Progressive DNA Hypomethylation as a Measure of Mitotic History and Potential Contributor to Replicative Senescence.

进行性 DNA 低甲基化作为有丝分裂历史的衡量标准和复制衰老的潜在贡献者。

• 批准号: 10450874
• 负责人: PETER W LAIRD
• 金额: $ 59.7万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10450874
• 关键词: Aging; Anatomy; Attention; Attenuated; Automobile Driving; Behavior; Biological Clocks; Biology; Cell Aging; Cell Culture Techniques; Cell Cycle; Cell Cycle Inhibition; Cell Line; Cell Lineage; Cell division; Cells; Chromatin; Chronology; Circadian Rhythms; Complex; Contact Inhibition; CpG Islands; CpG dinucleotide; Cues; Cytosine; DNA; DNA Maintenance; DNA Methylation; DNA Modification Methylases; DNA methyltransferase inhibition; Data; Degenerative Disorder; Dependence; Development; Disease; Epigenetic Process; Fibroblasts; Gene Expression; Genome; Genomics; Health; Histones; Human; Individual; Intervention; Link; Location; Longevity; Maintenance; Malignant Neoplasms; Measures; Metabolic; Methylation; Mitosis; Mitotic; Modeling; Molecular; Outcome; Phenotype; Physiological; Process; Recording of previous events; Reporting; Research; Resistance; Role; Serum; Techniques; Telomerase; Testing; Time; Tissues; base; cell type; deprivation; design; human disease; human tissue; inhibitor; molecular clock; overexpression; premalignant; premature; prevent; senescence; tool; ubiquitin-protein ligase

PROJECT SUMMARY / ABSTRACT Cellular and molecular alterations that accumulate during cell division are considered to be contributors to aging phenotypes. Changes in epigenetic marks, including DNA methylation, have been widely documented in aging. This has spurred the development of epigenetic molecular clocks, which rely primarily on replication- independent gain of methylation at CpG islands. As molecular clocks tuned to chronological time across different tissue types with varying mitotic rates and histories, these clocks are not directly linked to mitotic cell division. In contrast, we have found that loss of DNA methylation at lamina-attached, late-replicating regions of the genome is closely tied to apparent mitotic history. We propose to use primary human cell culture to experimentally validate that mitosis is a driver of hypomethylation, and to explore the underlying mechanisms and consequences of this hypomethylation, to define genomic and chromatin features driving hypomethylation, and use this data to construct a mitotic molecular clock. In Specific Aim 1 we propose to use primary human cell culture to provide experimental evidence for the contribution of mitosis to PMD hypomethylation, and to disentangle the time-dependency and mitosis- dependency of the phenomenon using cell cycle inhibition. We will also investigate whether enhancing maintenance methylation machinery is able to counteract the progressive loss of DNA methylation. In Specific Aim 2 we propose to investigate whether DNA hypomethylation contributes to replicative senescence or associated phenotypes in primary human cell culture. We will investigate whether inhibition of maintenance methylation accelerates senescence, and whether progressive hypomethylation is extended in telomerase- immortalized primary human fibroblasts, contributing to premalignant phenotypes associated with such immortalization. We will also investigate whether accelerated senescence by progerin expression or supra- physiologic O2 leads to accelerated DNA hypomethylation. In Specific Aim 3 we will characterize the genomic and chromatin features that influence individual CpG hypomethylation rates. We will measure the rates of DNA hypomethylation of individual CpGs in six different primary human cell types and use this information to identify cell-type-specific, as well as universal genomic and chromatin drivers of hypomethylation. Finally, we will use elastic net regression on the assembled data to construct cell-type-specific and universal epigenetic mitotic clocks in Specific Aim 4. The preliminary studies strongly support the concept and feasibility of a DNA hypomethylation-based mitotic clock. The outcome of this proposed research could have important impacts on our understanding of the contribution of widespread hypomethylation to aging phenotypes, with potential implications for aging interventions. The availability of accurate molecular clocks specifically designed to measure mitotic history would provide a valuable molecular tool to characterize cells and tissues in human health and disease.

项目摘要 /摘要 在细胞分裂期间积累的细胞和分子改变被认为是促成 老化表型。表观遗传标记的变化，包括DNA甲基化，已广泛记录在 老化。这刺激了表观遗传分子钟的发展，这些时钟主要依赖于复制 - CPG岛上甲基化的独立增长。随着分子钟调整为按时间顺序排列的时间 不同的组织类型具有不同的有丝分裂速率和历史，这些时钟与有丝分裂细胞没有直接相关 分配。相比之下，我们发现在椎板连接的，延迟重复区域的DNA甲基化损失的损失 基因组与明显的有丝分裂史密切相关。我们建议将原发性人细胞培养 实验验证有丝分裂是降低甲基化的驱动器，并探索潜在的机制 以及这种降压甲基化的后果，以定义驱动低甲基化的基因组和染色质， 并使用这些数据构建有丝分子分子时钟。 在特定目的1中，我们建议使用原发性人类细胞培养以提供实验证据 有丝分裂对PMD低甲基化的贡献，并消除时间依赖性和有丝分裂 - 使用细胞周期抑制作用的现象依赖性。我们还将调查是否增强 维持甲基化机制能够抵消DNA甲基化的进行性丧失。具体 目的2我们建议研究DNA降压甲基化是否有助于复制性衰老或 原代人细胞培养中的相关表型。我们将调查是否抑制维护 甲基化会加速衰老，以及端粒酶中进行性低甲基化是否扩展 永生的原代人成纤维细胞，有助于与此类相关的前态表型 永生化。我们还将调查是否通过孕激素表达或超质量加速衰老 生理O2导致加速DNA降压甲基化。在特定目标3中，我们将表征基因组 染色质特征会影响单个CpG降压率。我们将测量DNA的速率 在六种不同的主要人类细胞类型中单个CPG的甲基化降压甲基化，并使用此信息来识别 细胞型特异性以及降压甲基化的通用基因组和染色质驱动因素。最后，我们将使用 组装数据上的弹性净回归，以构建细胞型特异性和通用表观遗传学 特定目的4中的时钟。初步研究强烈支持DNA的概念和可行性 基于低甲基化的有丝分裂时钟。这项拟议的研究的结果可能会对 我们对广泛降低甲基化对衰老表型的贡献的理解，潜力 对衰老干预措施的影响。专门设计的精确分子时钟的可用性 测量有丝分裂史将提供有价值的分子工具来表征人类的细胞和组织 健康与疾病。