Epigenetics and transcriptional control in African trypanosomes

2010 ◽  
Vol 48 ◽  
pp. 201-219 ◽  
Author(s):  
Gloria Rudenko
Keyword(s):  
Transcriptional Control ◽  
Surface Glycoprotein ◽  
Small Subset ◽  
Rna Pol Ii ◽  
Protein Coding ◽  
African Trypanosomes ◽  
Protein Coding Genes ◽  
Pol I ◽  
Telomere Binding Protein ◽  
Polycistronic Rna

The African trypanosome Trypanosoma brucei is a unicellular parasite which causes African sleeping sickness. Transcription in African trypanosomes displays some unusual features, as most of the trypanosome genome is transcribed as extensive polycistronic RNA Pol II (polymerase II) transcription units that are not transcriptionally regulated. In addition, RNA Pol I is used for transcription of a small subset of protein coding genes in addition to the rDNA (ribosomal DNA). These Pol I-transcribed protein coding genes include the VSG (variant surface glycoprotein) genes. Although a single trypanosome has many hundreds of VSG genes, the active VSG is transcribed in a strictly monoalleleic fashion from one of approx. 15 telomeric VSG ESs (expression sites). Originally, it was thought that chromatin was not involved in the transcriptional control of ESs; however, this view is now being re-evaluated. It has since been shown that the active ES is depleted of nucleosomes compared with silent ESs. In addition, a number of proteins involved in chromatin remodelling or histone modification and which play a role in ES silencing {including TbISWI [T. brucei ISWI (imitation-switch protein)] and DOT1B} have recently been identified. Lastly, the telomere-binding protein TbRAP1 (T. brucei RAP1) has been shown to establish a repressive gradient extending from the ES telomere end up to the ES promoter. We still need to determine which epigenetic factors are involved in ‘marking’ the active ES as part of the counting mechanism of monoallelic exclusion. The challenge will come in determining how these multiple regulatory layers contribute to ES control.

scholarly journals The Trypanosomatid-Specific N Terminus of RPA2 Is Required for RNA Polymerase I Assembly, Localization, and Function

2012 ◽  
Vol 11 (5) ◽  
pp. 662-672 ◽  
Author(s):  
Jan-Peter Daniels ◽  
Keith Gull ◽  
Bill Wickstead
Keyword(s):  
Rna Polymerase ◽  
Nuclear Localization ◽  
Rna Polymerase I ◽  
Growth Defect ◽  
Protein Coding ◽  
African Trypanosomes ◽  
Protein Coding Genes ◽  
Pol I ◽  
N Terminus ◽  
Polymerase I

ABSTRACT African trypanosomes are the only organisms known to use RNA polymerase I (pol I) to transcribe protein-coding genes. These genes include VSG , which is essential for immune evasion and is transcribed from an extranucleolar expression site body (ESB). Several trypanosome pol I subunits vary compared to their homologues elsewhere, and the question arises as to how these variations relate to pol I function. A clear example is the N-terminal extension found on the second-largest subunit of pol I, RPA2. Here, we identify an essential role for this region. RPA2 truncation leads to nuclear exclusion and a growth defect which phenocopies single-allele knockout. The N terminus is not a general nuclear localization signal (NLS), however, and it fails to accumulate unrelated proteins in the nucleus. An ectopic NLS is sufficient to reinstate nuclear localization of truncated RPA2, but it does not restore function. Moreover, NLS-tagged, truncated RPA2 has a different subnuclear distribution to full-length protein and is unable to build stable pol I complexes. We conclude that the RPA2 N-terminal extension does not have a role exclusive to the expression of protein-coding genes, but it is essential for all pol I functions in trypanosomes because it directs trypanosomatid-specific interactions with RPA1.

scholarly journals RNA Polymerase I Transcribes Procyclin Genes and Variant Surface Glycoprotein Gene Expression Sites in Trypanosoma brucei

2003 ◽  
Vol 2 (3) ◽  
pp. 542-551 ◽  
Author(s):  
Arthur Günzl ◽  
Thomas Bruderer ◽  
Gabriele Laufer ◽  
Bernd Schimanski ◽  
Lan-Chun Tu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Protein C ◽  
Gene Promoter ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Pol I

ABSTRACT In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to α-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, αβ-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

scholarly journals Functional Characterization of a Trypanosoma brucei TATA-Binding Protein-Related Factor Points to a Universal Regulator of Transcription in Trypanosomes

2004 ◽  
Vol 24 (21) ◽  
pp. 9610-9618 ◽  
Author(s):  
Jia-peng Ruan ◽  
George K. Arhin ◽  
Elisabetta Ullu ◽  
Christian Tschudi
Keyword(s):  
Trypanosoma Brucei ◽  
Binding Protein ◽  
Functional Characterization ◽  
Tata Binding Protein ◽  
Related Factor ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Pol I ◽  
Rna Genes

