Parasites employ sophisticated mechanisms to escape host defenses. Trypanosoma brucei is a unicellular parasite that, in its mammalian host, lives in the bloodstream and interstitial spaces, causing the fatal disease African sleeping sickness in humans. T. brucei evades its host's immune system by periodically and rapidly changing the coat of proteins displayed on its surface—the variant surface glycoproteins (VSGs)—through a process called antigenic variation (Figure 1). The main focus of my laboratory is directed at understanding the molecular mechanism(s) of antigenic variation.
Each parasite expresses only one type of VSG coat at a time. The T. brucei genome contains hundreds of VSG genes, meaning that the parasite has the potential to change the "look" it presents to the immune system again and again. However, if VSGs were expressed simultaneously, the mammalian host would be able to mount an effective immune response and wipe out the infection (Figure 2). Antigenic variation is possible because only one VSG is transcribed at a time. For a VSG to be transcribed, it must be positioned at a specialized, subtelomeric locus called the bloodstream expression site (BES). There are approximately 15 BESs, only one of which is transcriptionally active at a time. How does the parasite activate one BES, while keeping the remaining 14 silent? How is this information inherited when parasites divide into daughter cells? Identifying and deciphering the molecular mechanism(s) that allow for such monoallelic expression is not only one of the fundamental questions in the field of trypanosome biology, but also of malaria parasite P. falciparum biology (var genes) and of mammalian neurobiology (olfactory receptor genes).
There are two mechanisms by which T. brucei changes its VSG coat: by inserting a silent VSG gene from somewhere in the genome into the active BES or by transcriptionally silencing the active BES and activating a new BES, which contains a different VSG gene. The first mechanism, gene conversion by homologous recombination, appears to be the most common one. In both cases, switching needs to be tightly regulated: switching too rarely would allow the immune system to eliminate all trypanosomes and clear the infection; switching too frequently would quickly exhaust the supply of new VSGs.
The explosion of epigenetics studies has revealed that, in most eukaryotes, chromatin structure is dynamic and imposes profound effects on almost all DNA-related processes, especially transcription. Chromatin structure is tightly regulated through multiple mechanisms, including chromatin remodeling, histone eviction, and histone posttranslational modifications. In trypanosomes, several chromatin-modifying enzymes have been shown to be necessary for VSG silencing. My laboratory and others have recently shown that the chromatin of the actively transcribed BES shows a very peculiar chromatin structure: the active BES is essentially devoid of regularly spaced nucleosomes, suggesting that chromatin of active BES is more open than that of silent BESs. How are these different chromatin structures established and maintained? What are the players involved? Is open chromatin structure at the active BES a cause or consequence of transcription?
Transcription and Chromatin Structure
It is remarkable that the polymerase responsible for BES transcription is RNA polymerase I, which in other organisms is exclusively dedicated to transcribing ribosomal RNA genes (rRNA). In yeast and mammalian cells, actively transcribed rRNA genes have an open chromatin structure, which seems to briefly assume a closed structure only during DNA replication. Is the open chromatin structure of the active BES a consequence of high levels of transcription of this locus? We are developing reporter systems to conditionally initiate and terminate transcription at BESs. Our goal is to follow the chromatin dynamics as soon as transcription is halted or initiated. These studies will allow us to begin understanding the order of events in VSG switching; namely, if transcription determines chromatin structure or vice versa.
Players Involved in Chromatin Dynamics
How is monoallelic expression achieved? Expression of only one VSG at a time is crucial; otherwise, the parasite becomes more vulnerable to the host defenses. We know that chromatin is important for VSG monoallelic expression because lack of a histone methyltransferase, DOT1B, and a telomeric protein, RAP1, results in loss of VSG monoallelic expression. In order to identify new players, we are using a combination of genetic and biochemical approaches. First, we are using reverse genetics to study the role of putative chromatin-related genes in VSG gene regulation. Second, after recent technological advances, it is now possible to undertake forward genetic screens in T. brucei bloodstream forms, the stage that causes disease in humans. In a collaborative project, we will perform an RNA interference screen to identify genes involved in VSG expression. Finally, because as much as 30 percent of human genes are regulated by noncoding RNAs, we are also interested in exploring the role of noncoding RNAs in antigenic variation in trypanosomes. Technologies such as high-throughput sequencing of RNA or DNA will be used to achieve some of these goals.
Grants from the European Molecular Biology Organization, Marie Curie Action, the Foundation for Science and Technology, and the Calouste Gulbenkian Foundation provided partial support for these projects.
As of January 17, 2012