Characterisation of TbSmee1 suggests endocytosis allows surface-bound cargo to enter the trypanosome flagellar pocket.

All endocytosis and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the single flagellum of the cell. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, was proposed to affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of T. brucei (Tb)MORN1, a hook complex component, resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form T. brucei and found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement and impaired access to the flagellar pocket membrane by surface-bound cargo, similar to depletion of TbMORN1. Unexpectedly, inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket.

A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling.

A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed.

Genome organization and DNA accessibility control antigenic variation in trypanosomes.

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei.

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes.

During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form (BSF) cells relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is transcribed from only 1 of the 15 subtelomeric expression sites (ESs). During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. We used mass spectrometry-based interactomics to determine the composition of telomere protein complexes in T. brucei BSF and PCF stages to learn more about the structure and functions of telomeres in trypanosomes. Our data suggest a different telomere complex composition in the two forms of the parasite. One of the novel telomere-associated proteins, TelAP1, forms a complex with telomeric proteins TbTRF, TbRAP1 and TbTIF2 and influences ES silencing kinetics during developmental differentiation.

A quorum sensing-independent path to stumpy development in Trypanosoma brucei.

For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival.

Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei.

Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes.

Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei.

The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes.

Fragment ion patchwork quantification for measuring site-specific acetylation degrees.

We introduce fragment ion patchwork quantification as a new mass spectrometry-based approach for the highly accurate quantification of site-specific acetylation degrees. This method combines (13)C1-acetyl derivatization on the protein level, proteolysis by low-specificity proteases and quantification on the fragment ion level. Acetylation degrees are determined from the isotope patterns of acetylated b and y ions. We show that this approach allows to determine site-specific acetylation degrees of all lysine residues for all core histones of Trypanosoma brucei. In addition, we demonstrate how this approach can be used to identify substrate sites of histone acetyltransferases.

Comparative proteomics of two life cycle stages of stable isotope-labeled Trypanosoma brucei reveals novel components of the parasite's host adaptation machinery.

Trypanosoma brucei developed a sophisticated life cycle to adapt to different host environments. Although developmental differentiation of T. brucei has been the topic of intensive research for decades, the mechanisms responsible for adaptation to different host environments are not well understood. We developed stable isotope labeling by amino acids in cell culture in trypanosomes to compare the proteomes of two different life cycle stages. Quantitative comparison of 4364 protein groups identified many proteins previously not known to be stage-specifically expressed. The identification of stage-specific proteins helps to understand how parasites adapt to different hosts and provides new insights into differences in metabolism, gene regulation, and cell architecture. A DEAD-box RNA helicase, which is highly up-regulated in the bloodstream form of this parasite and which is essential for viability and proper cell cycle progression in this stage is described as an example.

DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei.

Cell-cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell-cycle regulators are evolutionarily conserved in the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes.

Heterologous expression reveals distinct enzymatic activities of two DOT1 histone methyltransferases of Trypanosoma brucei.

Dot1 is a highly conserved methyltransferase that modifies histone H3 on the nucleosome core surface. In contrast to yeast, flies, and humans where a single Dot1 enzyme is responsible for all methylation of H3 lysine 79 (H3K79), African trypanosomes express two DOT1 proteins that methylate histone H3K76 (corresponding to H3K79 in other organisms) in a cell-cycle-regulated manner. Whereas DOT1A is essential for normal cell cycle progression, DOT1B is involved in differentiation and control of antigenic variation of this protozoan parasite. Analysis of DOT1A and DOT1B in trypanosomes or in vitro, to understand how H3K76 methylation is controlled during the cell cycle, is complicated by the lack of genetic tools and biochemical assays. To eliminate these problems, we developed a heterologous expression system in yeast. Whereas Trypanosoma brucei DOT1A predominantly dimethylated H3K79, DOT1B trimethylated H3K79 even in the absence of dimethylation by DOT1A. Furthermore, DOT1A activity was selectively reduced by eliminating ubiquitylation of H2B. The tail of histone H4 was not required for activity of DOT1A or DOT1B. These findings in yeast provide new insights into possible mechanisms of regulation of H3K76 methylation in Trypanosoma brucei.

Regulation and spatial organization of PCNA in Trypanosoma brucei.

As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.

A histone methyltransferase modulates antigenic variation in African trypanosomes.

