Elucidating Mechanisms of Histone H2B Dynamic Modification in Mammalian Apoptosis

描述（由申请人提供）：染色质由DNA和相关组蛋白组成，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使用组蛋白变体，如细胞凋亡。细胞凋亡是所有后生动物中必不可少的保守途径，其形态学特征包括染色质凝聚和DNA断裂。获得性抗凋亡是几乎所有类型癌症的一个主要特征。我们实验室之前的研究表明，哺乳动物细胞中核心组蛋白H2B（以下简称H2BS14）中的丝氨酸14和酿酒酵母中丝氨酸10 （H2BS10）在响应凋亡刺激时发生动态磷酸化。这种磷酸化是酵母程序性细胞死亡的关键调控步骤，因为酵母组蛋白H2B被一种不可磷酸化的形式所取代，从而导致对细胞凋亡的抵抗。最近，在酿酒酵母中，Hbs 3（一种组蛋白去乙酰化酶（HDAC））对H2B （H2BK11）中赖氨酸11的去乙酰化已被证明是H2BS10磷酸化发生的先决条件，从而在两个组蛋白H2B标记之间形成单向“串导”。基于这些初步的酵母实验数据，我拟进一步研究组蛋白H2B在哺乳动物细胞凋亡中的动态修饰调控机制。我建议使用生化分析和质谱分析来确定细胞生长和凋亡过程中H2B共价修饰的“特征谱”；我建议在哺乳动物细胞凋亡过程中使用体外激酶测定和体内功能细胞测定来检验“串扰”调节。我进一步建议使用体外肽拉下实验和体内单核体共诱导诱导肽（co-IPs）来鉴定和表征哺乳动物凋亡中磷酸化H2BS14下游的“效应物”。这些研究将揭示动态组蛋白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对人类生物学和人类疾病，特别是癌症产生影响的治疗策略。

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.