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

动质体寄生虫是引起包括恰加斯病、利什曼病和非洲锥虫病在内的几种重要热带疾病的主要病原体。锥虫寄生虫在撒哈拉以南非洲的人类（昏睡病）和牛（Nagana）中引起致命疾病，造成毁灭性的社会和经济后果。当锥虫在宿主血流中繁殖时（细长型），一部分分化成静止的短粗型。在舌蝇的取食过程中，它们会存活下来，并在舌蝇的中肠中繁殖，然后在唾液腺中迁移和成熟，最后在再次取食时感染新的宿主。锥虫的细胞核中含有大量的惰性染色质，在细胞学上看起来很致密，在大多数真核生物中，这种染色质被称为异染色质。锥虫异染色质的一种形式介导了逃避宿主免疫系统所需的可变表面基因（VSG）的许多未表达拷贝的抑制。然而，锥虫异染色质及其对发育和VSG基因调控、转座因子控制、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。这部分是由于锥虫及其组蛋白与主要真核生物范式的极端进化分歧。组蛋白经历过多的翻译后修饰（PTM;乙酰化、甲基化等），特别是在赖氨酸残基上，在从每个核小体突出的非结构化N-末端尾部内。总的来说，这种修饰被称为“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压实和进入封闭的DNA以进行转录、复制、修复和重组过程。在大多数常规的真核生物中，组蛋白H3（K3 K9，H3 K27）的赖氨酸9或27的甲基化形成抑制性异染色质，而H3 K4和H3 K36甲基化辅助转录。组蛋白赖氨酸乙酰化也促进转录。因此，存在于构成每个核小体的八个组蛋白亚基上的PTM的模式监督来自封闭的DNA的信息的释放。在动质体如布氏锥虫中，保守的核心组蛋白与明显的真核生物标准特别不同;在主流真核生物中通常受到PTM的等同保守残基要么不存在（例如H3 K9），要么周围环境如此不同，以至于比对是暂时的。因此，对锥虫中特定组蛋白PTM或它们吸引的蛋白质的功能进行了很少的研究，因为由针对组蛋白PTM产生的商业抗体识别的表位，并且在大多数真核生物中保守，不与这些高度不同的锥虫组蛋白交叉反应。实施尖端的质谱方法，以确定和定量染色质相关蛋白和组蛋白PTM在三个易处理的发展形式的T。brucei（血流：纤细和短粗;昆虫：原轮）。通过向导RNA将CRISPR/Cas9-GFP靶向几个异染色质区域，并利用这些区域通过亲和选择富集特定基因座。定量富含重复元件异染色质和沉默VSG基因染色质的蛋白质和组蛋白PTM.描述异染色质功能和布氏锥虫分化和发育中鉴定的特定PTM和蛋白质的作用。

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.