Molecular dynamics of genome and epigenome integrity in Trypanosoma brucei

项目总结 布鲁氏锥虫，锥虫病的病原体，威胁着6000万人和原因 撒哈拉以南非洲的经济负担。只有少数几种药物可用于治疗其感染，所有这些药物都是 副作用严重，难以管理。因此，本质的识别和表征 布氏毛滴虫独特的细胞过程是开发更好的抗寄生虫剂的关键 未来。DNA复制对细胞增殖和基因组完整性至关重要。复制启动和 伸长必须与核小体的拆解和组装很好地协调。重要的是，我们发现 同时缺失区域特异性染色质标记、组蛋白变体H3v和H4v，以及 动态体特异的DNA修饰，碱基J，会导致严重的生长缺陷。H3v∆H4v∆J∆突变体 表现出复制应激表型，包括核TbRPA1灶的形成(异常的迹象 复制应激导致的单链DNA暴露)，以及G2细胞和不完全 复制的DNA。因此，这些染色质标记对于正确的DNA复制是重要的。有可能是 这些染色质标记只在它们通常所在的位置影响DNA复制(局部效应)。它也是 染色质结构的变化可能会以线性方式影响遥远区域的DNA复制 (全局效果)。在这个项目中，我们将通过检查染色体上的DNA复制来检验这些假设 H3v∆H4v∆J∆突变体的内部区域(Aim 1)和端粒(Aim 2)。在目标1中，我们将研究原始发射。 目标1.1中MFA-SEQ和REPLICI-SEQ的配置文件和复制延伸(分叉迁移模式)，检查 通过确定TbORC1在AIM 1.2的起始处的结合来启动复制，确定染色质标记是否 突变导致AIM 1.3中的染色体脆性和/或AIM 1.5中的转录谱改变。我们还将 确定3D染色体组织，以获得对染色质如何标记的机械理解 在AIM 1.4中控制核空间内的DNA复制。这将有助于我们在以下方面建立逻辑联系 染色体内部区域的染色质结构、DNA复制和转录状态。布氏毛滴虫 端粒为研究染色质结构、复制和转录相互作用提供了特殊的益处， 因为布鲁氏毛滴虫的亚端粒要么在早期被高度转录和复制，要么被紧紧地抑制和复制。 复制的时间较晚。端粒的这些独特功能将帮助我们揭示更多关于端粒的机械细节 染色质结构、复制和转录之间的相互作用。我们将研究端粒/亚端粒 在AIM 2.1中使用2D凝胶分析和染色质纤维FISH进行复制。DNA断裂和重组将 也将在目标2.2中进行审查。从这个提案中，我们将为定义染色质如何标记奠定基础 协同工作，控制复制和转录。我们的研究将帮助我们通过 这种表观基因组和基因组之间的协同作用可以成功地引导到基因组稳定 以及细胞生存所需的基因多样化。

PROJECT SUMMARY Trypanosoma brucei, the causative pathogen of trypanosomiasis, threatens >60 million people and causes economic burdens in sub-Saharan Africa. Only a few drugs are available for treating its infection, all with severe side effects and are difficult to administer. Therefore, identification and characterization of essential cellular processes with unique features in T. brucei is essential for developing better anti-parasite agents in the future. DNA replication is essential for cell proliferation and genome integrity. Replication initiation and elongation must coordinate well with nucleosome disassembly and assembly. Importantly, we have discovered that simultaneous deletion of region-specific chromatin marks, histone variants H3v and H4v, and a Kinetoplastid-specific DNA modification, base J, results in severe growth defects. H3v∆ H4v∆ J∆ mutants exhibit replication stress phenotypes, including formation of nuclear TbRPA1 foci (an indicative of abnormal exposure of ssDNA resulting from replication stress), and accumulation of G2 cells and cells with incompletely replicated DNA. Therefore, these chromatin marks are important for proper DNA replication. It is possible that these chromatin marks influence DNA replication only at loci where they normally reside (local effect). It is also possible that changes in chromatin structure may affect DNA replication at regions far away on a linear scale (global effect). In this project, we will test these hypotheses by examining DNA replication at chromosome internal regions (Aim 1) and telomeres (Aim 2) in H3v∆ H4v∆ J∆ mutants. In Aim 1, we will examine origin firing profiles and replication elongation (fork migration pattern) by MFA-seq and Repli-seq in Aim 1.1, examine replication initiation by determining TbORC1 binding at origins in Aim 1.2, determine whether chromatin mark mutations cause chromosome fragility in Aim 1.3 and/or change transcription profile in Aim 1.5. We will also determine the 3D chromosome organization to obtain mechanistic understanding of how chromatin marks control DNA replication inside the nuclear space in Aim 1.4. This will help us to establish logical links between chromatin structure, DNA replication, and transcription status at the chromosome internal regions. T. brucei telomeres provide special benefits for studying chromatin structure, replication, and transcription interplay, because T. brucei subtelomeres are either `highly transcribed and replicated early' or `tightly repressed and replicated late'. These unique features at telomeres will help us unveil additional mechanistic details on the interaction among chromatin structure, replication, and transcription. We will study telomere/subtelomere replication using 2D gel analysis and Chromatin Fiber FISH in Aim 2.1. DNA breaks and recombination will also be examined in Aim 2.2. From this proposal, we will establish foundation to define how chromatin marks work together and control replication and transcription. Our studies will help us understand mechanisms by which this collaborative action between epigenome and genome can successfully channel into genome stability and gene diversification for cellular survival.