THE ROLE OF HISTONE H4 IN GENOME STABILITY

维持基因组的完整性对于遗传信息的准确传递和防止肿瘤发生至关重要。事实上，DNA序列和染色体的不稳定性都与人类癌症的高比例和多样性有关。染色体损伤的修复必须在染色质结构的背景下进行，但实际上对修复机制与染色质成分的相互作用一无所知。我们已经发现，组蛋白H4的n端结构域或“尾巴”对于有效的DNA损伤修复是必需的，而这一功能需要乙酰化赖氨酸残基的活性。在遗传和生化观察的基础上，我们假设H4通过赖氨酸乙酰化信号的蛋白质-蛋白质相互作用促进双链染色体断裂时DNA损伤的修复。我们进一步假设特定的组蛋白乙酰转移酶和/或组蛋白去乙酰化酶通过可逆的H4乙酰化直接参与DNA修复。为了检验这些模型，我们将关注三个主要的研究问题。首先，我们将研究可逆乙酰化缺陷组蛋白H4突变体的结构、加工和双链断裂修复的保真度。我们将测试H4乙酰化在非同源DNA末端连接和同源双链断裂修复中的作用。其次，我们将研究组蛋白H4乙酰化和DNA修复蛋白之间的遗传和生化相互作用。我们将利用我们发现的组蛋白H4乙酰化位点突变体对拟放射药物喜树碱过敏的发现来筛选相互作用的基因，我们将直接纯化酵母ku70相关蛋白来鉴定H4依赖性修复途径的新组分。最后，我们将使用遗传筛选和体内交联结合染色质免疫沉淀来表征特定的组蛋白乙酰转移酶在DNA损伤修复中的作用，以及它在确定的双链断裂时对组蛋白H4乙酰化状态的影响。我们观察到，DNA修复需要可逆的组蛋白H4乙酰化，这一重要的染色质修饰以前未被认识到。

The maintenance of genome integrity is essential for the accurate transmission of genetic information and to prevent tumorigenesis. Indeed, both DNA sequence and chromosome instabilities are associated with a high percentage and wide variety of human cancers. The repair of chromosomal damage must take place in the context of chromatin structure, but virtually nothing is known about the interactions of the repair machinery with the components of chromatin. We have discovered that the N-terminal domain, or "tail", of histone H4 is required for efficient DNA damage repair and that this function requires the activity of an acetylatable lysine residue. On the basis of genetic and biochemical observations, we hypothesize that H4 facilitates DNA damage repair at double-strand chromosome breaks by interacting with components of the repair machinery through protein-protein interactions signaled by lysine acetylation. We further hypothesize that specific histone acetyl transferases and/or histone deacetylases participate directly in DNA repair via reversible H4 acetylation. To test these models we will focus on three major research questions. First, we will examine the structure, processing and fidelity of repair at double-strand breaks in histone H4 mutants defective for reversible acetylation. We will test the role of H4 acetylation in both nonhomologous DNA end joining and homologous double-strand break repair. Second, we will examine the genetic and biochemical interactions between histone H4 acetylation and DNA repair proteins. We will exploit our finding that histone H4 acetylation site mutants are hypersensitivity to the radiomimetic drug camptothecin to screen for interacting genes, and we will purify yeast Ku70-associated proteins directly to identify new components of the H4-dependent repair pathway. Finally, we will use genetic screens and in vivo cross-linking coupled with chromatin immunoprecipitation to characterize a specific histone acetyl transferase for its role in DNA damage repair and its influence on the state of histone H4 acetylation at a defined double-strand break. Our observation that reversible histone H4 acetylation is required for DNA repair represents a previously unrecognized role for this important chromatin modification.