Role of Histone H2B Ubiquitylation in DNA Replication

描述（由申请人提供）：我们的实验室之前已经证明，在转录过程中，组蛋白H2B （H2Bub1）的单酚基化对于延长RNA聚合酶II （Pol II）后核小体的有效重组很重要。该标记是通过泛素化机制（Rad6和Bre1）与Pol II的关联共转录建立的。酵母中由Spt16和Pob3组成的组蛋白伴侣复合物FACT可促进H2Bub1的形成，并有助于组蛋白在转录过程中的再沉积。H2Bub1是动态的，标记被泛素蛋白酶Ubp8去除，Ubp8也与Pol II一起移动。一些证据表明H2Bub1/FACT关系可能对DNA复制也很重要。首先，Spt16定位于复制起点，并与RNA引物酶（Pol1）结合。其次，Pob3与复制蛋白A （RPA）相互作用，RPA对于结合和保护复制叉上产生的ssDNA至关重要。此外，Spt16/Pob3都被证明是一个更大的复制体进展复合体的组成部分。我的初步数据表明H2Bub1参与DNA复制。具体来说，我发现H2Bub1在s期早期HU阻断后恢复DNA合成中起作用。我发现MCM解旋酶在G1-S阶段不能被泛素化的htb-K123R细胞中从模板上脱落。在第一个具体目标中，我建议确定依赖于H2Bub1的精确复制步骤。另外，我将定义Spt16 （FACT）在流程中的角色。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，为这个奖项的独立阶段做准备。我还发现，在htb-K123R突变体中，其他复制体成分不能有效地招募到s期的复制起点。与此相一致的是s阶段的缓慢完成和缓慢的分叉进程。这种观察结果的一种可能性是，在复制叉的核小体动力学中存在缺陷。也许H2Bub1对核小体位移或重组很重要。因此，第二个目标主要涉及标记的动态调节以及它如何影响DNA复制过程中的核小体动力学。

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.