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 Nucleosomes Are Depleted at the VSG Expression Site Transcribed by RNA Polymerase I in African Trypanosomes

2009 ◽  
Vol 9 (1) ◽  
pp. 148-154 ◽  
Author(s):  
Luisa M. Figueiredo ◽  
George A. M. Cross
Keyword(s):  
Rna Polymerase ◽  
Antigenic Variation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Micrococcal Nuclease ◽  
Rrna Genes ◽  
Host Immune System ◽  
African Trypanosomes ◽  
Pol I ◽  
Polymerase I

ABSTRACT In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein VSG genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one VSG is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.

scholarly journals Single cell transcriptomic analysis of bloodstream form Trypanosoma brucei reconstructs cell cycle progression and differentiation via quorum sensing

2020 ◽  
Author(s):  
Emma Marie Briggs ◽  
Richard McCulloch ◽  
Keith Roland Matthews ◽  
Thomas Dan Otto
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Trypanosoma Brucei ◽  
Expression Patterns ◽  
Life Cycles ◽  
Bloodstream Form ◽  
Cycle Phase ◽  
Mammalian Host ◽  
Marker Genes ◽  
African Trypanosomes

The life cycles of African trypanosomes are dependent on several differentiation steps, where parasites transition between replicative and non-replicative forms specialised for infectivity and survival in mammal and tsetse fly hosts. Here, we use single cell transcriptomics (scRNA-seq) to dissect the asynchronous differentiation of replicative slender to transmissible stumpy bloodstream form Trypanosoma brucei. Using oligopeptide-induced differentiation, we accurately modelled stumpy development in vitro and captured the transcriptomes of 9,344 slender and stumpy stage parasites, as well as parasites transitioning between these extremes. Using this framework, we detail the relative order of biological events during development, profile dynamic gene expression patterns and identify putative novel regulators. Using marker genes to deduce the cell cycle phase of each parasite, we additionally map the cell cycle of proliferating parasites and position stumpy cell cycle exit at early G1, with subsequent progression to a distinct G0 state. We also explored the role of one gene, ZC3H20, with transient elevated expression at the key slender to stumpy transition point. By scRNA-seq analysis of ZC3H20 null parasites exposed to oligopeptides and mapping the resulting transcriptome to our atlas of differentiation, we identified the point of action for this key regulator. Using a developmental transition relevant for both virulence in the mammalian host and disease transmission, our data provide a paradigm for the temporal mapping of differentiation events and regulators in the trypanosome life cycle.

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 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 Quantification of RNA Polymerase I transcriptional attenuation at the active VSG expression site in Trypanosoma brucei

2021 ◽  
Author(s):  
Nadine Weisert ◽  
Klara Thein ◽  
Helena Reis ◽  
Christian J Janzen
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Transcriptional Elongation ◽  
Pol I ◽  
Expression Site ◽  
Polymerase I ◽  
Large Repertoire

The cell surface of the extracellular pathogen Trypanosoma brucei consists of a dense coat of variant surface glycoprotein (VSG), which enables the parasite to evade the immune system of the vertebrate host. Only one VSG gene from a large repertoire is expressed from a so-called bloodstream form expression site (BES) at a given timepoint. There are several BES in every parasite but only one is transcriptionally active. Other BES are silenced by transcriptional attenuation. Periodic activation of a previously-silenced BES results in differential VSG transcription and escape from the immune response. A process called antigenic variation. In contrast to gene transcription in other eukaryotes, the BES is transcribed by RNA polymerase I (Pol I). It was proposed that this highly-processive polymerase is needed to provide a sufficiently high transcription rate at the VSG gene. Surprisingly, we discovered a position-dependent Pol I activity and attenuation of transcriptional elongation also at the active BES. Transcription rates at the VSG gene appear to be comparable to Pol II-mediated transcription of house-keeping genes. Although these findings are in contradiction to the long-standing concept of continuously high transcription rates at the active BES in Trypanosoma brucei, they are complementary to recent groundbreaking findings about transcriptional regulation of VSG genes.

scholarly journals Essential Roles for GPI-anchored Proteins in African Trypanosomes Revealed Using Mutants Deficient in GPI8

2003 ◽  
Vol 14 (3) ◽  
pp. 1182-1194 ◽  
Author(s):  
Simon Lillico ◽  
Mark C. Field ◽  
Pat Blundell ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Trypanosoma Brucei ◽  
Cell Cycle Progression ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Surface Coat ◽  
Immune Challenge ◽  
Gene Encoding ◽  
Cycle Progression ◽  
African Trypanosomes

The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion ofGPI8 (to give Δgpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non–protein-linked GPI molecules, some of which are surface located. Procyclic Δgpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression.

scholarly journals In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

2006 ◽  
Vol 5 (8) ◽  
pp. 1276-1286 ◽  
Author(s):  
Sara D. Faulkner ◽  
Monika W. Oli ◽  
Rudo Kieft ◽  
Laura Cotlin ◽  
Justin Widener ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Trypanosoma Brucei ◽  
Density Lipoprotein ◽  
High Density ◽  
Surface Glycoprotein ◽  
Trypanosoma Brucei Brucei ◽  
African Trypanosomes ◽  
Expression Site ◽  
Human High Density Lipoprotein

ABSTRACT The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei

1992 ◽  
Vol 12 (3) ◽  
pp. 1218-1225
Author(s):  
P Paindavoine ◽  
S Rolin ◽  
S Van Assel ◽  
M Geuskens ◽  
J C Jauniaux ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trypanosoma Brucei ◽  
Membrane Fraction ◽  
Transcription Unit ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Yeast Cells ◽  
Variant Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Expression Site

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.

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