Cell-to-Cell Heterogeneity in Trypanosomes.

Recent developments in single-cell and single-molecule techniques have revealed surprising levels of heterogeneity among isogenic cells. These advances have transformed the study of cell-to-cell heterogeneity into a major area of biomedical research, revealing that it can confer essential advantages, such as priming populations of unicellular organisms for future environmental stresses. Protozoan parasites, such as trypanosomes, face multiple and often hostile environments, and to survive, they undergo multiple changes, including changes in morphology, gene expression, and metabolism. But why does only a subset of proliferative cells differentiate to the next life cycle stage? Why do only some bloodstream parasites undergo antigenic switching while others stably express one variant surface glycoprotein? And why do some parasites invade an organ while others remain in the bloodstream? Building on extensive research performed in bacteria, here we suggest that biological noise can contribute to the fitness of eukaryotic pathogens and discuss the importance of cell-to-cell heterogeneity in trypanosome infections.

Smoother: on-the-fly processing of interactome data using prefix sums.

Nucleic acid interactome data, such as chromosome conformation capture data and RNA-DNA interactome data, are currently analyzed via pipelines that must be rerun for each new parameter set. A more dynamic approach is desirable since the optimal parameter set is commonly unknown ahead of time and rerunning pipelines is a time-consuming process. We have developed an approach fast enough to process interactome data on-the-fly using a sparse prefix sum index. With this index, we created Smoother, a flexible, multifeatured visualization and analysis tool that allows interactive filtering, e.g. by mapping quality, almost instant comparisons between different normalization approaches, e.g. iterative correction, and ploidy correction. Further, Smoother can overlay other sequencing data or genomic annotations, compare different samples, and perform virtual 4C analysis. Smoother permits a novel way to interact with and explore interactome data, fostering comprehensive, high-quality data analysis. Smoother is available at https://github.com/Siegel-Lab/BioSmoother under the MIT license.

H2B.V demarcates divergent strand-switch regions, some tDNA loci, and genome compartments in Trypanosoma cruzi and affects parasite differentiation and host cell invasion.

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in tissue-derived trypomastigote (TCT) life forms, a nonreplicative stage of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using epimastigotes and TCT life forms, and we found that H2B.V was preferentially located at the edges of divergent transcriptional strand switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of TCT forms was depleted of H2B.V-enriched peaks in comparison to epimastigote forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle.

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.

GT-rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes.

Genome-wide transcription studies are revealing an increasing number of "dispersed promoters" that, unlike "focused promoters", lack well-conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well-defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite Trypanosoma brucei, in which RNA polymerase II transcription initiation occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site-specific and genome-wide assays, we identified GT-rich promoters that can drive transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context-dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.

Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria.

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.

Exploiting CRISPR-Cas9 technology to investigate individual histone modifications.

Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.

Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei.

Base J, ß-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In Trypanosoma brucei, J is enriched, along with histone H3 variant (H3.V), at sites involved in RNA Polymerase (RNAP) II termination and telomeric sites involved in regulating variant surface glycoprotein gene (VSG) transcription by RNAP I. Reduction of J in T. brucei indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the H3.V knockout. mRNA-seq indicates gene derepression includes VSG genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in T. brucei, depending on chromatin context (and transcribing polymerase). As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination.

Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination.

The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription.

Genome-wide analysis of chromatin structures in Trypanosoma brucei using high-resolution MNase-ChIP-seq.

Specific DNA-protein interactions are the basis for many important cellular mechanisms like the regulation of gene expression or replication. Knowledge about the precise genomic locations of DNA-protein interactions is important because it provides insight into the regulation of these processes. Recently, we have adapted an approach that combines micrococcal nuclease (MNase) digestion of chromatin with chromatin immunoprecipitation in Trypanosoma brucei. Here, we describe in detail how this method can be used to map the genome-wide distribution of nucleosomes or other DNA-binding proteins at high resolution in T. brucei.

Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve.

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.

Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei.

Unusually for a eukaryote, genes transcribed by RNA polymerase II (pol II) in Trypanosoma brucei are arranged in polycistronic transcription units. With one exception, no pol II promoter motifs have been identified, and how transcription is initiated remains an enigma. T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V. Using chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) to examine the genome-wide distribution of chromatin components, we show that histones H4K10ac, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold at probable pol II transcription start sites (TSSs). We also show that nucleosomes containing H2AZ and H2BV are less stable than canonical nucleosomes. Our analysis also identifies >60 unexpected TSS candidates and reveals the presence of long guanine runs at probable TSSs. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable pol II transcription termination sites. Our 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 pol II transcription factors have been elusive.

Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages.

While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5' UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.

Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei.

Base J, ß-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase (RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.

Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei.

Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.

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.

Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum.

BACKGROUND: Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets. RESULTS: To gain insights into the genome-wide distribution of NATs in P. falciparum, we performed RNA-ligation based strand-specific RNA sequencing at unprecedented depth. Our data indicate that 78.3% of the genome is transcribed during blood-stage development. Moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of NATs change during the intraerythrocytic developmental cycle (IDC), they do not correlate with the corresponding mRNA levels. Interestingly, antisense transcription is not evenly distributed across coding regions (CDSs) but strongly clustered towards the 3'-end of CDSs. Furthermore, for a significant subset of NATs, transcript levels correlate with mRNA levels of neighboring genes.Finally, we were able to identify the polyadenylation sites (PASs) for a subset of NATs, demonstrating that at least some NATs are polyadenylated. We also mapped the PASs of 3443 coding genes, yielding an average 3' untranslated region length of 523 bp. CONCLUSIONS: Our strand-specific analysis of the P. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in P. falciparum. For a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other NATs appear to be regulated independently of mRNA transcription. Our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. In addition, the extensive novel data on 3' UTR length will allow others to perform searches for regulatory motifs in the UTRs and help understand post-translational regulation in P. falciparum.

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites.

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation.

Decoding the impact of nuclear organization on antigenic variation in parasites.

Antigenic variation as a strategy to evade the host adaptive immune response has evolved in divergent pathogens. Antigenic variation involves restricted, and often mutually exclusive, expression of dominant antigens and a periodic switch in antigen expression during infection. In eukaryotes, nuclear compartmentalization, including three-dimensional folding of the genome and physical separation of proteins in compartments or condensates, regulates mutually exclusive gene expression and chromosomal translocations. In this Review, we discuss the impact of nuclear organization on antigenic variation in the protozoan pathogens Trypanosoma brucei and Plasmodium falciparum. In particular, we highlight the relevance of nuclear organization in both mutually exclusive antigen expression and genome stability, which underlie antigenic variation.

Two thymidine hydroxylases differentially regulate the formation of glucosylated DNA at regions flanking polymerase II polycistronic transcription units throughout the genome of Trypanosoma brucei.

Base J is a hypermodified DNA base localized primarily to telomeric regions of the genome of Trypanosoma brucei. We have previously characterized two thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2 is a chromatin re-modeling protein that induces de novo J-synthesis, allowing JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and high-throughput sequencing. This genome-wide analysis revealed an enrichment of base J at regions flanking polymerase II polycistronic transcription units (Pol II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the telomeric regions. However, the maintenance of J at JBP1-specific regions is dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania major also contain base J highlights the functional importance of the modified base at Pol II PTUs within members of the kinetoplastid family. The regulation of J synthesis/localization by two THs and potential biological function of J in regulating kinetoplastid gene expression is discussed.