To evade the host immune system, several pathogens periodically change their cell-surface epitopes. In the African trypanosomes, antigenic variation is achieved by tightly regulating the expression of a multigene family encoding a large repertoire of variant surface glycoproteins (VSGs). Immune evasion relies on two important features: exposing a single type of VSG at the cell surface and periodically and very rapidly switching the expressed VSG. Transcriptional switching between resident telomeric VSG genes does not involve DNA rearrangements, and regulation is probably epigenetic. The histone methyltransferase DOT1B is a nonessential protein that trimethylates lysine 76 of histone H3 in Trypanosoma brucei. Here we report that transcriptionally silent telomeric VSGs become partially derepressed when DOT1B is deleted, whereas nontelomeric loci are unaffected. DOT1B also is involved in the kinetics of VSG switching: in DeltaDOT1B cells, the transcriptional switch is so slow that cells expressing two VSGs persist for several weeks, indicating that monoallelic transcription is compromised. We conclude that DOT1B is required to maintain strict VSG silencing and to ensure rapid transcriptional VSG switching, demonstrating that epigenetics plays an important role in regulating antigenic variation in T. brucei.

A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei.

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 RNase 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 RNase 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. IMPORTANCE Trypanosoma brucei is a unicellular parasite that causes devastating diseases like sleeping sickness in humans and the "nagana" disease in cattle in Africa. Fundamental to the establishment and prolongation of a trypanosome infection is the parasite's ability to escape the mammalian host's immune system by antigenic variation, which relies on periodic changes of a protein surface coat. The exact mechanisms, however, which mediate these changes are still elusive. In this work, we describe a novel protein complex consisting of the histone methyltransferase DOT1B and RNase H2 which is involved in antigenic variation.

Trimethylation of histone H3K76 by Dot1B enhances cell cycle progression after mitosis in Trypanosoma cruzi.

Dot1 enzymes are histone methyltransferases that mono-, di- and trimethylate lysine 79 of histone H3 to affect several nuclear processes. The functions of these different methylation states are still largely unknown. Trypanosomes, which are flagellated protozoa that cause several parasitic diseases, have two Dot1 homologues. Dot1A catalyzes the mono- and dimethylation of lysine 76 during late G2 and mitosis, and Dot1B catalyzes trimethylation, which is a modification found in all stages of the cell cycle. Here, we generated Trypanosoma cruzi lines lacking Dot1B. Deletion of one allele resulted in parasites with increased levels of mono- and dimethylation and a reduction in H3K76me3. In the full knockout (DKO), no trimethylation was observed. Both the DKO and the single knockout (SKO) showed aberrant morphology and decreased growth due to cell cycle arrest after G2. This phenotype could be rescued by caffeine in the DKO, as caffeine is a checkpoint inhibitor of the cell cycle. The knockouts also phosphorylated γH2A without producing extensive DNA breaks, and Dot1B-depleted cells were more susceptible to general checkpoint kinase inhibitors, suggesting that a lack of H3K76 trimethylation prevents the initiation and/or completion of cytokinesis.

Trypanosome H2Bv replaces H2B in nucleosomes enriched for H3 K4 and K76 trimethylation.

Some inroads have been made into characterizing histone variants and post translational modifications of histones in Trypanosoma brucei. Histone variant H2BV lysine 129 is homologous to Saccharomyces cerevisiae H2B lysine 123, whose ubiquitination is required for methylation of H3 lysines 4 and 79. We show that T. brucei H2BV K129 is not ubiquitinated, but trimethylation of H3 K4 and K76, homologs of H3 K4 and K79 in yeast, was enriched in nucleosomes containing H2BV. Mutation of H2BV K129 to alanine or arginine did not disrupt H3 K4 or K76 methylation. These data suggest that H3 K4 and K76 methylation in trypanosomes is regulated by a novel mechanism, possibly involving the replacement of H2B with H2BV in the nucleosome.

Histone modifications in Trypanosoma brucei.

Several biological processes in Trypanosoma brucei are affected by chromatin structure, including gene expression, cell cycle regulation, and life-cycle stage differentiation. In Saccharomyces cerevisiae and other organisms, chromatin structure is dependent upon posttranslational modifications of histones, which have been mapped in detail. The tails of the four core histones of T. brucei are highly diverged from those of mammals and yeasts, so sites of potential modification cannot be reliably inferred, and no cross-species antibodies are available to map the modifications. We therefore undertook an extensive survey to identify posttranslational modifications by Edman degradation and mass spectrometry. Edman analysis showed that the N-terminal alanine of H2A, H2B, and H4 could be monomethylated. We found that the histone H4 N-terminus is heavily modified, while, in contrast to other organisms, the histone H2A and H2B N-termini have relatively few modifications. Histone H3 appears to have a number of modifications at the N-terminus, but we were unable to assign many of these to a specific amino acid. Therefore, we focused our efforts on uncovering modification states of H4. We discuss the potential relevance of these modifications.

The nuclear proteome of Trypanosoma brucei.

Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes.