Molecular dynamics of genome and epigenome integrity in Trypanosoma brucei

布氏锥虫基因组和表观基因组完整性的分子动力学

• 批准号: 9592250
• 负责人: Hee-Sook Kim
• 金额: $ 33.13万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9592250
• 关键词: Molecular; dynamics; genome; epigenome; integrity

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 these drugs have severe side effects and some are difficult to administer. Therefore, identification and characterization of essential cellular processes with unique features in T. brucei will be invaluable for developing better anti-parasite agents in the future. DNA replication is essential for cell proliferation and genome integrity. Replication initiation and elongation must be tightly regulated in accordance 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, are lethal with terminal phenotypes associated with replication defects, including accumulation of nuclear TbRPA1 foci, an indicative of abnormal exposure of ssDNA resulting from replication stress. Therefore, we hypothesize that dynamic interactions between replication and chromatin at specific loci are essential in maintaining genome and epigenome integrity. In this project, we will characterize the H3v∆ H4v∆ J∆ mutant in DNA replication at both chromosome internal regions (Aim 1) and telomere regions (Aim 2), focusing on changes in binding of TbORC1 with origins, origin firing choices, and fork migration. Our preliminary results suggest that H3v, H4v, and base J have roles in telomere maintenance. Thus, we will also examine whether deletion of these epigenetic marks disrupts telomere integrity (Aim 2). Transcription in T. brucei occurs polycistronically. Transcription start or termination sites (TSSs and TTSs) at boundaries of polycistronic transcription units are marked by specific nucleosome modifications and histone variants: H3K4me3, H4K10ac, H2Az, and H2Bv are at TSSs, H3v and H4v are at TTSs. Base J is located at both TSSs and TTSs. We have recently shown that simultaneous deletion of two chromatin marks, H3v and base J, disrupted transcription termination. Hence, T. brucei transcription relies greatly on chromatin structures. Furthermore, DNA replication is closely linked with transcription, because transcription and replication share their initiation sites. Depletion of TbMCM-BP, a replication protein, shares similar defects in replication and transcription termination as H3v∆ H4v∆ J∆ and H3v∆ J∆ mutant. Finally, in Aim 3, we will investigate whether the overall chromatin structure changes at both chromosome internal and telomere regions account for DNA replication and transcription defects in mutants lacking epigenetic marks or replication factors. This aim will reveal mechanistic relationship between DNA replication and transcription that is mediated by the same epigenetic factors. Our studies will reveal unique features in regulation of DNA replication and transcription termination in T. brucei, which will help develop better means for eventual eradication of T. brucei in the future.

项目概要 布氏锥虫是锥虫病的致病病原体，威胁着超过 6000 万人并导致 撒哈拉以南非洲地区的经济负担只有少数药物可用于治疗其感染。 一些药物具有严重的副作用，因此难以识别和服用。 T. brucei 中具有独特特征的基本细胞过程的表征对于以下方面将具有无价的价值： 未来开发更好的抗寄生虫药物对于细胞增殖和DNA复制至关重要。 基因组完整性。复制起始和延伸必须根据核小体进行严格调控。 重要的是，我们发现区域特定的同时删除。 染色质标记、组蛋白变体 H3v 和 H4v 以及动质体特异性 DNA 修饰（碱基 J） 与复制缺陷相关的终末表型致死，包括核 TbRPA1 的积累 病灶，表明复制应激导致 ssDNA 异常暴露。 特定位点的复制和染色质之间的动态相互作用对于 保持基因组和表观基因组的完整性 在这个项目中，我们将表征 H3vΔ H4vΔ JΔ 突变体。 染色体内部区域（目标 1）和端粒区域（目标 2）的 DNA 复制，重点关注 TbORC1 与起源、起源激发选择和分叉迁移的结合发生变化。 表明 H3v、H4v 和碱基 J 在端粒维持中发挥作用，因此，我们还将检查是否具有端粒维持作用。 删除这些表观遗传标记会破坏端粒完整性（目标 2）。 T. brucei 中的转录以多顺反子方式发生。 多顺反子转录单元的边界处有特定的核小体修饰和组蛋白标记 变体：H3K4me3、H4K10ac、H2Az 和 H2Bv 位于 TSS，H3v 和 H4v 位于 TTS 处。 我们最近证明了两个染色质标记 H3v 和 TTS 的同时删除。 碱基 J，转录终止被破坏，因此，T. brucei 转录很大程度上依赖于染色质。 此外，DNA 复制与转录密切相关，因为转录和复制。 复制蛋白 TbMCM-BP 的缺失具有相似的缺陷。 作为 H3vΔ H4vΔ JΔ 和 H3vΔ JΔ 突变体的复制和转录终止 最后，在目标 3 中，我们将。 研究染色体内部和端粒区域的整体染色质结构是否发生变化 解释缺乏表观遗传标记或复制因子的突变体中的 DNA 复制和转录缺陷。 这一目标将揭示DNA复制和转录之间由DNA复制和转录介导的机制关系。 相同的表观遗传因素。 我们的研究将揭示 T. 中 DNA 复制和转录终止调节的独特特征。 布氏锥虫，这将有助于开发更好的方法，在未来最终根除布氏锥虫。