Characterization of a novel histone H3 phosphorylation mark in DNA replication

摘要 真核细胞细胞核中的DNA被组蛋白包装成高阶染色质结构 蛋白质。因此，DNA模板化的细胞过程需要改变染色质结构以动态 方便获取打包的DNA。一般说来，这是由依赖于ATP的染色质完成的 组蛋白的重塑、组蛋白交换或化学修饰。磷酸化 特别是丝氨酸/苏氨酸残基已被证明在许多细胞过程中起作用， 包括转录、有丝分裂、细胞凋亡和产孢子。我们已经分离出一种新的组蛋白激酶 酵母中含有保守的S期调控蛋白的复合体。这个建筑群有能力 使游离组蛋白磷酸化，但在完整的组蛋白八聚体的背景下靶向组蛋白H3。我们的 初步数据表明，磷酸化映射到以前未描述的组蛋白位置 可能在组蛋白-DNA相互作用中发挥关键作用的修饰，并可能对 DNA复制的过程。这项提案的目标是提纯和充分描述 多蛋白组蛋白激酶活性的组成，以表征这一新的功能 在活体中进行表观遗传学标记，并验证其对有问题的激酶复合体的依赖性。我们还建议 将这种修饰映射到人类基因组的1%上，以评估其稳定性、定位及其适合性 复制和增殖细胞类型的新表观遗传标记。鉴于我们的假设这是 在复制许可中的修改功能，我们还将比较其在 细胞周期及其对细胞周期进程的要求。因为不受控制的细胞分裂和DNA复制 与癌细胞的增殖有关，我们预计这种表观遗传标记将提供 疾病状态的生物标记物，将为癌症治疗提供一个新的靶点。

Abstract DNA in the nucleus of eukaryotic cells is packaged into the higher-order chromatin structure by histone proteins. Thus, DNA templated cellular processes require alteration of chromatin structure to dynamically facilitate access to packaged DNA. In general, this is accomplished by ATP-dependent chromatin remodeling, histone exchange or chemical modification of histone proteins. Phosphorylation of serine/threonine residues in particular has been shown to function in a number of cellular processes, including transcription, mitosis, apoptosis, and sporulation. We have isolated a novel histone kinase complex in yeast containing conserved S-phase regulatory proteins. This complex is capable of phosphorylating free histones, but targets histone H3 in the context of the intact histone octamer. Our preliminary data indicate that phosphorylation maps to a previously undescribed site of histone modification that may play a critical role in histone-DNA interactions and is likely of great significance to the process of DNA replication. The goals of this proposal are to purify and fully characterize the components of the multi-protein histone kinase activity, to characterize the function of this novel epigenetic mark in vivo and verify its dependence on the kinase complex in question. We also propose to map this modification on 1% of the human genome to assess its stability, localization and its suitability as a new epigenetic marker of replicating and proliferating cell types. Given our hypothesis that this modification functions in licensing of replication, we will also compare its relative abundance across the cell cycle and its requirement for cell cycle progression. As uncontrolled cell division and DNA replication is associated with the proliferation of cancer cells we anticipate that this epigenetic mark will provide a biomarker of diseased states and will offer a novel target for cancer therapeutics.