Role of Histone H2B Ubiquitylation in DNA Replication

组蛋白 H2B 泛素化在 DNA 复制中的作用

• 批准号: 8223072
• 负责人: Kelly Miguel Trujillo
• 金额: $ 11.5万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-21 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8223072
• 关键词: Affect; Area; Award; Binding; Cell Cycle; Cells; Chromatin Structure; Complex; Critiques; DNA Primase; DNA biosynthesis; Data; Defect; Development; Epigenetic Process; Gene Expression; Genes; Genetic; Genetic Transcription; Health; Histone H2B; Histones; Human; Individual; Lead; Link; Malignant Neoplasms; Molecular Chaperones; Nucleosomes; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Phase Transition; Phosphotransferases; Play; Polymerase; Post-Translational Protein Processing; Process; RNA; RNA Polymerase II; Recruitment Activity; Regulation; Replication Origin; Role; S Phase; Staging; Travel; Tumor Suppressor Proteins; Ubiquitin; Work; Writing; Yeasts; base; cancer therapy; career; falls; helicase; histone modification; leukemia; meetings; mutant; novel; replication factor A; uncontrolled cell growth

DESCRIPTION (provided by applicant): Our lab had previously demonstrated that during transcription, the monoubiquitylation of histone H2B (H2Bub1) is important for the efficient reassembly of nucleosomes in the wake of elongating RNA polymerase II (Pol II). The mark is established co-transcriptionally, via the association of the ubiquitylation machinery (Rad6 and Bre1) with Pol II. The histone chaperone complex, FACT, which consists of Spt16 and Pob3 in yeast, promotes the formation of H2Bub1, and aids in histone redeposition during transcription. H2Bub1 is dynamic, with the mark being removed by the ubiquitin protease, Ubp8, which also travels with Pol II. Several lines of evidence suggest that the H2Bub1/FACT relationship might also be important for DNA replication. First, Spt16 localizes to origins of replication and associates with the RNA primase (Pol1). Second, Pob3 interacts with Replication Protein A (RPA), which is essential for binding and protecting ssDNA generated at replication forks. In addition, both Spt16/Pob3 were shown to be components of a larger Replisome Progression Complex. My preliminary data have implicated H2Bub1 in DNA replication. Specifically, I find that H2Bub1 plays a role in the resumption of DNA synthesis following an HU block early in S-phase. I have discovered that the MCM helicase falls off the template in htb-K123R cells that cannot be ubiquitylated at the G1-S phase transition. In the first specific aim, I propose to identify the precise replication steps that are dependent on H2Bub1. Also, I will define the role of Spt16 (FACT) in the process. Lastly, I will begin my search for novel epigenetic marks that influence DNA replication so as to expand my area of study for the independent phase of this award. I have also discovered that in the htb-K123R mutant, other replisome components are not efficiently recruited to origins of replication in S-phase. Consistent with that, is a slow completion of S-phase and slow fork progression. One possibility for this observation is that there is a defect in nucleosome dynamics at a replication fork. Perhaps H2Bub1 is important for nucleosome displacement or reassembly. Therefore, the second aim deals largely with the dynamic regulation of the mark and how it influences nucleosome dynamics during DNA replication. PUBLIC HEALTH RELEVANCE: H2B ubiquitylation is a histone modification with significant relevance to human health, especially cancer. A key regulator of H2B ubiquitylation is a tumor suppressor, and several downstream histone modifications that are regulated by H2B ubiquitylation are linked to the inappropriate expression of developmentally important genes in leukemia. Understanding the fundamental role of H2B in chromatin structure and function could lead to the development of new therapies in this cancer. Aside from its role in gene expression, my current work describes a novel role of H2B ubiquitylation in DNA replication. Because a hallmark of cancer is uncontrolled cell growth, many chemotherapeutic strategies have been developed that target DNA replication. These strategies minimize the deleterious effects on non-dividing, terminally differentiated cells. However, most of these drugs only target the replication machinery itself, rather than the epigenetic regulators of DNA replication. Therefore, the work proposed herein would be invaluable towards identifying a completely new class of targets for the treatment of cancer. ! The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the "Critique" section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The "Resume and Summary of Discussion" section above summarizes the final opinions of the committee.

描述（由申请人提供）：我们的实验室先前证明，在转录过程中，组蛋白H2b（H2BUB1）的单液化对于在拉长RNA聚合酶II（POL II）后的有效重新组装而言，对核小体的有效重新组装很重要。该商标是通过泛素化机制（RAD6和BRE1）与Pol II的关联共同转录的。由SPT16和POB3组成的组蛋白伴侣复合物在酵母中促进了H2BUB1的形成，并在转录过程中有助于组蛋白重新沉积。 H2BUB1是动态的，其标记被泛素蛋白酶ubp8删除，ubp8也与Pol II一起传播。几条证据表明，H2BUB1/事实关系对于DNA复制也可能很重要。首先，SPT16本地化为复制的起源，并与RNA Primase（POL1）相关。其次，POB3与复制蛋白A（RPA）相互作用，这对于在复制叉时结合和保护ssDNA至关重要。此外，SPT16/POB3均显示为较大的重置型进程复合物的组成部分。我的初步数据已在DNA复制中暗示H2BUB1。具体而言，我发现H2BUB1在S期早期HU块后的DNA合成中起作用。我发现MCM解旋酶从HTB-K123R细胞中的模板上掉落，而在G1-S相跃迁上无法泛滥。在第一个特定目的中，我建议确定取决于H2BUB1的精确复制步骤。另外，我将定义SPT16（事实）在此过程中的作用。最后，我将开始寻找影响DNA复制的新型表观遗传标记，以扩大我的研究领域，以换取该奖项的独立阶段。我还发现，在HTB-K123R突变体中，其他重置组成分没有被有效地募集到S期复制的起源。与此相一致的是，S期和较慢的叉进程的完成缓慢。该观察结果的一种可能性是，复制叉处的核小体动力学存在缺陷。也许H2BUB1对于核小体位移或重新组装很重要。因此，第二个目标在很大程度上涉及标记的动态调节及其在DNA复制过程中如何影响核小体动力学。 公共卫生相关性：H2B泛素化是一种与人类健康，尤其是癌症相关的组蛋白修饰。 H2B泛素化的关键调节剂是肿瘤抑制剂，由H2B泛素化调节的几种下游组蛋白修饰与白血病中发育重要基因的不适当表达有关。了解H2B在染色质结构和功能中的基本作用可能导致该癌症中新疗法的发展。除了其在基因表达中的作用外，我目前的工作还描述了H2B泛素化在DNA复制中的新作用。由于癌症的标志是不受控制的细胞生长，因此已经开发出许多针对DNA复制的化学治疗策略。这些策略最大程度地减少了对非分裂的终末分化细胞的有害影响。但是，这些药物中的大多数仅针对复制机制本身，而不是DNA复制的表观遗传调节剂。因此，本文提出的工作对于确定癌症治疗的全新目标是无价的。呢 以下“批评”部分提供了基本未经编辑的审稿人的书面批评和标准评分。请注意，这些批评和标准分数是在会议之前准备的，并且在审查会议上的任何讨论之后可能没有修订。上面的“简历和摘要”部分总结了委员会的最终意见。