Mechanisms of RAP1 functions in monoallelic VSG expression in Trypanosoma brucei

项目总结 全球有800多万人感染了三种密切相关的动质体寄生虫： 布氏锥虫、克氏锥虫和利什曼原虫。其中，布氏毛滴虫导致致命的非洲人 威胁数百万人的锥虫病。可用的治疗方法很少，而且都有严重的副作用 效果。布鲁氏毛滴虫通过定期切换其主要表面抗原VSG来逃避宿主的免疫反应。 VSG是从亚端粒VSG表达位点(ES)以严格的单等位基因方式表达的，这也是 帮助协调寄生虫的增殖和发育。此外，许多微生物病原体经历了 抗原变异以单等位基因表达其主要表面抗原。例如，在疟原虫中 导致人类致命疟疾的恶性疟原虫，var基因的单等位基因表达(许多在 亚端粒区)是其抗原性变异所必需的。然而，基因表达的单等位基因 取得的成就还不完全清楚。我们已经证明，布鲁氏毛滴虫RAP1，一种重要的端粒蛋白，可以沉默 然而，许多问题仍然没有得到回答：RAP1是怎样的 被招募到端粒？如何防止RAP1沉默表达的VSG？在目标1中，我们将 确定新近鉴定的RAP1和RAP1的DNA结合活性与S的相互作用是否已知 端粒结合因子帮助将RAP1靶向端粒。在目标2中，我们将确定最近的 已知的RAP1与活性VSG RNA之间的相互作用有助于防止RAP1沉默活性 VSG。在RAP1缺失的细胞中，Terra(端粒重复序列的转录本)表达升高导致更多 DNA在亚端粒VSG位点断裂，这对细胞活力是有害的。因此，我们将研究如何 Rap1在AIM 3中调节Terra的表达。我们还发现Terra对完整的VSG很重要 并将在AIM 3中探讨其机制。最后，许多因素参与VSG ES的表达 监管。RAP1如何与其他ES调节子协调以实现单等位VSG表达 未知。我们发现RAP1与一个大的支架蛋白Not1相互作用，Not1的酵母和人类 同源基因在多个水平上参与基因表达调控。我们假设Not1在 RAP1和其他ES调节器之间的协调，这将在目标4中进行审查。我们拟议的研究 将揭示RAP1介导的VSG沉默和等位基因排除VSG表达的详细机制， 强调活动和静默VSG Ess之间的通信，目前研究较少。基座 根据我们的初步结果，我们希望了解RAP1在布鲁氏锥虫中所独有的功能，而不是在 哺乳动物宿主，这将有助于开发一种消除布氏毛滴虫和其他寄生虫的方法，采用 类似的抗原变异机制。由于RAP1是一种重要的端粒蛋白，从我们的 研究将很容易推断出密切相关的动质体寄生虫，并有助于根除它们。

Project summary More than eight million people worldwide are infected by three closely related Kinetoplastid parasites: Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. Among these, T. brucei causes fatal human African trypanosomiasis that threatens millions of people. Few treatments are available, and all have severe side effects. T. brucei evades its host's immune responses by regularly switching its major surface antigen, VSG. VSG is expressed from subtelomeric VSG expression sites (ESs) in a strict monoallelic manner, which also helps coordinate parasite proliferation and development. In addition, many microbial pathogens that undergo antigenic variation express their major surface antigens monoallelically. For example, in Plasmodium falciparum that causes deadly malaria in humans, monoallelic expression of var genes (many are at subtelomeric region) is essential for its antigenic variation. However, how monoallelic gene expression is achieved is not completely clear. We have shown that T. brucei RAP1, an essential telomere protein, silences VSGs in a telomere proximity-dependent manner, yet, many questions remain unanswered: How is RAP1 recruited to the telomere? How is RAP1 prevented from silencing the one expressed VSG? In Aim 1, we will determine whether the recently identified DNA binding activity of RAP1 and RAP1's interaction with known telomere binding factors help target RAP1 to the telomere. In Aim 2, we will determine whether the recently identified interaction between RAP1 and the active VSG RNA helps prevent RAP1 from silencing the active VSG. Elevated TERRA (the transcript of telomere repeats) expression in RAP1-depleted cells causes more DNA breaks at subtelomeric VSG loci, which is detrimental to cell viability. We will therefore examine how RAP1 regulates TERRA expression in Aim 3. We also found TERRA to be important for complete VSG silencing and will investigate its mechanisms in Aim 3. Finally, many factors participate in VSG ES expression regulation. How RAP1 coordinates with other ES regulators to achieve monoallelic VSG expression is unknown. We found that RAP1 interacts with a large scaffold protein, NOT1, whose yeast and human homologs are involved in gene expression regulation at multiple levels. We hypothesize that NOT1 mediates coordination between RAP1 and other ES regulators, which will be examined in Aim 4. Our proposed studies will reveal detailed mechanisms of RAP1-mediated VSG silencing and allelic exclusion VSG expression, with an emphasis on communications between active and silent VSG ESs, which is currently poorly studied. Based on our preliminary results, we expect to understand functions of RAP1 that are unique in T. brucei but not in mammalian hosts, which will help develop a means for eliminating T. brucei and other parasites employing similar antigenic variation mechanisms. As RAP1 is an essential telomere protein, knowledge gleaned from our studies will be easily extrapolated to closely related Kinetoplastid parasites and aid in their eradication.