50 research outputs found
Keeping Balance Between Genetic Stability and Plasticity at the Telomere and Subtelomere of Trypanosoma brucei
Telomeres, the nucleoprotein complexes at chromosome ends, are well-known for their essential roles in genome integrity and chromosome stability. Yet, telomeres and subtelomeres are frequently less stable than chromosome internal regions. Many subtelomeric genes are important for responding to environmental cues, and subtelomeric instability can facilitate organismal adaptation to extracellular changes, which is a common theme in a number of microbial pathogens. In this review, I will focus on the delicate and important balance between stability and plasticity at telomeres and subtelomeres of a kinetoplastid parasite, Trypanosoma brucei, which causes human African trypanosomiasis and undergoes antigenic variation to evade the host immune response. I will summarize the current understanding about T. brucei telomere protein complex, the telomeric transcript, and telomeric R-loops, focusing on their roles in maintaining telomere and subtelomere stability and integrity. The similarities and differences in functions and underlying mechanisms of T. brucei telomere factors will be compared with those in human and yeast cells
Regulation of Gene Expression in Trypanosoma brucei
The protozoan parasite Trypanosoma brucei is one of the most divergent well-studied eukaryotes and many discoveries of general interest have been made in T. brucei. Atypical for a eukaryote, in T. brucei genes transcribed by RNA polymerase II (RNA pol II) are arranged in polycistronic transcription units (PTUs). mRNAs are separated post-transcriptionally by coupled splicing and polyadenylation reactions. During the splicing reaction a 39 nt ‘sliced-leader’ is added to every mRNA, a process termed trans splicing. The arrangement of generally unrelated genes in PTUs has lead to the assumption that little gene regulation occurs at the level of transcription initiation but that gene expression is regulated at the levels of mRNA maturation and stability. To better understand gene regulation in T. brucei, I performed a systematic analysis to correlate trans-splicing efficiency with specific DNA sequence motifs and observed large variations in trans-splicing efficiency depending on the DNA sequence upstream of the 3´ splice site. With one exception, no RNA pol II promoter motif has been identified in T. brucei, and how transcription is initiated remains an enigma. The second part of my thesis was based on the assumption that, given the apparent lack of RNA pol II promoter motifs, RNA pol II transcription start sites (TSS) are marked by distinct chromatin structures that facilitate recruitment of the transcription machinery. T. brucei has four histone variants: H2AZ, H2BV, H3V and H4V. Using ChIP-seq to examine the genome-wide distribution of chromatin components, I showed that H2AZ, H2BV, the K10-Ac form of H4, and the bromodomain factor BDF3 are significantly enriched at probable RNA pol II TSSs and used this mark to identify more than 60 previously unanticipated TSS candidates. Co-IP experiments with tagged H2A, H2AZ, H2B, and H2BV indicated that less histone H3 and histone H4 coimmunoprecipitated with variant histones than with core histones suggesting that variant nucleosomes are less stable than canonical nucleosomes. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable RNA pol II transcription termination sites. These findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general RNA pol II transcription factors have been elusive
Genetic and Epigenetic Determinants of Transcription in the Divergent Eukaryote Leishmania major
Leishmania spp. and other trypanosomatid protozoa use a highly unusual mechanism to generate functional messenger RNAs (mRNAs) in which protein-coding genes are transcribed polycistronically. Here, transcription initiates primarily in divergent strand switch regions (dSSRs), where two polycistronic gene clusters are oriented head-to-head. These regions lack all known eukaryotic cis-regulatory elements, and it is not known how genetic and epigenetic factors cooperate to define dSSRs as regions of productive initiation. To quantitatively identify regulatory elements and to study the contribution of epigenetic factors to dSSR function, we combined genome-wide studies of chromatin structure with a focused interrogation of a single dSSR using a novel integrated bidirectional, dual-luciferase reporter. Chromatin-based studies demonstrated that Leishmania lack well-positioned nuclease-hypersensitive sites associated with promoters in other eukaryotes. Rather, nuclease-hypersensitive sites are positioned heterogeneously across