Epigenetic Regulation of Cell and Tissue Aging

细胞和组织衰老的表观遗传调控

基本信息

批准号：
7476015
负责人：
ESTELA E MEDRANO
金额：
$ 31.47万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
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/7476015
关键词：
Acids Aging Animals Appearance Biological Assay Biological Markers CDKN1A gene CDKN2A gene Cell Aging Cells Characteristics Chromatin Chromatin Remodeling Factor Class Co-Immunoprecipitations Complex Cultured Cells 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 H2AFX gene 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 Localized 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 Numbers Oncogenes Oncogenic Pathology Pathway interactions Phenotype Phosphorylation Play Population Principal Investigator Process Proliferating Protein Overexpression 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 remodeling histone acetyltransferase human H2AX protein in vivo melanocyte oncoprotein p21 prevent programs protein function repaired response senescence size telomere transcription factor tumor

项目摘要

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.

正常人体细胞会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阻止具有称为细胞的特征表型的生长来激活癌基因衰老。这个过程首先在细胞培养中被识别出来，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，但不是 p53，途径调节培养物中的人类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造成的，因为衰老产生的 RB 复合物黑素细胞的组蛋白脱乙酰酶活性和束缚的 HDAC1 水平升高。我们发现中等水平的 HDAC1 表达驱动染色质的顺序和协作活动重塑效应子，包括将 Brahma 瞬时招募到 RB/HDAC1 大型复合物中，稳定 RB 与染色质的关联、yH2AX DNA 损伤灶的形成和整体异染色质化。这些染色质变化与典型衰老标志物的表达一致。基于这些研究结果我们假设基因组维护中的表观遗传变化导致细胞和有机体老化。具体来说，我们建议：目标 1：A) 确定 Brm1 相关染色质重塑活性是否是衰老相关异染色质灶 (SAHF) 和 RB 诱导的细胞衰老的组装人类黑色素细胞。该假设将使用原代培养物中的病毒表达系统进行测试，基于功能细胞的检测，包括 HDAC1 诱导系统和基于染色质的检测。 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 的尺寸排除分级分离、通过 HPLC 分级分离对复合物进行免疫分离、GST 下拉和质量光谱测定。 C) 确定肝脏中是否存在染色质重塑复合物的“保守核心” 来自古老的动物。目标 2：定义衰老前的表观遗传变化与 DNA 之间的分子联系损害反应。我们将确定 H2AX 是否磷酸化和/或特定的甲基化组蛋白 H3 残基表示染色质重塑触发的“DNA 反应”。总之，本提案中包含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功能。如果成功的话，我们的研究将帮助定义“细胞衰老的组蛋白代码”；从而开辟新的干预途径以人类表观基因组为目标。