Transcriptional control of telomeric expression sites and antigenic variation

变异表面糖蛋白（VSG）基因的等位基因排斥对于原生动物寄生虫布氏锥虫是必需的。通过抗原变异逃避宿主免疫反应。t.布氏杆菌一次仅表达其约2，000个VSG基因中的一个，其仅在20个端粒表达位点（ES）中的一个处转录，并且其周期性地切换VSG基因表达以逃避宿主抗体识别。这种VSG等位基因排斥机制确保T细胞上仅表达一种VSG蛋白。布氏杆菌表面，并且通过端粒ES之间的转录转换或通过VSG基因重组发生抗原性变异。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知之甚少。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基因转换的表观遗传和信号传导机制。 我们以前发现，VSG等位基因排斥和开关的控制涉及一个多蛋白复合物，并通过磷酸肌醇系统调节。这种蛋白质复合物与端粒ES染色质相关，包括在DNA结合、RNA加工、核纤层、蛋白激酶和磷酸酶中起作用的蛋白质。我们将研究这种端粒调控复合物的蛋白质组成及其在VSG等位基因排斥中的功能作用。我们将集中在这种蛋白质复合物的沉默活性的磷酸肌醇的表观遗传作用，和ES染色质组织和VSG转录的蛋白质复合物的功能。具体而言，我们将（目标1）使用化学交联和质谱来绘制端粒调节蛋白复合物的组成，并确定该组成在VSG基因沉默和转录过程中如何变化。此外（目的2），使用CRISPR-Cas9诱变和生物化学测定，我们将确定磷酸肌醇调控系统如何控制VSG基因的沉默。最后（目标3），我们将确定这种蛋白质复合物如何影响端粒ES染色质结构组织，以及ES染色质如何在VSG基因沉默和转录过程中发生变化。 这些研究将促进我们对T细胞中控制等位基因排斥和抗原变异的基本分子过程的理解。布鲁氏菌和其它生物，例如，疟原虫属，它也可能推进我们的知识等位基因排斥基因在其他系统，如嗅觉神经元受体在哺乳动物。我的研究计划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.