Epigenetic Regulation of Cell and Tissue Aging

细胞和组织衰老的表观遗传调控

• 批准号: 7666127
• 负责人: ESTELA E MEDRANO
• 金额: $ 30.84万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7666127
• 关键词: Acids; Age; Aging; Animals; Appearance; Biological Assay; Biological Markers; CDKN2A gene; Cell Aging; Cell Culture Techniques; Cells; Characteristics; Chromatin; Chromatin Remodeling Factor; Co-Immunoprecipitations; Complex; Cyclin E; DNA; DNA Damage; DNA Double Strand Break; DNA lesion; Data; Deacetylase; Deposition; Dominant-Negative Mutation; Double Strand Break Repair; Down-Regulation; Ectopic Expression; Engineering; Epigenetic Process; Euchromatin; Event; Exclusion; Fatty Liver; Fractionation; Frozen Sections; Genome; Growth; HDAC1 gene; Heterochromatin; High Pressure Liquid Chromatography; Higher Order Chromatin Structure; Histone Code; Histone Deacetylase; Histone H3; Histone H4; Histones; Homologous Gene; Human; Immunofluorescence Immunologic; Incidence; Infection; Insulin; Intervention; Label; Laboratories; Lead; Link; Liver; Longevity; Lysine; Maintenance; Malignant - descriptor; Mass Spectrum Analysis; Measures; Melanocytic nevus; Melanoma Cell; Methods; Methylation; Modeling; Modification; Molecular; Molecular Chaperones; Morphology; Mus; Nuclear; Nuclear Pleomorphism; Oncogenes; Oncogenic; Pathology; Pathway interactions; Phenotype; Phosphorylation; Play; Population; Principal Investigator; Process; Proliferating; Proteins; Reading; Recruitment Activity; Regulation; Research Personnel; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Somatomedins; Stress; Structure; Sum; System; TP53 gene; Technology; Testing; Tetanus Helper Peptide; Tissues; Variant; Viral; Western Blotting; Work; Yeasts; base; beta-Galactosidase; brahma; brahma protein; chromatin modification; chromatin remodeling; histone acetyltransferase; histone modification; in vivo; melanocyte; overexpression; prevent; programs; protein function; regenerative; repaired; response; senescence; telomere; transcription factor; tumor

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.

正常的人类细胞对潜在的致癌事件做出反应，例如短端粒，DNA损伤和 通过不可逆地以称为细胞的特征表型来激活癌基因 衰老。该过程最初在细胞培养中鉴定出来，最近在体内被证实为关键 减少人类肿瘤的恶性进展的机制。 RB/P16INK4A，但不是P53， 途径调节体内培养物和黑素细胞内黑素细胞的衰老。这 衰老反应可能是由于染色质修饰所致，因为RB复合物来自衰老 黑色素细胞含有增加的组蛋白脱乙酰基酶活性和束缚的HDAC1。我们找到了 中等水平的HDAC1表达驱动染色质的顺序和合作活性 重塑效应子，包括将梵天的短暂募集到RB/HDAC1大型综合体中，稳定 RB与染色质，YH2AX DNA损伤焦点的形成和全局异染色性的缔合。 这些染色质变化与典型衰老标记的表达相吻合。基于这些 调查结果我们假设基因组维持的表观遗传变化导致细胞和生物 老化。具体来说，我们建议： 目标1：a）确定是否需要与BRM1相关的染色质重塑活性 衰老相关的异质灶（SAHF）和RB诱导的细胞衰老的组装 人类黑色素细胞。该假设将在原代培养物中使用病毒表达系统进行检验， 基于功能细胞的测定法，包括HDAC1诱导系统和基于染色质的测定法。 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 通过HPLC分级，GST扣和质量，排除分馏，复合物的免疫异化 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” 来自老动物。 目标2：定义衰老之前的表观遗传变化与DNA之间的分子联系 伤害响应。我们将确定H2AX的磷酸化和/或特定的甲基化是否是否 组蛋白H3残基表示由染色质重塑触发的“ DNA响应”。 总之，该提案中包括的研究旨在揭示顺序和合作的作用 组蛋白在细胞衰老和组织衰老中修饰蛋白质的功能。如果成功，我们的学习将 有助于定义“细胞衰老的组蛋白代码”；因此为干预措施开辟了新的途径 靶向人类表观基因组。