Regulation of histone genes upon replication arrest and DNA damage

描述(申请人提供)：研究摘要：所有真核细胞中的基因组DNA都包裹在称为组蛋白的碱性蛋白质周围，形成称为染色质的核蛋白细丝。组蛋白是必不可少的，因为它们不仅包装DNA，而且还调节对DNA中包含的遗传信息的访问。缺乏组蛋白的酵母细胞是不能存活的，而组蛋白基因在S期的抑制会触发人类细胞的自发DNA损伤和细胞周期停滞。由于它们的正电荷，组蛋白也可以非特异性地结合到带负电荷的DNA上，并对需要接触DNA的过程产生不利影响。毫不奇怪，组蛋白水平的升高会导致酵母基因组的不稳定，这是人类癌细胞的一个标志。因此，细胞面临的一个主要挑战是严格协调组蛋白的合成和它们在DNA上的有序沉积。细胞进化了各种机制来实现组蛋白和DNA合成之间的这种非常微妙的平衡。组蛋白的合成与增殖细胞中的DNA复制紧密相连。此外，S期的DNA损伤导致DNA合成速率迅速下降，并伴随着组蛋白mRNA水平的急剧下降。然而，导致复制抑制时组蛋白mRNA水平下调的分子机制尚不清楚。我们已经发现，DNA损伤后必需的DNA损伤检查点激酶Rad53(人类肿瘤抑制基因Chk2的萌芽酵母同源物)对于DNA损伤后的组蛋白基因调控是必要的。此外，我们发现在DNA复制被抑制的细胞中，多亚基依赖于ATP的染色质重塑复合体RSC(重塑染色质的结构)以Rad53依赖的方式被招募到组蛋白基因启动子。我们的长期目标是了解细胞确保组蛋白和DNA合成之间微妙平衡的分子机制。为了解决这个问题，我们将剖析Rad53在DNA损伤和复制受阻后调节组蛋白转录水平的途径。以发芽酵母为模型系统，我们计划重点研究以下问题：(1)在S期的正常发展过程中，Rad53下调组蛋白mRNA水平的潜在潜力是如何调节的？(2)RSC在组蛋白基因启动子上的定位与什么相关，这种招募是如何调节的？在复制受阻时组蛋白转录水平下调的过程中，Rad53、RSC和组蛋白调节因子HIR复合体(已知参与组蛋白基因调控)之间的功能相互作用是什么？组蛋白和染色质结构调节DNA新陈代谢的各个方面，越来越多的证据表明，它们在调节基因组稳定性和可能的癌症方面发挥着重要作用。因此，彻底了解组蛋白等基本染色质成分的调节对于理解导致基因组不稳定并最终导致癌症的过程至关重要。我们的目标是了解细胞在复制过程中确保组蛋白合成和组蛋白并入DNA之间的微妙平衡的分子机制，并应用这一知识更好地了解可能参与人类癌症预防的过程。

DESCRIPTION (provided by applicant): RESEARCH ABSTRACT The genomic DNA in all eukaryotic cells is wrapped around basic proteins known as histones to form nucleoprotein filaments called chromatin. Histones are essential as they not only package the DNA, but also regulate access to the genetic information contained in the DNA. Yeast cells lacking histones are inviable, whereas histone gene repression during S-phase triggers spontaneous DNA damage and cell cycle arrest in human cells. Due to their positive charge, histones can also bind non-specifically to the negatively charged DNA and adversely affect processes that require access to DNA. Not surprisingly, elevated histone protein levels lead to genomic instability in yeast, which is a hallmark of human cancer cells. Hence, a major challenge for the cell is to strictly coordinate histone synthesis with their orderly deposition onto the DNA. Cells have evolved a variety of mechanisms to achieve this very delicate balance between histone and DNA synthesis. Histone synthesis is tightly coupled to DNA replication in proliferating cells. Further, DNA damage during S- phase leads to a rapid decrease in the rate of DNA synthesis that is accompanied by dramatic reduction of histone mRNA levels. However, the molecular mechanisms responsible for this downregulation of histone mRNA levels upon replication inhibition are unknown. We have discovered that the essential DNA damage checkpoint kinase Rad53 (the budding yeast homolog of the human tumor suppressor Chk2) is necessary for histone gene regulation following DNA damage. Furthermore, we find that the multi-subunit ATP-dependent chromatin remodeling complex RSC (Remodel the Structure of Chromatin) is recruited to the histone gene promoters in a Rad53 dependent manner in cells where DNA replication has been inhibited. Our long term goal is to understand the molecular mechanisms by which cells ensure a delicate balance between histone and DNA synthesis. To address this issue, we will dissect the pathway by which Rad53 regulates histone transcript levels following DNA damage and replication arrest. Using the budding yeast as a model system, we plan to focus on the following questions: (1) How is the latent potential of Rad53 to downregulate histone mRNA levels regulated during the normal progression of S-phase? (2) What is the relevance of RSC localization at the histone gene promoters and how is this recruitment regulated? (3.) What are the functional interactions between factors such as Rad53, RSC and the histone regulator Hir complex (which are known to be involved in histone gene regulation), in the downregulation of histone transcript levels upon replication arrest? Histones and chromatin structure regulate all aspects of DNA metabolism and mounting evidence suggests that they play a major role in regulating genomic stability and possibly cancer. As such, a thorough understanding of the regulation of essential chromatin components such as the histones is essential for the understanding the processes that lead to genomic instability and ultimately cancer. Our objective is to understand the molecular mechanisms by which cells ensure a delicate balance between histone synthesis and their incorporation into DNA during replication, and apply this knowledge to get a better understanding of the processes that may be involved in cancer prevention in humans.