Elucidating Mechanisms of Histone H2B Dynamic Modification in Mammalian Apoptosis

描述（由申请人提供）：由DNA和相关组蛋白组成的染色质是我们基因组的生理模板。越来越多的证据表明，染色质可以经历动态变化，包括共价组蛋白修饰，ATP依赖性复合物的重塑，或在不同的生物过程中利用组蛋白变体，如凋亡。细胞凋亡是所有后生动物中必不可少的保守途径，其特征在于包括染色质浓缩和DNA片段化的形态学标志。对细胞凋亡的获得性抗性是几乎所有类型癌症的主要特征。本实验室先前的研究已经证明了哺乳动物细胞中核心组蛋白H2B中丝氨酸14（以下简称H2BS 14）和S.酿酒酵母对凋亡刺激的反应。这种磷酸化是酵母中程序性细胞死亡的关键调节步骤，因为用不可磷酸化形式替换酵母组蛋白H2B导致对细胞凋亡的抗性。最近，在S.在酿酒酵母中，H2B（H2BK11）中的赖氨酸11被Hbs 3（一种组蛋白脱乙酰酶（HDAC））脱乙酰化已被证明是H2BS10磷酸化发生的先决条件，因此在两个组蛋白H2B标记之间形成单向“串扰”。基于这些在酵母中的初步数据，我建议研究组蛋白H2B在哺乳动物细胞凋亡中的动态修饰的调控机制。我建议，以确定“签名档案”的H2B共价修饰细胞生长和凋亡过程中使用生化分析和质谱法，我建议检查“串扰”调节在哺乳动物细胞中使用体外激酶测定和体内功能细胞凋亡过程中的测定。我进一步提出，以确定和表征的“效应”下游磷酸化H2BS14在哺乳动物细胞凋亡，在体外肽下拉测定，并在体内单核细胞共IP。这些研究将阐明动态组蛋白H2B修饰参与哺乳动物细胞凋亡调控的机制。在深入了解这些基本过程方面取得的进展将导致影响人类生物学和人类疾病，特别是癌症的治疗策略。

DESCRIPTION (provided by applicant): Chromatin comprised of DNA and associated histone proteins is the physiological template of our genome. An increasing body of evidence suggests that Chromatin can undergo dynamic changes, including covalent histone modifications, remodeling by ATP-dependent complexes, or utilization of histone variants during different biological processes, such as apoptosis. Apoptosis is a conserved pathway essential in all metazoans, characterized by morphological hallmarks including Chromatin condensation, and DNA fragmentation. Acquired resistance toward apoptosis is a major characteristic in nearly all types of cancer. Previous studies in our laboratory have demonstrated a dynamic phosphorylation of serine 14 in core histone H2B (hereafter H2BS14) in mammalian cells and of serine 10 (H2BS10) in S. cerevisiae in response to apoptotic stimuli. This phosphorylation is a key regulatory step of programmed cell death in yeast as replacement of yeast histone H2B with a non-phosphorylable form results in resistance to apoptosis. Recently, in S. cerevisiae, deacetylation of lysine 11 in H2B (H2BK11) by Hbs 3, a histone deacetylase (HDAC), has been shown to be a prerequisite for H2BS10 phosphorylation to occur, thus forming a unidirectional "cross-talk" between the two histone H2B marks. Based on these preliminary data in yeast, I propose to investigate the regulatory mechanisms of dynamic modifications of histone H2B in mammalian apoptosis. I propose to identify the "signature profiles" of H2B covalent modifications during cell growth and apoptosis using biochemical assays and mass spectrometry; I propose to examine the "crosstalk" regulation in mammalian cells using both in vitro kinase assays and in vivo functional cellular assays during apoptosis. I further propose to identify and characterize the "effectors" downstream of phosphorylated H2BS14 in mammalian apoptosis, using in vitro peptide pull-down assays, and in vivo mononucleosomal co-IPs. These studies will shed light on the mechanisms of dynamic histone H2B modifications involved in the regulation of mammalian apoptosis. Advances made in gaining insights into these fundamental processes will lead to therapeutic strategies that impact on human biology and human diseases, notably cancer.