null allele in the Tbp gene by insertion of a hygromycin resistance cassette in exon III and a floxed allele, in which exon III is surrounded by LoxP sites. From these mice, we generated an embryonic fibroblast Tbplox/- cell line in which TBP expression can be inactivated by expression of the Cre recombinase leading to cell death. We adopted a two-step strategy to generate Tbp-/- cell lines expressing human (h)TBP. Cells were first infected with pBABE retrovirus vectors expressing Wt hTBP or a series of mutants in the TBP core region, all of which carry a Flag-HA tag on their N-terminus. Cells expressing endogenous mTBP and exogenous hTBP were then infected with a second retrovirus expressing the 4 hydroxy-tamoxifen (OHT) inducible Cre-ERT2. Subsequently, multiple clonal populations from the OHT treated cells were isolated and genotyped by PCR to identify the Tbp-/-clones. At least two independent clones where expression of the endogenous mTBP was lost and replaced by the exogenous hTBP were isolated.

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Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum

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