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Nuclear Architecture Underlying Epigenetic Regulation of Antigenic Variation in Trypanosoma brucei
Summary: Miguel Navarro is examining the way parasites such as Trypanosoma brucei, which causes African sleeping sickness, elude the host’s immune response by changing their protein coats. His research uses molecular and cell biology tools, including nuclear architecture analysis through three-dimensional microscopy, proteomics, and functional complementation of promoter activity.
Trypanosoma brucei is the extracellular protozoan parasite responsible for African trypanosomiasis, known as sleeping sickness in humans. The infective or bloodstream form multiplies in the vascular system of mammals. The entire surface of the bloodstream form is covered with a dense coat consisting mainly of one type of a variant surface glycoprotein (VSG). By changing its surface VSG type, the parasite is able to elude host immune antibody responses; such antigenic variation ensures a persistent infection. The expressed VSG gene is always located at the end of one of 20 telomeric loci, known as expression sites (ES). However, only one ES is transcribed at a time, so that each trypanosome displays a single VSG type on its surface—a genuine example of monoallelic expression from a multigene family. Once active, this ES is maintained over many generations. In addition to DNA recombination involving replacement of VSG genes in an active ES, antigenic variation can occur by transcriptional activation or inactivation of ESs, which is called in situ switching. In both cases, it is essential to achieve transcription of only one ES to display a single VSG type at any time. This raises the question of how a cell accomplishes the expression of a single VSG-ES from 20 loci that share homologous sequences in the promoter region.
Previous work has shown that ES in situ switching occurs epigenetically. One model proposed to explain monoallelic expression in T. brucei is nuclear compartmentalization of the active ES. RNA polymerase I (pol I), which is highly compartmentalized in the nucleolus of eukaryotic cells, mediates transcription of ESs. In T. brucei, pol I transcribes not only ribosomal loci but also those encoding the two main surface proteins characteristic of each developmental stage: (1) Procyclins, the surface glycoprotein gene family of the procyclin form, with tandem genes organized in two diploid loci that are constitutively transcribed and (2) the VSG-ES, which shows monoallelic exclusion, with the ability to switch transcription between ESs.
To investigate whether VSG-ES regulation is mediated by nuclear compartmentalization, we developed antibodies against the large subunit of pol I; this allowed us to identify an extranucleolar body containing pol I. We found that the body, named ES body (ESB), was associated with the active VSG-ES chromosomal site that we had tagged with green fluorescent protein (GFP). Furthermore, the ESB remained stable upon treatment with DNase and was duplicated along with the active ES, suggesting that it has a coherent structure. We proposed a model whereby ESB-dependent ES recruitment leads to the activation of a single ES, whereas inactive ESs are excluded from this structure by the presence of the active one. We recently showed that the Procyclin family of surface protein genes localizes to the nucleolar periphery and is constitutively transcribed at a similar level for all allelic variants. In contrast, we found that the monoallelically expressed VSG-ES promoter segregated to the ESB, which serves as a unique recruitment transcription site. The architecture of the ESB may define the singularity of the active ES and enable a high level of both transcription and RNA processing by efficiently recruiting transcriptional and RNA-processing machinery.
To address the questions that arise from this model, we are focusing our research on the nuclear architecture of this unique transcription site. The ESB represents a specialized transcription complex that, in addition to the pol I basal machinery, may contain transcriptional activators specific to the bloodstream (and/or modified common components). Isolating transcription factors of the ESB will involve the use of two complementary approaches: nuclear architectural and proteomic. We will combine them with functional studies of ES promoter activity.
The methodology used for our research is based on the excellent molecular tools available in T. brucei, which allow us to carry out complex reverse genetic experiments. Stable transformants occur by homologous recombination; in addition, RNA interference has been developed in trypanosomes, providing rapid generation of phenotypes produced by ablation of a particular mRNA in a tetracycline-inducible manner. We recently adapted in vivo GFP tagging of chromosomes in trypanosomes for nuclear positional analysis of a particular position in a chromosome, based on the GFP-LacI system. The expression of GFP-LacI, inducible by tetracycline, has allowed us to localize a particular DNA sequence by GFP-LacI binding to chromosomally inserted Lac operator repeats. The technology necessary to localize nuclear transcriptional activity in situ, by labeling nascent RNA with bromouridine triphosphate, is also available.
During its life cycle, the parasite alternates between its mammalian host and the tsetse fly vector. In the parasite's procyclic form (the midgut insect stage of the parasite), developmentally regulated chromatin remodeling of all ESs ensures that no VSG is expressed and that the invariant glycoprotein procyclin covers the surface. Our recent analyses by three-dimensional microscopy showed that, upon differentiation, the active VSG-ES promoter repositions to the nuclear envelope, in contrast to inactive ESs. At the same time, extranucleolar pol I ESB is no longer detected. These data suggest that, during the initial stages of differentiation to the procyclic form, the promoter region of the active VSG-ES telomere is selectively subjected to a rapid, nuclear position-dependent silencing mechanism.
We will conduct a functional analysis of candidate genes involved in VSG-ES regulation with regard to their specific effects on either the ESB or the ES promoter activity in vivo. We will examine factors associating with the ESB by immunocytochemistry and coimmunoprecipitation experiments together with anti–pol I antibodies. We will also study the functional role of candidates in vivo by RNA interference experiments in bloodstream-form cell lines containing a reporter gene downstream of the ES promoter.
Based on current data, we favor a model that suggests two mechanisms for VSG-ES regulation, both influenced by nuclear position. The first is recruitment-dependent activation of a single ES by the ESB, which allows monoallelic expression and transcriptional switching by ES replacement with a transcriptionally competent, inactive ES. Later the ES promoter repositions to the nuclear envelope; this repositioning is followed by chromatin condensation during development to the insect stage and during which no VSG is expressed.
The combination of molecular and cell biology approaches in vivo has given us new insights into epigenetic regulation of gene expression in this early branched eukaryote and promises to reveal novel findings. We will evaluate the relevance of identified genes using molecular and cellular tools together with the transgenic cell lines available to our group. In this way, we hope to identify the key players and contribute to the basic understanding of developmentally regulated gene expression and antigenic variation in this parasite.
Last updated October 2008
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