Epigenetic Regulation of Cell and Tissue Aging

正常的人类细胞对潜在的致癌事件，如短端粒，DNA损伤和 通过不可逆地抑制生长激活癌基因，具有称为细胞的特征表型， 衰老这一过程首先在细胞培养中被发现，最近在体内被证实是一个关键的过程。 抑制人类肿瘤恶性进展的机制。RB/p16INK4a，而不是p53， 该途径调节培养的人黑素细胞和体内黑素细胞痣的衰老。这 衰老反应可能是由于染色质修饰，因为来自衰老反应的RB复合物 黑素细胞含有增加水平的组蛋白脱乙酰酶活性和束缚的HDAC 1。我们发现 中等水平HDAC1的表达驱动了染色质的顺序和合作活动， 重塑效应，包括短暂招募Brahma进入RB/HDAC 1 mega复合物，稳定 RB与染色质的结合、yH2AX DNA损伤灶的形成和整体异染色质化。 这些染色质变化与衰老的典型标志物的表达相一致。基于这些 我们假设基因组维护中的表观遗传变化导致细胞和生物体的 衰老具体而言，我们建议： 目的1：A）确定Brm1相关的染色质重塑活性是否是细胞凋亡所必需的。 衰老相关异染色质灶（SAHF）的组装和RB诱导的细胞衰老， 人类黑素细胞该假设将在原代培养物中使用病毒表达系统进行测试， 基于功能性细胞的测定，包括HDAC 1诱导系统和基于染色质的测定。B）定义 Brm1/RB复合物组成的衰老依赖性变化。我们将使用基于HPLC的，大小 排阻分级分离、通过HPLC分级分离的复合物的免疫分离、GST-下拉和质谱 光谱法C）确定肝脏中是否存在染色质重塑复合物的"保守核心" 古老的动物。 目的2：确定衰老前的表观遗传变化与DNA之间的分子联系 损伤响应我们将确定H2AX的磷酸化和/或特异性磷酸化的甲基化是否与H2AX的磷酸化有关。 组蛋白H3残基指示由染色质重塑触发的"DNA反应"。 总之，本建议中所包括的研究旨在阐明顺序和合作的作用， 组蛋白修饰蛋白在细胞衰老和组织衰老中的作用。如果成功，我们的研究将 帮助定义"细胞衰老的组蛋白密码"，从而为干预开辟新的途径， 针对人类表观基因组

Normal human cells respond to potentially oncogenic events such as short telomeres, DNA damage and activating oncogenes by irreversibly arresting growth with a characteristic phenotype termed cellular senescence. This process, first identified in cell culture, has been recently confirmed in vivo as a critical mechanism that curtails the malignant progression of human tumors. The RB/p16INK4a, but not p53, pathway regulates the senescence of human melanocytes in culture and melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase activity and tethered HDAC1. We have found that expression of moderate levels of HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma into RB/HDAC1 mega complexes, stable association of RB with chromatin, formation of yH2AX DNA damage foci and global heterochromatization. These chromatin changes coincided with expression of typical markers of senescence. Based on these findings we hypothesize that epigenetic changes in genome maintenance lead to cellular and organismal aging. Specifically, we propose to: Aim 1: A) To determine whether the Brm1-associated chromatin remodeling activity is required for the assembly of senescent-associated heterochromatic foci (SAHF) and RB-induced cellular senescence in human melanocytes. This hypothesis will be tested using viral expression systems in primary cultures, functional cell-based assays including an HDAC1-inducible system and chromatin-based assays. B) Define senescent-dependent changes in the composition of Brm1/RB complexes. We will use HPLC-based, size exclusion fractionation, immuno-isolation of complexes by HPLC fractionation, GST-pull-downs and Mass Spectrometry. C) Determine whether a "conserved core" of chromatin remodeling complexes exists in livers from old animals. Aim 2: To define molecular links between epigenetic changes that precede senescence and the DNA damage response. We will determine whether phosphorylation of H2AX and/or methylation of specific Histone H3 residues are indicative of a "DNA response" triggered by chromatin remodeling. Together, the studies included in this proposal are aimed at unraveling the role of sequential and cooperative functions of histone modifying proteins in cellular senescence and tissue aging. If successful, our studies will help define a "histone code for cellular aging"; and consequently open new avenues for interventions that target the human epigenome.