Role of Histone H2B Ubiquitylation in DNA Replication

描述（由申请人提供）：我们的实验室先前已经证明，在转录过程中，组蛋白H2 B（H2 Bub 1）的单泛素化对于延长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（Rad 6和Bre 1）与Pol II的关联共转录建立的。组蛋白伴侣复合物FACT由Spt 16和Pob 3组成，在酵母中促进H2 Bub 1的形成，并在转录过程中帮助组蛋白再沉积。H2 Bub 1是动态的，标记被泛素蛋白酶Ubp 8去除，Ubp 8也与Pol II一起移动。一些证据表明，H2 Bub 1/FACT的关系也可能是重要的DNA复制。首先，Spt 16定位于复制起点并与RNA引发酶（Pol 1）相关联。其次，Pob 3与复制蛋白A（RPA）相互作用，这对于结合和保护复制叉产生的ssDNA至关重要。此外，Spt 16/Pob 3都被证明是更大的复制体进展复合物的组分。我的初步数据表明H2 Bub 1参与了DNA复制。具体来说，我发现，H2 Bub 1在恢复DNA合成后，在S期早期的HU块中发挥作用。我已经发现，MCM解旋酶福尔斯在htb-K123 R细胞中从模板上脱落，在G1-S相变时不能被泛素化。在第一个具体的目标，我建议确定精确的复制步骤是依赖于H2 Bub 1。此外，我将定义Spt 16（FACT）在此过程中的角色。最后，我将开始寻找影响DNA复制的新的表观遗传标记，以便为这个奖项的独立阶段扩展我的研究领域。我还发现，在htb-K123 R突变体中，其他复制体组分不能有效地募集到S期的复制起点。与此相一致的是，S期的缓慢完成和缓慢的分叉进展。这种观察的一种可能性是，在复制叉的核小体动力学中存在缺陷。H2 Bub 1可能在核小体移位或重组中起重要作用。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。 公共卫生相关性：H2 B泛素化是一种组蛋白修饰，与人类健康，特别是癌症有着重要的相关性。H2 B泛素化的关键调节因子是肿瘤抑制因子，并且由H2 B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H2 B在染色质结构和功能中的基本作用可能会导致这种癌症的新疗法的开发。除了在基因表达中的作用，我目前的工作描述了H2 B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，因此已经开发了许多靶向DNA复制的化疗策略。这些策略使对非分裂的终末分化细胞的有害影响最小化。然而，这些药物中的大多数只针对复制机制本身，而不是DNA复制的表观遗传调节因子。因此，本文提出的工作对于鉴定用于治疗癌症的全新类别的靶标将是非常宝贵的。! 个别评审员的书面评论和标准评分在下面的“评论”部分以基本上未经编辑的形式提供。请注意，这些评论和标准评分是在会议之前准备的，在审查会议上进行任何讨论之后可能没有进行修订。上文“讨论的简历和摘要”部分总结了委员会的最终意见。

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.