broad regions associated with epigenetic marks indicative of active transcription, suggesting that transcription initiation events occur promiscuously within regions associated with a transcriptionally-permissive epigenetic state. Our studies using the bidirectional reporter validate these observations and strongly suggest that Leishmania do not require cis-regulatory elements for efficient bidirectional transcription initiating in dSSRs, as a large region of the dSSR can be replaced with unrelated sequences without altering bidirectional reporter gene expression. In addition to these genetic studies we also focused on epigenetic determinants of transcriptional activity in Leishmania, with respect to both transcription initiation and transcription termination. We showed that the histone variants H2A.Z and H2B.V, which are associated with transcriptionally permissive regions in T. brucei, are essential in L. major, while the transcription termination-associated histone variant H3.V is not. Interestingly, unlike Leishmania lacking the DNA modification base J, H3.V-null L. major shows no defects in transcription termination. Although the study of essential genes in Leishmania is challenging at this time, we present preliminary data describing elements of inducible gene expression systems which may improve our ability to study essential genes. Together, the data in this thesis show that transcription of protein-coding genes is primarily determined epigenetically, and suggest that chromatin-related processes may be an attractive target for therapeutic intervention
Analysis of Metacyclic Telomeres in Trypanosoma brucei
Trypanosoma brucei spp. are the agents responsible for African sleeping sickness in man and Nagana in cattle. The organisms have the ability to evade the host's immune system by antigenic variation of their surface coat. The surface coat of the infective forms is composed of a single molecular species, the variant surface glycoprotein (VSG). Each specific VSG is encoded by a separate gene, expression of which occurs in a loose hierarchical order. T. brucei has the coding capacity for approximately 10e3 VSG genes, which are found either in chromosomal clusters or at telomeric loci; it is only from the latter that the gene may be expressed. Telomeric expression sites (ESs) utilized during bloodstream infection are complex, typically between 40 - 60 Kb long, containing several non-VSG expression site associated genes (ESAGs) and preceded by a long barren region devoid of restriction sites. Transcription of telomeric ESs is insensitive to the toxin alpha-amanitin and transcription of a given ES appears to be mutually exclusive of other ESs in vivo. By virtue of their location, chromosomal internal VSG genes need to be transposed to a telomere for expression. The transposition event is duplicative and produces an expression linked copy of the gene. Telomeric VSG genes, however, can be expressed either by duplicative transposition or reciprocal recombination to an active ES, or by in situ activation. The metacyclic stage of the trypanosome life cycle is the first to express VSG genes. The metacyclic form utilizes a highly predictable subset of VSG genes (M-VSGs), comprising only 1-2% of the entire VSG gene repertoire, which appear to be expressed by a distinct and dominant mechanism to that employed during chronic bloodstream infection. Direct molecular analysis of M-VSG gene expression is precluded by the paucity of metacyclic forms in the salivary exudate of the tsetse fly vector and the transient nature of this developmental stage which cannot be cultured in vitro. M-VSG gene expression, however, is still extant in the host bloodstream in the first few days following fly bite. Analysis during this period is compounded by the polyclonal origin of the M-VSG genes expressed by individual organisms and instability of VSG expression. The work described in this thesis focuses on attempts to clone an M-VSG gene telomere and to gain insight into the predictability and stability of the M-VSG repertoire, by analysis of the telomere in a model trypanosome line which circumvents some of the problems associated with previous systems. Cloning and analysis of the BC telomeres for the M-VSG genes for GUTat 7.1 and ILTat 1.22 revealed that each had a remarkably small barren region and shared no sequence homology with ESs used in chronic bloodstream infection, apart from the 70 bp repeat sequence constituting the barren region 5'of the VSG gene. Transcriptional analysis of the ILTat 1.22 metacyclic ES, utilising the model line of trypanosomes expressing the gene in situ, revealed that the ES is extremely short in comparison to bloodstream ESs, extending