Project description:Chromatin immunoprecipitation of genomic loci in Trypanosoma brucei where Scc1 is deposited. This was achieved by deletion of one Scc1 allele and N-terminal tagging of the second Scc1 allele with a Ty1-tag. During the ChIP experiment, the DNA was crosslinked and cells were permeabilized with digitonin. DNA was fragmented by sonication with a Covaris instrument. DNA bound to Ty1-Scc1 was pulled down by using a BB2 anti-Ty1 antibody. Cross links are reversed and the DNA was purified and prepared for Illumina sequencing.
Project description:Chromatin immunoprecipitation of genomic loci in Trypanosoma brucei where histone variant H3.V is deposited. This was achieved by deletion of one H3.V allele and N-terminal tagging of the second H3.V allele with a Ty1 tag. During the ChIP experiment, the DNA was digested with MNase to obtain mononucleosomes. Nucleosomes containing H3.V were pulled down by using a BB2 anti-Ty1 antibody. Cross links are reversed and mononucleosomal DNA is purified and prepared for Illumina sequencing.
Project description:Chromatin immunoprecipitation of genomic loci in Trypanosoma brucei where histone variant H4.V is deposited. A previously generated cell line (Siegel et al., 2009) in which both endogenous H4.V alleles are knockout out and ectopic overexpression of a Ty1-tagged version of H4.V can be induced was used. During the ChIP experiment, the DNA was digested with MNase to obtain mononucleosomes. Nucleosomes containing H4.V were pulled down by using a BB2 anti-Ty1 antibody. Cross links are reversed and mononucleosomal DNA is purified and prepared for Illumina sequencing.
Project description:Trypanosomes diverged from the main eukaryotic lineage about 600 million years ago, and display some unusual genomic and epigenetic properties that provide valuable insight into the early processes employed by eukaryotic ancestors to regulate chromatin-mediated functions. We sequenced Trypanosoma brucei core histones by high mass accuracy middle-down mass spectrometry to map core histone post-translational modifications (PTMs) and elucidate cis‑histone combinatorial PTMs (cPTMs). T. brucei histones are heavily modified and display intricate cPTMs patterns, with numerous hypermodified cPTMs that could contribute to the formation of non‑repressive euchromatic states. The T. brucei H2A C‑terminal tail is hyperacetylated, containing up to 5 acetylated lysine residues. MNase-ChIP-seq revealed a striking enrichment of hyperacetylated H2A at Pol II transcription start regions, and showed that H2A histones that are hyperacetylated in different combinations localised to different genomic regions, suggesting distinct epigenetic functions. Our genomics and proteomics data provide insight into the complex epigenetic mechanisms used by this parasite to regulate a genome that lacks the transcriptional control mechanisms found in higher eukaryotes. The findings further demonstrate the complexity of epigenetic mechanisms that were probably shared with the last eukaryotic common ancestor.
Project description:Trypanosoma brucei gambiense is the causative agent of the fatal human disease African sleeping sickness. Using Digital Gene Expression we have compared the transcriptome of a group 1 T.b.gambiense (Eliane) and a T.b.brucei (STIB 247).
Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance
Project description:Eukaryotes have an array of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from Kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically characterised nucleosome core particles (NCPs) from the Kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened DNA binding overall. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.