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

基本信息

批准号：
10215251
负责人：
Hee-Sook Kim
金额：
$ 31.95万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10215251
关键词：
3-Dimensional Affect Africa Africa South of the Sahara African Trypanosomiasis Antigenic Switching Binding Binding Sites Biological Assay Blood Circulation Bromodeoxyuridine Cell Nucleus Cell Proliferation Cell physiology Cells ChIP-seq Chromatin Chromatin Fiber Chromatin Structure Chromatin Structure Alteration Chromosomal Instability Chromosome Fragility Chromosome Structures Chromosomes DNA DNA Modification Process DNA biosynthesis Data Defect Development Economic Burden Epigenetic Process Exhibits Fiber FISH Foundations Frequencies Future Gel Gene Expression Profile Generations Genes Genetic Recombination Genetic Transcription Genome Genome Stability Growth Heterochromatin Histones Human Immunity Impairment Infection Lesion Link Measures Modification Molecular Molecular Conformation Monitor Mutation Nuclear Nucleosomes Parasites Pattern Pharmaceutical Preparations Phenotype Ploidies Process Replication Initiation Replication Origin Sahara Site Structure Surface Antigens Testing Transcription Initiation Site Trypanocidal Agents Trypanosoma Trypanosoma brucei brucei Trypanosomiasis Variant Work base cell growth chromosome conformation capture epigenome experimental study gel electrophoresis genome integrity histone modification insight migration molecular dynamics mutant origin recognition complex pathogen preservation promoter replication stress side effect telomere transcription factor transcription termination

项目摘要

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. H3vΔ H4vΔ JΔ 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 H3vΔ H4vΔ JΔ 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.

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，而且全部都需要治疗。严重的副作用并且难以管理，因此，识别和表征至关重要。布氏锥虫具有独特特征的细胞过程对于开发更好的抗寄生虫药物至关重要 DNA复制对于细胞增殖和基因组完整性至关重要。重要的是，我们发现延伸必须与核小体分解和组装良好协调。同时删除区域特异性染色质标记、组蛋白变体 H3v 和 H4v，以及动质体特异性 DNA 修饰（碱基 J）会导致严重的生长缺陷。复制表现出应激表型，包括核 TbRPA1 灶的形成（异常的指示）复制应激导致的 ssDNA 暴露），以及 G2 细胞和不完全细胞的积累因此，这些染色质标记对于 DNA 的正确复制可能很重要。这些染色质标记仅影响它们通常所在的位点的 DNA 复制（局部效应）。染色质结构的变化可能会线性影响远处区域的 DNA 复制（全局效应）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。 H3vΔ H4vΔ JΔ 突变体中的内部区域（目标 1）和端粒（目标 2）在目标 1 中，我们将检查起源放电。 Aim 1.1 中 MFA-seq 和 Repli-seq 的概况和复制延伸（分叉迁移模式），检查 Aim 1.2中通过确定TbORC1在起点的结合来启动复制，确定是否染色质标记突变会导致 Aim 1.3 中的染色体脆弱和/或改变 Aim 1.5 中的转录谱。确定 3D 染色体组织，以获得对染色质标记方式的机械理解在目标 1.4 中控制核空间内的 DNA 复制这将帮助我们建立之间的逻辑联系。 T. brucei 染色体内部区域的染色质结构、DNA 复制和转录状态。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好处，因为布氏锥虫亚端粒要么是“高度转录和早期复制”，要么是“严格抑制和复制” 复制较晚”。端粒的这些独特特征将帮助我们揭示有关端粒的更多机制细节。我们将研究染色质结构、复制和转录之间的相互作用。使用 Aim 2.1 中的 2D 凝胶分析和染色质纤维 FISH 进行复制。也将在目标 2.2 中进行检查。根据该提案，我们将建立定义染色质标记的基础。我们的研究将帮助我们理解机制。表观基因组和基因组之间的这种协作作用可以成功地转化为基因组稳定性以及细胞生存的基因多样化。