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.

项目摘要 布氏锥虫是锥虫病的病原体，威胁着超过6000万人， 撒哈拉以南非洲的经济负担。只有少数药物可用于治疗其感染。所有这些 药物具有严重的副作用，并且一些药物难以施用。因此，识别和 在T.布鲁塞将是无价的， 在未来开发出更好的抗寄生虫药物。DNA复制对于细胞增殖是必不可少的， 基因组完整性复制的起始和延伸必须严格按照核小体进行调控 拆卸和组装。重要的是，我们已经发现，同时删除区域特异性 染色质标记，组蛋白变体H3 v和H4 v，以及动质体特异性DNA修饰，碱基J， 致死性，具有与复制缺陷相关的终末表型，包括细胞核TbRPA 1的积累 病灶，指示由复制应激引起的ssDNA的异常暴露。因此，我们假设 复制和染色质在特定位点的动态相互作用是必不可少的， 维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H3 v H4 v H4 v J 4v突变体， 在染色体内部区域（Aim 1）和端粒区域（Aim 2）的DNA复制中， TbORC 1与起源结合的变化，起源触发选择和分叉迁移。我们的初步结果 提示H3 v、H4 v和碱基J在端粒维持中具有作用。因此，我们还将研究是否 这些表观遗传标记的缺失破坏了端粒的完整性（Aim 2）。 转录在T.布氏杆菌以多顺反子方式发生。转录起始或终止位点（TSS和TTS） 在多顺反子转录单位的边界处，由特定的核小体修饰和组蛋白标记， 变体：H3 K4 me 3、H4 K10 ac、H2 Az和H2 Bv位于TSS，H3 v和H4 v位于TTS。基地J位于 TSS和TTS。我们最近发现，同时缺失两个染色质标记，H3 v和 碱基J，中断的转录终止。因此，T.布氏杆菌的转录在很大程度上依赖于染色质 结构.此外，DNA复制与转录密切相关，因为转录和 复制共享它们的起始位点。TbMCM-BP是一种复制蛋白，它的缺失与TbMCM-BP的缺失有相似的缺陷。 复制和转录终止作为H3 v H4 v H4 v H4 v H4 v和H3 v H4 v H4 v突变体。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复制和转录之间的机制关系，这是介导的 相同的表观遗传因素。 我们的研究将揭示T. brucei，这将有助于开发更好的手段，最终根除T。布鲁日在未来