Chromatin associated proteins and histone post-translational modification dynamics in the development and immune evasion of the sleeping sickness para

动体寄生虫是查加斯病、利什曼病和非洲锥虫病等几种重要热带病的主要病原体。锥虫寄生虫在整个撒哈拉以南非洲造成人类(昏睡病)和牛(Nagana)的致命性疾病，造成毁灭性的社会和经济后果。随着锥虫在宿主血流中繁殖(细长形态)，一部分分化为静止的粗壮形态。随后，矮小的采采蝇通过摄食采采蝇存活下来，在唾液腺迁移和成熟之前，它们最初聚集在中肠(原环状)，最后当苍蝇再次取食时感染新的宿主。锥体的核含有大量的惰性染色质，在细胞学上看起来很密集，像大多数真核生物一样，被称为异染色质。锥体异染色质的一种形式介导了许多逃避宿主免疫系统所需的可变表面基因(VSG)的未表达拷贝的抑制。然而，对锥虫异染色质及其对发育和VSG基因调控、转座元件的控制、着丝粒重复序列功能和一般核结构的分子理解仍处于起步阶段。这在一定程度上是由于锥虫及其组蛋白与主要的真核生物范例在进化上的极端分歧。组蛋白受到过多的翻译后修饰(PTM；乙酰化、甲基化等)，特别是在每个核小体突出的非结构化N末端尾巴中的赖氨酸残基上。这些修饰统称为“表观遗传标记”，因为它们可以赋予独立于潜在DNA序列的特性。这些标记调节染色质的紧凑和对转录、复制、修复和重组过程中封闭的DNA的访问。在大多数传统的真核生物中，组蛋白H3(K3K9，H3K27)的赖氨酸9或27的甲基化形成抑制性异染色质，而H3K4和H3K36的甲基化有助于转录。组蛋白赖氨酸乙酰化也促进转录。因此，组成每个核小体的8个组蛋白亚基上存在的PTM模式监督所附DNA的信息释放。在动质体中，如布鲁氏锥虫，保守的核心组蛋白特别不同于明显的真核标准；在主流真核生物中，通常受到PTMS影响的等效保守残基要么缺失(例如H3K9)，要么周围环境如此明显，以至于比对是试探性的。因此，很少有人对锥虫体内特定的组蛋白PTM的功能或它们吸引的蛋白质进行研究，因为商业抗体识别的抗组蛋白PTM的表位在大多数真核生物中是保守的，不会与这些高度分化的锥体组蛋白发生交叉反应。采用尖端质谱学方法，鉴定和定量布氏毛滴虫三种易驯化发育形式(血液：细长和粗壮；昆虫：原环状)中的染色质相关蛋白和组蛋白PTM。通过引导RNA将CRISPR/Cas9-GFP靶向到几个异染色质区域，并利用这些RNA通过亲和选择来丰富特定的基因座。定量检测富含重复元件异染色质和沉默的VSG基因染色质的蛋白质和组蛋白PTM。鉴定特定的PTM和蛋白质在异染色质功能和布氏锥虫分化和发育中的作用。

The kinetoplastid parasites are major pathogens responsible for several important tropical diseases including Chagas disease, Leishmaniasis and African trypanosomiasis. Trypanosome parasites cause lethal disease in humans (sleeping sickness) and cattle (Nagana) across sub-Saharan Africa with devastating social and economic consequences. As trypanosomes multiply in the host bloodstream (slender forms) a proportion differentiate into quiescent stumpy forms. Stumpy forms subsequently survive uptake by feeding tsetse flies where they initially populate the mid-gut (procyclic forms) prior to migration and maturation in the salivary gland, finally infecting a new host when the fly feeds again.The nucleus of trypanosomes contains a large amount of inert chromatin that cytologically appears dense and, as in most eukaryotes, is referred to as heterochromatin. One form of trypanosome heterochromatin mediates the repression of the many unexpressed copies of Variable Surface Genes (VSG) required to evade host immune systems. However, a molecular understanding of trypanosome heterochromatin and its contribution to developmental and VSG gene regulation, the control of transposable elements, centromere repeat sequence function and general nuclear architecture remains in its infancy. This is partly due to the extreme evolutionary divergence of trypanosomes and their histone proteins from the main eukaryotic paradigm. Histones are subject to a plethora of post-translational modifications (PTMs; acetylation, methylation etc), particularly on the lysine residues, within the unstructured N-terminal tails that protrude from each nucleosome. Collectively such modifications are referred to as 'epigenetic marks' as they can confer properties independent from the underlying DNA sequences. Such marks regulate chromatin compaction and access to the enclosed DNA for the processes of transcription, replication, repair and recombination. In most conventional eukaryotes methylation of lysine 9 or 27 of histone H3 (K3K9, H3K27) forms repressive heterochromatin whereas H3K4 and H3K36 methylation assist transcription. Histone lysine acetylation also promotes transcription. Thus, the pattern of PTMs present on the eight histone subunits comprising each nucleosome supervise the release of information from the enclosed DNA. In kinetoplastids such as Trypanosoma brucei the conserved core histones are particularly divergent from the apparent eukaryotic norm; the equivalent conserved residues that are usually subject to PTMs in mainstream eukaryotes are either absent (e.g. H3K9) or the surrounding context is so distinct that alignments are tentative. Consequently few studies have been performed on the function of specific histone PTMs in trypanosomes, or the proteins that they attract, because the epitopes recognised by commercial antibodies raised against histone PTMs, and conserved in most eukaryotes, do not cross-react with these highly divergent trypanosome histones.Aims1. Implement cutting edge mass spectrometry methods to identify and quantify chromatin associated proteins and histone PTMs in the three tractable developmental forms of T. brucei (Bloodstream: slender and stumpy; Insect: procyclic).2. Target CRISPR/Cas9-GFP to several heterochromatin regions via guide RNAs and utilize these to enrich specific loci by affinity selection. Quantify proteins and histone PTMs enriched in repeat element heterochromatin and silent VSG gene chromatin.3. Characterize the role of specific PTMs and proteins identified in heterochromatin function and Trypanosoma brucei differentiation and development.