Nuclear bodies play a crucial role in the compartmentalisation and regulation of gene expression. However, the importance of nuclear bodies in controlling gene expression in more diverse organisms is still largely unexplored. The early branching eukaryote Trypanosoma brucei has long been known to regulate expression of its Variant Surface Glycoprotein (VSG) genes using a unique nuclear body called the Expression Site Body (ESB). The ESB has been defined as an extra-nucleolar structure enriched in RNA polymerase I which is essential for high levels of VSG expression. Importantly, a single VSG gene is expressed at a time inside the ESB in a monoallelic manner from one of 15 VSG Expression Sites (ESs). How the ESB mechanistically controls VSG expression and monoallelic exclusion is not fully understood.
Here, I present the microscopic and functional analysis of the properties of nuclear bodies in T. brucei with a focus on the ESB. Using a “double expresser” T. brucei strain with two simultaneously active VSG-ESs, I show using DNA and RNA-FISH that these ESs dynamically share a single ESB. Unexpectedly, these two active ESs can only be simultaneously transcribed when located within this single ESB. Furthermore, double expresser cells are unable to stably produce more than one ESB, indicating that the ESB is a key limiting component in coordinating monoallelic exclusion.
In order to further understand the molecular components inside the ESB, the TrypTag database was mined to find potential nuclear body proteins. In doing so, I have discovered proteins located in three nuclear bodies; a NUFIP body, a Cajal body and a Spliced Leader Array Body (SLAB) that associate with the active ES in bloodstream form cells forming a “super-hub” for VSG expression. These separate nuclear bodies appear to be involved in the production of splicing components. Interestingly, I find that splicing is essential for transcription elongation at the active ES. These results suggest a model in which extraordinarily high levels of VSG mRNA production are facilitated by the association of a conglomerate of nuclear bodies with the active ES. The sequestration of these multiple nuclear bodies to the active ES may also serve as a mechanism to prevent their interaction and subsequent activation of silent ESs, thus facilitating monoallelic exclusion.