ABSTRACT Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3′ portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.

scholarly journals An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
James Budzak ◽  
Robert Jones ◽  
Christian Tschudi ◽  
Nikolay G. Kolev ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase I ◽  
Nuclear Bodies ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Cajal Bodies ◽  
African Trypanosomes ◽  
Spliced Leader ◽  
Pol I ◽  
Expression Site ◽  
Polymerase I

AbstractA Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated.

scholarly journals Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes

2009 ◽  
Vol 9 (1) ◽  
pp. 136-147 ◽  
Author(s):  
Tara M. Stanne ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Histone H3 ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Micrococcal Nuclease ◽  
Pcr Analysis ◽  
Marker Genes ◽  
African Trypanosomes ◽  
Pol I

ABSTRACT African trypanosomes regulate transcription differently from other eukaryotes. Most of the trypanosome genome is constitutively transcribed by RNA polymerase II (Pol II) as large polycistronic transcription units while the genes encoding the major surface proteins are transcribed by RNA polymerase I (Pol I). In bloodstream form Trypanosoma brucei, the gene encoding the variant surface glycoprotein (VSG) coat is expressed in a monoallelic fashion from one of about 15 VSG bloodstream form expression sites (BESs). Little is known about the chromatin structure of the trypanosome genome, and the chromatin state of active versus silent VSG BESs remains controversial. Here, we determined histone H3 occupancy within the genome of T. brucei, focusing on active versus silent VSG BESs in the bloodstream form. We found that histone H3 was most enriched in the nontranscribed 50-bp and 177-bp repeats and relatively depleted in Pol I, II, and III transcription units, with particular depletion over promoter regions. Using two isogenic T. brucei lines containing marker genes in different VSG BESs, we determined that histone H3 is 11- to 40-fold depleted from active VSG BESs compared with silent VSG BESs. Quantitative PCR analysis of fractionated micrococcal nuclease-digested chromatin revealed that the active VSG BES is depleted of nucleosomes. Therefore, in contrast to earlier views, nucleosome positioning appears to be involved in the monoalleleic control of VSG BESs in T. brucei. This may provide a level of epigenetic regulation enabling bloodstream form trypanosomes to efficiently pass on the transcriptional state of active and silent BESs to daughter cells.

scholarly journals Cell Biology of the Trypanosome Genome

2010 ◽  
Vol 74 (4) ◽  
pp. 552-569 ◽  
Author(s):  
Jan-Peter Daniels ◽  
Keith Gull ◽  
Bill Wickstead
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Nuclear Organization ◽  
Nuclear Architecture ◽  
Gene Clusters ◽  
Model Organisms ◽  
Protein Coding ◽  
African Trypanosomes ◽  
Protein Coding Genes ◽  
A Genome

SUMMARY Trypanosomes are a group of protozoan eukaryotes, many of which are major parasites of humans and livestock. The genomes of trypanosomes and their modes of gene expression differ in several important aspects from those of other eukaryotic model organisms. Protein-coding genes are organized in large directional gene clusters on a genome-wide scale, and their polycistronic transcription is not generally regulated at initiation. Transcripts from these polycistrons are processed by global trans-splicing of pre-mRNA. Furthermore, in African trypanosomes, some protein-coding genes are transcribed by a multifunctional RNA polymerase I from a specialized extranucleolar compartment. The primary DNA sequence of the trypanosome genomes and their cellular organization have usually been treated as separate entities. However, it is becoming increasingly clear that in order to understand how a genome functions in a living cell, we will need to unravel how the one-dimensional genomic sequence and its trans-acting factors are arranged in the three-dimensional space of the eukaryotic nucleus. Understanding this cell biology of the genome will be crucial if we are to elucidate the genetic control mechanisms of parasitism. Here, we integrate the concepts of nuclear architecture, deduced largely from studies of yeast and mammalian nuclei, with recent developments in our knowledge of the trypanosome genome, gene expression, and nuclear organization. We also compare this nuclear organization to those in other systems in order to shed light on the evolution of nuclear architecture in eukaryotes.

scholarly journals The holobiont transcriptome of teneral tsetse fly species of varying vector competence

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Miguel Medina Munoz ◽  
Caitlyn Brenner ◽  
Dylan Richmond ◽  
Noah Spencer ◽  
Rita V. M. Rio
Keyword(s):  
Control Strategies ◽  
Vector Competence ◽  
Tsetse Fly ◽  
Tsetse Flies ◽  
Protein Coding ◽  
African Trypanosomes ◽  
Animal African Trypanosomiasis ◽  
Protein Coding Genes ◽  
Sodalis Glossinidius ◽  
Immunological Protection

Abstract Background Tsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species. Results An active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, ‘translation, ribosomal structure and biogenesis’ followed by ‘coenzyme transport and metabolism’ were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways. Conclusions These transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

scholarly journals The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters.

