In Trypanosoma brucei, RNA interference (RNAi) and recombinant protein expression are established as powerful approaches for functional genomics, particularly when combined with inducible expression. The favoured methods involve exploiting homologous recombination to target expression cassettes to a chromosome sub-set to establish stable cell-lines. Unfortunately, bloodstream-form cells, those that cause disease in mammals, exhibit low efficiency stable transfection. Current expression systems can also exhibit other undesirable features, including variable position effects and leaky, inducible expression. We have developed systems in bloodstream-form cells that alleviate these problems. Using constructs for RNAi and expression of (GFP) tagged proteins, we target a (hyg) tagged ribosomal RNA (RRNA) locus which circumvents position effects and allows increased targeting efficiency. We also report a compatible double-inducible system for tight regulation of highly toxic products. This system exploits a new inducible RRNA promoter to drive T7 RNA polymerase (T7RNAP) transcription which then drives expression from inducible T7 promoters. The developments described should facilitate functional analysis and increased throughput.

SummarySilent information regulator 2 (Sir2)-related proteins or sirtuins function as NAD + -dependent deacetylases or ADP ribosylases that target a range of substrates, thereby influencing chromatin structure and a diverse range of other biological functions. Genes encoding three Sir2-related proteins (SIR2rp1-3) have been identified in the parasitic trypanosomatids, early branching protozoa with no previously reported transcriptional silencing machinery. Here we show that, in the mammalian-infective bloodstream-stage of the African trypanosome, Trypanosoma brucei, SIR2rp1 localizes to the nucleus while SIR2rp2 and SIR2rp3 are both mitochondrial proteins. The nuclear protein, SIR2rp1, controls DNA repair and repression of RNA polymerase I-mediated expression immediately adjacent to telomeres. Antigenic variation, however, which involves the silencing and Pol I-mediated transcriptional switching of subtelomeric variant surface glycoprotein genes, continues to operate independent of SIR2rp1.

African trypanosomes evade the host immune response through antigenic variation, which is achieved by periodically expressing different variant surface glycoproteins (VSGs). VSG expression is monoallelic such that only one of approximately 15 telomeric VSG expression sites (ESs) is transcribed at a time. Epigenetic regulation is involved in VSG control but our understanding of the mechanisms involved remains incomplete. Histone deacetylases are potential drug targets for diseases caused by protozoan parasites. Here, using recombinant expression we show that the essential Trypanosoma brucei deacetylases, DAC1 (class I) and DAC3 (class II) display histone deacetylase activity. Both DAC1 and DAC3 are nuclear proteins in the bloodstream stage parasite, while only DAC3 remains concentrated in the nucleus in insect-stage cells. Consistent with developmentally regulated localization, DAC1 antagonizes SIR2rp1-dependent telomeric silencing only in the bloodstream form, indicating a conserved role in the control of silent chromatin domains. In contrast, DAC3 is specifically required for silencing at VSG ES promoters in both bloodstream and insect-stage cells. We conclude that DAC1 and DAC3 play distinct roles in subtelomeric gene silencing and that DAC3 represents the first readily druggable target linked to VSG ES control in the African trypanosome.

SummaryChromatin modification is important for virtually all aspects of DNA metabolism but little is known about the consequences of such modification in trypanosomatids, early branching protozoa of significant medical and veterinary importance. MYST-family histone acetyltransferases in other species function in transcription regulation, DNA replication, recombination and repair. Trypanosoma brucei HAT3 was recently shown to acetylate histone H4K4 and we now report characterization of all three T. brucei MYST acetyltransferases (HAT1-3). First, GFP-tagged HAT1-3 all localize to the trypanosome nucleus. While HAT3 is dispensable, both HAT1 and HAT2 are essential for growth. Strains with HAT1 knock-down display mitosis without nuclear DNA replication and also specific de-repression of a telomeric reporter gene, a rare example of transcription control in an organism with widespread and constitutive polycistronic transcription. Finally, we show that HAT2 is responsible for H4K10 acetylation. By analogy to the situation in Saccharomyces cerevisiae, we discuss low-level redundancy of acetyltransferase function in T. brucei and suggest that two MYST-family acetyltransferases are essential due to the absence of a Gcn5 homologue. The results are also consistent with the idea that HAT1 contributes to establishing boundaries between transcriptionally active and repressed telomeric domains in T. brucei.

SummaryPost-translational histone modifications have been studied intensively in several eukaryotes. It has been proposed that these modifications constitute a 'histone code' that specifies epigenetic information for transcription regulation. With a limited number of histone-modifying enzymes, implying less redundancy, Trypanosoma brucei represents an excellent system in which to investigate the function of individual histone modifications and histone-modifying enzymes. In this study, we characterized the acetylation of lysine 4 of histone H4 (H4K4), the most abundant acetylation site in T. brucei histones. Because of the large sequence divergence of T. brucei histones, we generated highly specific antibodies to acetylated and unmodified H4K4. Immunofluorescence microscopy and Western blots with sorted cells revealed a strong enrichment of unmodified H4K4 in S phase and suggested a G1/G0-specific masking of the site, owing to non-covalently binding factors. Finally, we showed that histone acetyltransferase 3 (HAT3) is responsible for H4K4 acetylation and that treatment of cells with the protein synthesis inhibitor cycloheximide led to an almost instantaneous loss of unmodified H4K4 sites. As HAT3 is located inside the nucleus, our findings suggest that newly synthesized histone H4 with an unmodified K4 is imported rapidly into the nucleus, where it is acetylated, possibly irreversibly.

Graphical abstractTudor staphylococcal nuclease is a component of the major RNA interference effector complex, RISC. Using a novel reporter system, we show that Tudor-nuclease is dispensable for dsRNA, mRNA or siRNA processing.

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