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它的感染。所有这些都是 药物有严重的副作用，有些药物很难管理。因此，身份识别和 对布鲁氏毛滴虫特有的基本细胞过程的表征将对 未来开发更好的抗寄生虫剂。DNA复制对细胞增殖和 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，我们发现同时删除特定区域的 染色质标记、组蛋白变异体H3v和H4v，以及动质体特异性DNA修饰，碱基J 与复制缺陷相关的末端表型致死性，包括核TbRPA1的堆积 Foci，复制应激引起的单链DNA异常暴露的指标。因此，我们假设 复制和染色质之间在特定基因座上的动态相互作用在 保持基因组和表观基因组的完整性。在本项目中，我们将对H3v∆H4v∆J∆突变体进行鉴定 在染色体内部区域(目标1)和端粒区域(目标2)的DNA复制中，重点是 TbORC1与原点、原点激发选择和分叉迁移的结合变化。我们的初步结果 提示H3v、H4v和碱基J在端粒维持中起作用。因此，我们还将审查是否 这些表观遗传标记的缺失破坏了端粒的完整性(目标2)。 布氏毛滴虫的转录是以多顺反子方式进行的。转录起始或终止位点(TSSS和TTSS) 在多顺反子转录单位的边界上，标记有特定的核小体修饰和组蛋白 变种：H3K4me3、H4K10ac、H_2Az和H_2Bv在TSSS，H3v和H4v在TTSS。J基地位于 TSS和TTSS都是。我们最近发现同时缺失两个染色质标记H3v和H3v 碱基J，转录终止中断。因此，布氏锥虫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点。TbMCM-BP是一种复制蛋白，它的耗尽在 复制和转录终止为H3v∆H4v∆J∆和H3v∆J∆突变体。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，这种关系是由 相同的表观遗传因素。 我们的研究将揭示T.DNA复制和转录终止的独特调控特征。 这将有助于开发更好的手段，在未来最终根除布鲁氏菌。

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.