THE ROLE OF HISTONE H4 IN GENOME STABILITY

保持基因组的完整性对于准确传递遗传信息和预防肿瘤发生至关重要。事实上，DNA序列和染色体的不稳定性都与高比例和多种人类癌症有关。染色体损伤的修复必须在染色质结构的背景下进行，但对修复机制与染色质成分的相互作用几乎一无所知。我们已经发现，组蛋白H4的N-末端结构域，或“尾”，是有效的DNA损伤修复所必需的，并且这一功能需要一个可乙酰化的赖氨酸残基的活性。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，与修复机制的组成部分相互作用，促进双链染色体断裂的DNA损伤修复。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，我们将重点研究三个主要问题。首先，我们将研究组蛋白H4可逆乙酰化缺陷突变体的结构、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。我们将利用我们的发现，即组蛋白H4乙酰化位点突变体对拟放射药物喜树碱超敏，以筛选相互作用的基因，并将直接纯化酵母Ku70相关蛋白，以鉴定H4依赖的修复途径的新成分。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对组蛋白H4在确定的双链断裂时乙酰化状态的影响。我们观察到，DNA修复需要可逆组蛋白H4乙酰化，这代表了这一重要的染色质修饰之前未被认识到的作用。

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.