Transcriptional control of telomeric expression sites and antigenic variation

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Allelic exclusion of variant surface glycoprotein (VSG) genes is essential for the protozoa parasite Trypanosoma brucei spp. to evade the host immune response by antigenic variation. T. brucei expresses only one of its ~2,000 VSG genes at a time, which is exclusively transcribed at one of the 20 telomeric expression sites (ESs), and it periodically switches VSG gene expression to evade host antibody recognition. This VSG allelic exclusion mechanism ensures the expression of only one VSG protein on T. brucei surface, and the antigenic variation occurs by transcriptional switching between telomeric ESs or by VSG gene recombination. The molecular mechanisms that control VSG allelic exclusion and the periodic switching in VSG gene expression are poorly understood. My long-term goal is to understand the molecular basis underlying the control of antigenic variation with a focus on epigenetic and signalling mechanisms that control allelic exclusion, telomere silencing, and switching of VSG genes. We previously found that the control of VSG allelic exclusion and switching involves a multiprotein complex and is regulated by a phosphoinositol system. This protein complex associates with telomeric ES chromatin and includes proteins that function in DNA binding, RNA processing, the nuclear lamina, and protein kinases and phosphatases. We will study the protein composition of this telomere regulatory complex and its functional role in VSG allelic exclusion. We will focus on the epigenetic role of phosphoinositides in the silencing activity of this protein complex, and the protein complex function in ES chromatin organization and VSG transcription. Specifically, we will (aim 1) use chemical cross-linking and mass spectrometry to map the telomere regulatory protein complex composition and determine how this composition changes during silencing and transcription of VSG genes. Moreover (aim 2), using CRISPR-Cas9 mutagenesis and biochemical assays we will determine how the phosphoinositol regulatory system controls the silencing of VSG genes. Finally (aim 3), we will determine how this protein complex affects the telomeric ES chromatin structural organization, and how the ES chromatin changes during silencing and transcription of VSG genes. These studies will advance our understanding of the fundamental molecular processes that control allelic exclusion and antigenic variation in T. brucei and other organisms, e.g., Plasmodium spp., Giardia sp. It might also advance our knowledge of allelic exclusion of genes in other systems such as olfactory neuron receptors in mammals. My research program will also benefit economic and social sectors through the training of students in interdisciplinary, technological and innovative research.