RIP-Seq analysis of Trypanosoma brucei ELAV-like protein Tb927.8.6650
Ontology highlight
ABSTRACT:
ORGANISM(S): Trypanosoma brucei
SUBMITTER: Hamed Najafabadi
PROVIDER: E-GEOD-46160 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists as the proliferative slender-form or the non-proliferative, transmissible, stumpy-form. The transition from the slender to stumpy-form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. Here, we use a monomorphic reporter cell line in a whole-cell fluorescence-based assay to screen over 6000 small molecules from a kinase-focussed compound library for their ability to induce stumpy-like formation in a high-throughput screening programme. This identified one compound able to induce modest, yet specific, changes in gene expression indicative of a partial differentiation to stumpy forms. This not only provides a potential tool for the further understanding of stumpy formation, but also demonstrates the use of high throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes. Examination of gene expression in response to treatment with DDD00015314.
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
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Project description:Three potential ELAV-like proteins of T. brucei, including Tb927.3.2930, Tb927.7.5380, and Tb927.8.6650, were either inhibited by RNAi or phenotypically activated by over-expression, followed by microarray analysis of the transcriptome. The results indicated that these ELAV-like proteins regulate the abundance of a large number of T. brucei transcripts, potentially through regulation of mRNA stability. Each of the three ELAV-like proteins were either inhibited by RNAi or over-expressed, in stable transgenic procyclic form cell lines. Total RNA was extracted 48h after tetracycline induction of the constructs (except for total RNA from Tb927.8.6650 RNAi which was extracted 24h after tet-induction), and sent to NimbleGen for cDNA synthesis and hybridization. Non-induced cells were analyzed in parallel.
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Project description:The parasite Trypanosoma brucei periodically changes the expression of protective variant surface glycoproteins (VSGs) to evade its host’s immune system in a process known as antigenic variation. One route to change VSG expression is the transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B-interactors. One of these was the Ribonuclease H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage and ES switching events. Surprisingly, a similar pattern of VSG deregulation was observed in Ribonuclease H2 mutants. We propose that both proteins act together in resolving R-loops to ensure genome integrity and contribute to the tightly-regulated process of antigenic variation.
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Project description:The parasite Trypanosoma brucei periodically changes the expression of its protective variant surface glycoproteins (VSGs) to evade the host’s immune system in a process known as antigenic variation. One route to change VSG expression is by transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B interactors. One of these was the trimeric Ribonuclease H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage and recombination-based ES switching events. Surprisingly, the observed VSG deregulation was similar to Ribonuclease H2 mutants. We here propose that the epigenetic regulator DOT1B forms a complex with RNase H2 that acts in resolving R-loops to ensure genome integrity and contribute to the tightly regulated process of antigenic variation.
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