Analysis of the architecture and function of the nuclear DNA replication apparatus in Trypanosoma brucei

DNA replication is central to the propagation of life and initiates by the designation of genome sequences as origins, where synthesis of a copy of the genetic material begins once per cell division. Despite considerable progress in understanding mitochondrial (kinetoplast) DNA replication in kinetoplastid parasites, little is known about nuclear DNA replication. The mechanism and machinery of DNA replication initiation is well-conserved among characterised eukaryotes. The six protein origin recognition complex (ORC, Orc1-Orc6), Cdc6, and Cdt1 are recruited sequentially to DNA, and once bound, they load the replicative helicase (MCM, a heterohexamer; subunits Mcm2-7) to form a pre-replicative complex (pre-RC) at potential origins of replication. The largest subunit of ORC, Orc1, is related in sequence to Cdc6, indicative of derivation from a common ancestor. Such an ancestral molecule appears still to function in archaea. These prokaryotes lack Cdc6 and possess a protein named Orc1/Cdc6, which appears to provide all ORC functions, since orthologues of Orcs2-6 are absent. In addition to this, archaeal orthologues of Cdt1 have not been clearly described, though potentially related factor, named WhiP (winged helix initiator protein), has been found. Comparative genome analysis of Trypanosoma brucei and related trypanosomatids (Leishmania major and Trypanosoma cruzi) revealed, remarkably, only a single ORC protein that is equally related to eukaryotic Orc1 and Cdc6 (named here TbORC1/CDC6). In addition, no clear homologue of Cdt1 was found. These observations have been interpreted as suggesting that origin designation in trypanosomatids, although eukaryotic, may be archaeal-like, raising numerous mechanistic and evolutionary questions. To test this hypothesis, and to dissect the process of nuclear DNA replication, a number of experiments are described in this thesis. We used RNA interference (RNAi) to demonstrate that knockdown of TbORC1/CDC6 in procyclic form (PCF) T. brucei cells inhibits nuclear DNA synthesis, as revealed by cell cycle analysis and a BrdU incorporation assay. Immunofluorescence and GFP-tagging showed that in procyclic form (PCF) cells TbORC1/CDC6 is a nuclear protein. In PCF cells, based on the evidence gathered, we confirm that TbORC1/CDC6 acts in nuclear DNA synthesis. In contrast, RNAi knockdown of TbORC1/CDC6 in bloodstream form (BSF) T. brucei cells resulted in the rapid accumulation of cells with more than two nuclei and two kinetoplasts, indicating a deregulation of the cell cycle, which is then followed by cell rapid cell death. This RNAi result provides greater evidence that TbORC1/CDC6 provides an essential function in the parasite, since RNAi depletion of TbORC1/CDC6 in PCF cells has a less pronounced effect on growth. Nevertheless, attempts to generate TbORC1/CDC6 null mutants failed in PCF cells, consistent with an essential role in this life cycle stage also. To study the molecular interactors of TbORC1/CDC6, we performed immunoprecipitation analyses. From this, we have identified one protein (gene ID, Tb927.10.13380) that acts as a component of the T. brucei pre-replicative machinery, and suggest that this is a previously unidentified orthologue of Orc4. We also indentified a further protein (gene ID, Tb927.10.7980) that may also act in T. brucei DNA replication, but whose identity and function are unclear. TbORC1/CDC6 appears not to interact directly with the TbMCM helicase (for which orthologues of all subunits can be identified), consistent with previous observations from a number of eukaryotic organisms, and contrary to reports in some archaeal species. MCM subunits in T. brucei form at least one subcomplex (TbMCM2/4/6/7) homologous to that previously observed for human, yeast, Drosophila, Xenopus and mouse MCM proteins. Taken together, these data appears to refute the hypothesis that the DNA replication pre-RC machinery in T. brucei is analogous to archaea. Rather, we propose that TbORC contains at least two components, TbORC1/CDC6 and Tb927.10.13380, more analogous to the eukaryotic model, suggesting that origin designation is not carried out by a single protein. To identify potential replication origin sequences, we performed chromatin immunoprecipitation with functional, epitope-tagged TbORC1/CDC6 in PCF cells and, using a high-resolution tiling array (NimbleGen) for T. brucei, we have mapped TbORC1/CDC6 binding sites along all the megabase chromosomes in the genome. Analyses of chromosomes 1-10 showed that 278 binding sites are sparsely located within the core of chromosomes, of which 114 loci (40%) co-localise with probable RNA Polymerase II transcription start sites, perhaps consistent with an origin function. In addition, a further 330 binding sites are present as high density clusters in subtelomeric VSG arrays, and 81 binding sites are associated with sub-telomeric elements, perhaps consistent with a non-origin function. Consistent with these results, RNAi knockdown of TbORC1/CDC6 led to derepression of metacyclic Variant Surface Glycoprotein (VSG) genes, suggesting that TbORC1/CDC6 plays a role in the epigenetic silencing at VSG expression sites in PCF T. brucei. Similar analysis of VSG expression in BSF cells, and of BSF VSGs in PCF cells, was less conclusive, perhaps suggesting differential functions of TbORC1/CDC6 in different life cycle stages or at different VSG expression sites. These analyses shed new light on the architecture and potential function of TbORC1/CDC6 in T. brucei nuclear DNA replication in general, as well as a potential association between replication and antigenic variation in T. brucei

Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying

Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation

Identification of ORC1/CDC6-Interacting Factors in Trypanosoma brucei Reveals Critical Features of Origin Recognition Complex Architecture

DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1–6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing

Nuclear DNA replication initiation in kinetoplastid parasites: new insights into an ancient process

Nuclear DNA replication is, arguably, the central cellular process in eukaryotes, because it drives propagation of life and intersects with many other genome reactions. Perhaps surprisingly, our understanding of nuclear DNA replication in kinetoplastids was limited until a clutch of studies emerged recently, revealing new insight into both the machinery and genome-wide coordination of the reaction. Here, we discuss how these studies suggest that the earliest acting components of the kinetoplastid nuclear DNA replication machinery – the factors that demarcate sites of the replication initiation, termed origins – are diverged from model eukaryotes. In addition, we discuss how origin usage and replication dynamics relate to the highly unusual organisation of transcription in the genome of Trypanosoma brucei

TfR RNAi and functional complementation.

<p>The parental TfR RNAi cell line alone (Par), or complemented with RNAi^R E6^R and E7^R constructs (E6:E7), were cultured without (tet-) or with tetracycline (tet+). <b>A</b>. Cell density was measured by hemocytometer and cultures were adjusted to starting density daily. Data are means ± SEM (n = 3). All subsequent analyses were performed at 24 hrs of silencing. <b>B</b>. Total RNA was prepared from the Par or E6:E7 cell lines. Transcript levels of native RNAi-sensitive E6^N and E7^N and RNAi-resistant E6^R and E7^R were determined by qRT-PCR; n.a indicates not assayed. Specific primers are indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006366#ppat.1006366.s001" target="_blank">S1 Fig</a>. Results are normalized to un-induced controls and are presented as fold-change for three biological replicates (mean ± SEM). C. Un-induced and induced Par and E6:E7 TfR RNAi cells were pulse radiolabeled (1 hr) with [³⁵S]Met/Cys and polypeptides were specifically pull-downed with rabbit anti-TfR antibodies (anti-TfR), transferrin-conjugated beads (Tf-beads) or rabbit anti-HSP70 antibodies (anti-HSP70). Pull-downs were fractionated by SDS-PAGE (10⁷ cell equivalents/lane) and visualized by phosphorimaging. The mobilities of ESAG6 (E6), ESAG7 (E7) and HSP70 (H) are indicated on the left of the appropriate panels. Representative phosphorimages are presented (n = 3). <b>D</b>. Receptor mediated uptake of fluorescent transferrin (Tf) and tomato lectin (TL) was measured by flow cytometry. Data are presented as median fluorescent intensity (MFI ± SEM.) for three biological replicates and are normalized to un-silenced control cells. <b>E</b>. Localization of native TfR in cells without (Par-, E6:E7-) or with (E6:E7+) tetracycline. Cells were fixed, permeabilized and stained with mouse anti-BiP (green), rabbit anti-TfR (red), and DAPI (blue) to detect nucleus (n) and kinetoplast (k). In each case flagellar pocket localizations of TfR are indicated (fp). Deconvolved three-channel summed stack projections of representative cells (top panel) with the matched DIC images (bottom panel) are presented. Bar = 4 μm.</p

Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes

<div><p>Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI² homodimer; TfR is a GPI¹ heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI²) is a surface protein; truncated VSG (GPI⁰) is degraded in the lysosome; and native TfR (GPI¹) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI² ESAG6 homodimers that are unable to bind transferrin—thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI^0–2. Our results conform to the valence model: GPI⁰ ESAG7 homodimers traffick to the lysosome and GPI² ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI² TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI² TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.</p></div

Turnover of native and RNAi^R TfR.

<p>Cell lines as indicated were cultured (24 hr) without (<b>A</b>. Par) or with tetracycline for all other cell lines (<b>B-E</b>). Cells were pulse/chase radiolabeled (15 min/4 hr) in the absence (-, open circles) or presence (+, closed circles) of FMK024 (20 μM) to block lysosomal degradation. At the indicated times E6 and E7 polypeptides were immunoprecipitated from cell lysates with anti-TfR antibody and fractionated by SDS-PAGE (5x10⁶ cell equivalents/lane). The rates of turnover were quantified from phosphorimages as fraction of initial species (mean ± SEM) for multiple biological replicates (n values are inset in each graph). For Par (<b>A</b>) and E6:E7 (<b>B</b>) quantifications combined E6 and E7 values are presented, but identical results were obtained when quantified individually. Representative phosphorimages are presented below each corresponding decay curve. Mobilities of E6 and E7 subunits are indicated on the left and chase times (hr) are shown above each lane.</p

Expression and function of E6^R:E7^G.

<p>The parental TfR RNAi cell line containing RNAi resistant E6^R and E7^G (E6^R:E7^G) was cultured without (tet-) or with (tet+) tetracycline. All analyses are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006366#ppat.1006366.g003" target="_blank">Fig 3</a>. <b>A</b>. Cell density. <b>B</b>. Transcript levels by qRT-PCR. <b>C</b>. Biosynthesis and pull-down of TfR subunits. Note that the E6^R and E7^G ORFs/proteins are the same length/size. All phosphorimages are representative of three independent biological replicates. <b>D</b>. Receptor mediated endocytosis by flow cytometry. <b>E</b>. IFA of fixed permeabilized and non-permeabilized cells, as indicated. Permeable cells (left) were stained with anti-BiP (green), rabbit anti-TfR (red), and DAPI (blue) to detect nucleus and kinetoplast. Arrowheads indicate surface staining along the flagellar membrane. Non-permeable cells (right) were stained with anti-TfR alone. Deconvolved three-channel summed stack projections of representative cells (tet-) or (tet+) are shown. Bar = 4 μm.</p

Expression and function of E6^R alone.

<p>The parental TfR RNAi cell line containing RNAi resistant E6^R was cultured without (tet-) or with (tet+) tetracycline. All analyses are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006366#ppat.1006366.g003" target="_blank">Fig 3</a>. <b>A</b>. Cell density. <b>B</b>. Transcript levels by qRT-PCR. <b>C</b>. Biosynthesis and pull-down of TfR subunits. All phosphorimages are representative of three independent biological replicates. <b>D</b>. Receptor mediated endocytosis by flow cytometry. <b>E</b>. IFA of fixed permeabilized cells with mouse anti-BiP (green), rabbit anti-TfR (red), and DAPI (blue) to detect nucleus and kinetoplast. Cell outline (tet- only) was traced from matched transmitted light images. Deconvolved three-channel summed stack projections of representative cells are shown. Bar = 4 μm.</p