Transcriptional control of telomeric expression sites and antigenic variation

对布氏锥虫原虫进行变异表面糖蛋白(VSG)基因的等位基因排除是至关重要的。通过抗原变异逃避宿主免疫反应。布氏毛滴虫每次只表达其2,000个VSG基因中的一个，该基因仅在20个端粒表达位点(Ess)中的一个处转录，并周期性地切换VSG基因的表达以逃避宿主抗体的识别。这种VSG等位基因排斥机制保证了只有一种VSG蛋白在布氏毛滴虫表面表达，抗原变异是通过端粒Ess之间的转录切换或VSG基因重组发生的。控制VSG等位基因排斥和VSG基因表达周期性转换的分子机制尚不清楚。我的长期目标是了解控制抗原变异的分子基础，重点是控制等位基因排斥、端粒沉默和VSG基因切换的表观遗传学和信号机制。 我们以前发现，VSG等位基因排斥和转换的控制涉及一个多蛋白复合体，并受磷酸肌醇系统的调节。这种蛋白质复合体与端粒ES染色质结合，包括在DNA结合、RNA加工、核层以及蛋白激酶和磷酸酶中发挥作用的蛋白质。我们将研究这种端粒调节复合体的蛋白质组成及其在VSG等位基因排除中的功能作用。我们将重点介绍磷脂酰肌醇在该蛋白复合体沉默活性中的表观遗传学作用，以及该蛋白复合体在ES染色质组织和VSG转录中的功能。具体地说，我们将(目标1)使用化学交联和质谱学来绘制端粒调节蛋白复合体的组成图，并确定该组成在VSG基因的沉默和转录过程中如何变化。此外(目标2)，利用CRISPR-Cas9突变和生化分析，我们将确定磷酸肌醇调节系统如何控制VSG基因的沉默。最后(目标3)，我们将确定该蛋白复合体如何影响端粒ES染色质结构组织，以及ES染色质在VSG基因沉默和转录过程中的变化。 这些研究将促进我们对布氏毛滴虫和其他生物，如疟原虫、贾第鞭毛虫等控制等位基因排斥和抗原变异的基本分子过程的理解。这也可能促进我们对其他系统中基因的等位基因排斥的了解，例如哺乳动物的嗅觉神经元受体。我的研究项目还将通过对学生进行跨学科、技术和创新研究的培训，使经济和社会部门受益。

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.