The role of nuclear architecture in transcriptional regulation in the African Trypanosome, Trypanosoma brucei

布氏锥虫（T. brucei）是一种单细胞原生动物寄生虫，可在包括人类和牲畜在内的几种哺乳动物宿主中引起致命感染（Matthews, 2005）。在哺乳动物宿主体内，布鲁氏杆菌驻留在细胞外的血液中，在那里它不断暴露于免疫系统。为了避免免疫系统的清除，布鲁氏杆菌进化出了一种优雅的蛋白质外壳抗原变异系统——变异表面糖蛋白（VSG） （Pays, 2004）。基因组中有超过1000个VSG基因和假基因，其中大多数位于亚端粒阵列中。对于抗原变异过程至关重要的是，尽管在锥虫基因组中有大量的变异，但在任何时候只有一个VSG被表达。在锥虫的血流中，单个活性VSG总是从多个端粒表达位点中的一个表达（ESs）， （Taylor, 2006）。布鲁氏体的一个不寻常的特征是VSG基因是由RNA聚合酶I （Pol I）转录的，聚合酶通常与核核生物核糖体RNA转录的转录相关（daniels, 2012）。活性VSG ES已被证明与核外含Pol体共定位，该体被称为ES体（ESB） （Navarro和Gull， 2001）。有证据表明，端粒以及活性和非活性ESs分布在整个细胞核中（Horn, 2014）。这是一个不寻常的特征，在许多其他真核生物中，端粒通常被隔离在核外围。端粒和VSG ESs在布鲁氏T.细胞核内的定位非常有趣，因为基因在细胞核内的位置已被证明是其他生物调节的一个重要方面（Lemaitre, 2015）。尽管布鲁氏体中VSG严格的单等位基因表达，但通过药物选择可以迫使两个VSG ESs同时活跃。这些所谓的“双表达者”有一层由两种VSG变体组成的蛋白质涂层，但这些细胞的产生频率非常低。至于是什么让这两种ESs在布鲁氏t细胞中同时活跃，我们还知之甚少。这些双表达细胞可以用来验证亚核定位参与VSG严格转录控制的假设，并且双表达细胞中的活性ESs可能在细胞内共享相同的亚核位置。VSG位点的三维核组织可以解释沉默的VSG是如何被抑制的。然而，VSG基因在细胞核内的排列必须是动态的，以允许VSG通过原位开关或重组发生开关。在这个项目中，将研究布鲁氏T.细胞系中细胞核内svsg表达位点的组织和动态，这些细胞系已经选择了2个活跃的VSG表达位点。这将有助于深入了解布鲁氏t核的动力学，以及寄生虫如何利用这一点来抑制沉默的VSG并使VSG转换发生。在这个项目的过程中将要解决的问题包括：双表达细胞是否有两个功能齐全的esb，或者两个活性ESs是否共享转录所需的因子？如果无法检测到两个ESB，是否有证据表明两个活动ESB在双表达器中物理接近，表明它们共享一个ESB？VSG的核结构和亚核定位是否在转录调控和VSG严格的单等位基因表达中发挥作用？ ES在细胞核内定位的动力学是什么？这如何影响转录调控？

Trypanosoma brucei (T. brucei) is a unicellular protozoan parasite that can cause fatal infections in severalmammalian hosts including humans and livestock (Matthews, 2005). In a mammalian host, T. brucei residesextracellularly in the bloodstream, where it is continuously exposed to the immune system. To evadeclearance by the immune system, T. brucei has evolved an elegant system of antigenic variation of itsprotein coat - variant surface glycoprotein (VSG) (Pays, 2004).There are over 1000 VSG genes and pseudogenes in the genome, with most being located within sub-telomeric arrays. Crucial to the process of antigenic variation, only one VSG is expressed at any one time,despite the large repertoire of variants available in the trypanosome genome. In bloodstream formtrypanosomes, the single active VSG is always expressed from one of multiple telomeric expression sites(ESs), (Taylor, 2006).An unusual feature of T. brucei is that VSG genes are transcribed by RNA Polymerase I (Pol I), apolymerase usually associated with transcribing ribosomal RNA transcription within the nucleolus ineukaryotes (Daneils, 2012). The active VSG ES has been shown to co-localise with an extra-nucleolar Pol Icontaining body, which has been termed the ES body (ESB) (Navarro and Gull, 2001). There is evidencethat telomeres, and thereby active and inactive ESs, are distributed throughout the nucleus (Horn, 2014).This is an unusual feature, and in many other eukaryotes telomeres are commonly sequestered at thenuclear periphery. The positioning of telomeres and VSG ESs within the nucleus of T. brucei is veryinteresting as the location of genes within the nucleus has been shown to be an important aspect ofregulation in other organisms (Lemaitre, 2015).Despite the strict monoallelic expression of VSG in T. brucei, it has been possible to force two VSG ESs tobe simultaneously active using drug selection. These so called 'double-expressors' have a protein coatmade up of two VSG variants, but these cells are generated at a very low frequency. Much remainsunknown about what allows these two ESs to be simultaneously active in T. brucei. These doubleexpressor cells can be used to test the hypothesis that sub-nuclear localisation is involved in the stringenttranscriptional control of VSG, and that active ESs in double-expressors may share the same sub-nuclearlocation within the cell.The 3D nuclear organisation of VSG loci could provide some explanation as to how silent VSGs arerepressed. However, VSG gene arrangement within the nucleus must be dynamic to allow VSG switchingto occur, either by in situ switching or recombination. During this project the organisation and dynamicsVSG expressions sites within the nucleus will be investigated in T. brucei cell lines which have beenselected for 2 active VSG expressions sites. This should give insight into the dynamics of the T. bruceinucleus, and how this is utilised by the parasite to repress silent VSGs and to enable VSG switching tooccur. Questions that will be addressed during the course of this project include:Do double-expressor cells have two fully functional ESBs, or do the two active ESs share factorsrequired for transcription?If two ESBs can not be detected, is there evidence for the physical proximity of the two active ESsin a double-expressor, suggesting they are sharing an ESB?Does the nuclear architecture and sub-nuclear localisation of the VSG ESs play a role intranscriptional regulation and the stringent mono-allelic expression of VSGWhat are the dynamics of ES positioning within the nucleus and how does this affect transcriptionalregulation?