1992 ◽  
Vol 12 (6) ◽  
pp. 2644-2652 ◽  
Author(s):  
S D Brown ◽  
J Huang ◽  
L H Van der Ploeg
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Pol I ◽  
Pol Iii ◽  
Repetitive Protein

All eukaryotic protein-coding genes are believed to be transcribed by RNA polymerase (Pol) II. An exception may exist in the protozoan parasite Trypanosoma brucei, in which the genes encoding the variant surface glycoprotein (VSG) and procyclic acidic repetitive protein (PARP) are transcribed by an RNA polymerase that is resistant to the Pol II inhibitor alpha-amanitin. The PARP and VSG genes were proposed to be transcribed by Pol I (C. Shea, M. G.-S. Lee, and L. H. T. Van der Ploeg, Cell 50:603-612, 1987; G. Rudenko, M. G.-S. Lee, and L. H. T. Van der Ploeg, Nucleic Acids Res. 20:303-306, 1992), a suggestion that has been substantiated by the finding that trypanosomes can transcribe protein-coding genes by Pol I (G. Rudenko, H.-M. Chung, V. P. Pham, and L. H. T. Van der Ploeg, EMBO J. 10:3387-3397, 1991). We analyzed the sequence elements of the PARP promoter by linker scanning mutagenesis and compared the PARP promoter with Pol I, Pol II, and Pol III promoters. The PARP promoter appeared to be of limited complexity and contained at least two critical regions. The first was located adjacent to the transcription initiation site (nucleotides [nt] -69 to +12) and contained three discrete domains in which linker scanning mutants affected the transcriptional efficiency: at nt -69 to -56, -37 to -11, and -11 to +12. The second region was located between nt -140 and -131, and a third region may be located between nt -228 and -205. The nucleotide sequences of these elements, and their relative positioning with respect to the transcription initiation site did not resemble those of either Pol II or Pol III promoter elements, but rather reflected the organization of Pol I promoters in (i) similarity in the positioning of essential domains in the PARP promoter and Pol I promoter, (ii) strong sequence homology between the PARP core promoter element (nt -37 to -11) and identically positioned nucleotide sequences in the trypanosome rRNA and VSG gene promoters, and (iii) moderate effects on promoter activity of mutations around the transcription initiation site.

scholarly journals LncRNAs Are Differentially Expressed between Wildtype and Cell Line Strains of African Trypanosomes

2022 ◽  
Vol 8 (1) ◽  
pp. 7
Author(s):  
Hyung Chul Kim ◽  
Emmitt R. Jolly
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Cell Lines ◽  
Differentially Expressed ◽  
Protein Coding ◽  
African Trypanosomes ◽  
Transcriptomic Data ◽  
Protein Coding Genes ◽  
Parasitic Protist ◽  
Non Coding Rnas

Trypanosoma brucei is a parasitic protist that causes African sleeping sickness. The establishment of T. brucei cell lines has provided a significant advantage for the majority of T. brucei research. However, these cell lines were isolated and maintained in culture for decades, occasionally accumulating changes in gene expression. Since trypanosome strains have been maintained in culture for decades, it is possible that difference may have accumulated in fast-evolving non-coding RNAs between trypanosomes from the wild and those maintained extensively in cultures. To address this, we compared the lncRNA expression profile of trypanosomes maintained as cultured cell lines (CL) to those extracted from human patients, wildtype (WT). We identified lncRNAs from CL and WT from available transcriptomic data and demonstrate that CL and WT have unique sets of lncRNAs expressed. We further demonstrate that the unique and shared lncRNAs are differentially expressed between CL and WT parasites, and that these lncRNAs are more evenly up-regulated and down-regulated than protein-coding genes. We validated the expression of these lncRNAs using qPCR. Taken together, this study demonstrates that lncRNAs are differentially expressed between cell lines and wildtype T. brucei and provides evidence for potential evolution of lncRNAs, specifically in T. brucei maintained in culture.

scholarly journals VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes

2016 ◽  
Vol 113 (26) ◽  
pp. 7225-7230 ◽  
Author(s):  
Lucy Glover ◽  
Sebastian Hutchinson ◽  
Sam Alsford ◽  
David Horn
Keyword(s):  
Antigenic Variation ◽  
Negative Regulation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Allelic Exclusion ◽  
Positive Regulation ◽  
African Trypanosomes ◽  
Pol I ◽  
Winner Takes All ◽  
Polymerase I

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I–transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I–transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I–transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a “winner-takes-all” mechanism of allelic exclusion.

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