ReviewControl of VSG gene expression sites
Introduction
Trypanosoma brucei is covered by a dense surface coat consisting of a single glycoprotein, the Variant Surface Glycoprotein (VSG) [1]. VSGs are translated from mRNAs transcribed from VSG genes located in telomeric expression sites. There are two types of expression sites (ESs), schematically depicted in Fig. 1, metacyclic ESs (M-ESs) and bloodstream form ESs (B-ESs). The M-ESs are used in metacyclic trypanosomes in the salivary gland of the tsetse fly and in the first few days after the tsetse fly introduces the trypanosomes with its saliva in a mammalian host. Then the B-ESs take over and they are used throughout a chronic infection.
Trypanosomes can change the VSG gene transcribed in a B-ES in two ways.
By replacing the VSG gene in an active expression site by a different one. This is quantitatively by far the most important source of VSG antigenic variation and it requires a DNA rearrangement.
By switching expression sites. This involves switching off the active site and activating an earlier silent site. As there are only about 20 B-ESs per trypanosome nucleus, the switching of ESs makes a very modest contribution to antigenic variation of the VSG coat. It is, therefore, likely that multiple B-ESs exist to allow expression of different sets of the ESAGs present in each ES [2], [3]. (This point will be discussed by Pays et al. in an upcoming review.)
How trypanosomes switch between expression sites and how they keep silent sites silent (allelic exclusion) is the subject of this review.
Section snippets
Control of metacyclic expression sites
In preparation for their entry into the mammalian host, trypanosomes that have reached the salivary gland of the tsetse fly acquire a VSG coat. The VSG mRNAs used for this coat are transcribed from metacyclic expression sites, M-ESs, shown in Fig. 1. M-ESs probably arose from B-ESs in evolution [4]. They lack functional ESAGs and the promoter is directly upstream of the VSG gene. The VSG genes in these sites are rarely replaced and antigenic variation in the metacyclic phase of the life cycle
Control of bloodstream expression sites: old facts and misinterpretations
Two types of models have been considered to explain control of bloodstream expression sites, cross-talk and non-cross talk models. In the cross-talk models, the activation of a new ES-A and the inactivation of the old one B are coupled events. In the non-cross-talk model, the two events are unrelated and switching from A to B is helped by selection. If site A is switched off before B is switched on, the trypanosome becomes coatless and dies; if site B is switched on without switching off A, the
Control of bloodstream expression sites; recent developments
Two recent developments have changed thinking on expression site control. First, it has become clear that there is cross-talk between ESs, and that activation of a new ES is coupled to inactivation of the earlier active site. Secondly, it has been shown that silent sites are not completely silent and that control of expression sites is at least in part at the level of transcription attenuation. The data are complex and not entirely unambiguous, and we shall present them here in some detail.
The B-ES promoter
The minimal B-ES promoter still maximally active in transient assays is remarkably small, only about 70 bp [42], [43]. The promoter sequences required for regulated expression in vivo have been analyzed by promoter remodeling in intact trypanosomes [30], [44], [45]. Rudenko et al. [30] showed that the ES core promoter itself is dispensable and that a B-ES in which this promoter is replaced by a ribosomal DNA promoter can be maximally activated and also silenced and reactivated at a normal rate.
Chromatin structure of active and inactive B-ESs
The chromatin structure of active and inactive B-ESs has been analyzed by exposure to nucleases in isolated nuclei or permeabilized trypanosomes. The VSG gene in an active B-ES is slightly more sensitive to DNase I than the same gene in an inactive ES (Pays et al., 1981)
Much larger differences in sensitivity were observed when the VSG genes were exposed to single-strand DNA specific endonucleases [46]. The presence of single-stranded DNA regions within VSG gene chromatin was interpreted as an
Silencing of expression sites
Early studies on silencing of B-ESs suggested that shut-off is complete. No mRNA corresponding to VSG genes of silent ESs was detected and this has been confirmed by recent experiments using more sensitive techniques, such as RT-PCR [18], [47]. These early studies appeared to be substantiated by the analysis of silenced ESs tagged with drug resistance genes or a luciferase gene inserted just downstream of the ES promoter. Silencing of the ES reduced the transcript level of these genes more than
How does it work?
We do not know yet how the control of trypanosome ESs works, but it may be useful to briefly summarize the main elements that should find a place in any future model. The requirements for maximal transcription of a B-ES and for the ability to switch are minimal, only a 70 bp core promoter will do as long as this is in the context of an ES and sufficiently distant from the upstream 50-bp repeats. Even the 70-bp core sequence is not essential, as it can be replaced by a ribosomal DNA promoter
Acknowledgements
We thank Dr G. Rudenko (Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, UK) and Dr R. Mussmann for helpful comments on the manuscript. The experimental work on trypanosomes in our lab is supported in part by grants from The Netherlands Foundation for Chemical Research (SON), with financial support of The Netherlands Organization for Scientific Research (NWO). S. Ulbert. is supported by a grant from The Boehringer Ingelheim Fonds.
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