no more than 3.5 - 4 Kb 5' of the VSG gene. One other region of the ILTat 1.22 BC and GUTat 7.1-2 BC telomeres appeared to be transcriptionally active. This comprised a genomic repetitive element which also was transcribed in procyclic tiypanosomes. The structural individuality of these telomeres was proposed as underlying the stability and physical distinction of the M- VSG repertoire. This hypothesis is supported by an epidemiological analysis of the ILTat 1.22 BC telomere over a 24 year period and spanning diverse epidemic foci of infection. Within Kenyan epidemic foci this telomere is present unaltered in all the stocks investigated over the the period examined and suggests that spread of the disease in East Africa is principally by mechanical transmission; this has important consequences for tackling the disease at source. Cloning and analysis of these telomeres now facilitates characterisation of metacyclic ES control elements and comparisons with other M-ES to be made
The significance of duplicative VSG gene activation during antigenic variation in African trypanosomes
African trypanosomes can survive prolonged exposure to the immune responses of their mammalian hosts by constantly changing the variant surface glycoprotein (VSG) expressed in their surface coat in a process known as antigenic variation. Each parasite has a repertoire of approximately 1000 silent VSG genes, which are expressed differentially. The VSG genes are expressed exclusively at specialised transcriptional units known as the bloodstream expression sites (BBSs). Approximately 20 of these sites exist in the trypanosome nucleus, although only one is maximally active at a time, ensuring that a single VSG is expressed in the parasite's surface coat. VSG switching is mediated by replacing the VSG gene in the active BES, or by silencing the active BES and initiating transcription from a new BES. Investigations into trypanosome antigenic variation have described several different VSG switching mechanisms, although the majority of these studies were performed with laboratory attenuated trypanosome lines. Following repeated syringe passaging, these "monomorphic" lines have lost the ability to differentiate from the proliferative bloodstream form. Under normal circumstances these trypanosomes cannot be transmitted by the tsetse fly and do not develop through their life cycle. In addition, monomorphic trypanosomes display a marked reduction in their VSG switch rates, which are up to 4 or 5 orders of magnitude lower than those of non-adapted lines. This raises questions about the significance of the switching mechanisms observed in these lines. It has been proposed that non-adapted, or pleomorphic, trypanosomes normally have an active VSG switch mechanism, involving gene duplication, that is depressed, or from which a component is absent, in monomorphic lines. The main aim of this thesis was to examine this hypothesis by analysing the switching mechanisms used during VSG activation during the early stages of a chronic, pleomorphic infection. Additionally, this investigation examined the chromosomal environment of the basic copy VSG genes, to determine whether a chromosome position effect influenced the early order of VSG gene expression. It appears that, during the early stages of infection, trypanosome antigenic variation is predominated by duplicative transposition of telomeric VSGs, many of which reside on the minichromosomes. These results contrast strongly with the outcome of analyses of monomorphic trypanosomes, which utilize several different switching mechanisms, and most commonly display transcriptional switching during the early stages of infection. The work presented in this thesis therefore supports the hypothesis of a dedicated duplicative switching mechanism that is reduced in (or even absent from) laboratory adapted, monomorphic lines
Insights into the function of short interspersed degenerated retroposons in the protozoan parasite Leishmania
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal
Molecular cloning and characterization of a glucose transporter-related gene from Trypanosoma brucei
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Developmentally regulated genes in Trypansoma Cruzi
Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted by blood feeding triatomine bugs. As T. cruzi cycles between the insect vector and vertebrate host, it goes through several distinct developmental stages. The molecular mechanism that regulates these differentiation steps is poorly understood. We have initiated a systematic dissection of metacyclogenesis, the differentiation step from replicating non-infective epimastigotes to non-replicating infective metacyclic trypomastigotes. Our aim is to functionally characterise novel stage-regulated genes, using transfection-based approaches. We demonstrate that one of these genes, MET3, encodes a protein that localises to the nucleus and associates with nucleolar antigens. During metacyclogenesis the structure of the nucleus is modified: heterochromatin spreads as the nucleus changes shape from round to elongated, and nucleolar antigens are dispersed in the metacyclic nucleus. Our data show that while dispersal of nucleolar antigens occurs in all cells of a stationary phase culture, MET3 protein is expressed only in the subset of cells that have differentiated to metacyclics or to intermediate forms. By expressing MET3-GFP fusion proteins in the parasite, we identified two sequence elements that can independently direct localisation. To address function, we generated MET3 null mutants. These knockout cells produced significantly fewer fully developed metacyclics than wild-type controls. However, this phenotype could not be complemented with an ectopic copy of MET3. The reduced rate of metacyclogenesis may therefore be unrelated to the loss of MET3. The knockout cells are able to complete the entire life-cycle in vitro. This demonstrates that MET3 is not essential for development of the infective stage. RNA transcripts of the second gene, (MET2) are induced during metacyclogenesis. Recombinant MET2 protein associates with the kinetoplast when expressed in epimastigotes. We analysed in detail its genomic locus near a putative centromere on chromosome 3, and generated deletion mutants for further investigating its role in differentiation
Kinetoplastid Phylogenomics and Evolution
This Special Issue, Kinetoplastid Phylogenomics and Evolution, unites a series of research and review papers related to kinetoplastid parasites. The diverse topics represented in this collection display a variety of scientific questions and methodological approaches currently used to study these fascinating organisms
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Methods to Probe the Function of Modified Bases in DNA
This thesis is focused on the development and utilisation of chemical and biological tools to probe the function of modified bases in DNA with specific exploration of the less well-studied T-modifications: 5-hmU, 5-fU and Base J.
LCMS/MS techniques are first utilised to enable the accurate global quantification of T-modifications (5-hmU, 5-fU and Base J) in both trypanosomatids and mammalian DNA.
A chemical affinity-enrichment sequencing method for the T-modifications is next described, which allows their chemoselective tagging over their C-modification counterparts. DNA fragments containing 5-fU are selectively tagged and enriched via oxime, hydrazine or benzimidazole formation using a biotinylated probe, and DNA fragments containing 5-hmU can be first chemically oxidised to 5-fU using KRuO4. .Proof-of-principle T-modification enrichment is demonstrated by DNA sequencing.
In the following chapter, sequencing methods are employed to investigate the role of T-modifications in both trypanosomatids and mammalian samples. In T.Brucei, Base J formation is probed by artificial incorporation of 5-hmU and subsequent Base J chemical sequencing. Base J is preferentially formed or depleted at certain genomic loci; suggesting that Base J formation is sequence-specific. This may imply a distinct role for the 5-hmU sites which are not further glucosylated. Next, 5-hmU enrichment sequencing is performed in SMUG1 knockdown HEK293T cells to determine the genomic location of 5-hmU in mammals. An increase in 5-hmU loci is observed upon SMUG1 knockdown. 5-hmU enriched regions are found to be T-rich and depleted in exons and promoters. Furthermore, 5-hmU sites show poor overlap with known TET-enzyme binding sites, indicating that 5-hmU is formed via a TET-independent mechanism in HEK293T cells. Next, mass spectrometry-based proteomics studies are utilised to determine 5-fU protein-binders in mammals. Pulldown of proteins using biotinylated baits enables the identification of proteins which are enriched or suppressed in the presence of the 5-fU modification compared to a non-modified control. Enriched proteins include those associated with DNA-damage, consistent with the current understanding that 5-fU is a product of oxidative damage in mammalian DNA.
Finally, a mechanistic insight into the effect of formylated bases on nucleosomal structure is described. Schiff base formation between formylated nucleobases and histone protein lysine side-chains is demonstrated. This provides a molecular mechanism for the association of 5-fC with increased nucleosomal occupancy in vivo.CHESS scholarshi