Epigenetic Dysregulation as a Driver of Skeletal Muscle Aging

摘要 衰老的“染色质修饰物的再定位”理论假定， 衰老是表观遗传噪声终生积累的结果， 信号通路，引发组织功能障碍和健康恶化的症状。证据表明 特别是DNA甲基化，可能会在几种哺乳动物组织中设定衰老时钟的速度，但它是 尚不清楚这些变化是否可逆。骨骼肌是一种特别感兴趣的组织，因为它 高代谢负荷、生物量和对称为肌肉减少症年龄相关功能障碍的易感性。 我的方法是首先调查从年轻人和成年人中分离的单个细胞核的表观基因组景观， 使用snATAC-seq和整合snRNA-seq数据来测试染色质区域是否 每种离散细胞类型的可接近性与来自这些区域的基因表达相关。我会整合整个 使用5 hmC捕获数据进行基因组亚硫酸氢盐测序，以找到年轻人中羟甲基化的CpG位点， 但在年老时仍保持甲基化，看看这些是否与基因表达相关。为了检验这一假设 与年龄相关的肌肉功能障碍是表观遗传环境变化的结果， 到TET 2驱动的细胞身份丧失，我将使用单核和5 hmC数据来确定年龄是否 表观基因组的相关变化与细胞身份的丧失相关，如通过显著性差异所测量的， 老年肌肉细胞转录组的变化。接下来，我将重点介绍TET 2双加氧酶在成人中的作用。 年轻和年老动物的骨骼肌。我将执行CHiP-seq以在健康的年轻肌肉中定位TET 2 然后确定这个位置在老骨骼肌中是否发生了改变。我还将测试TET 2活动，包括 5 mC氧化，和稳定性，在培养和在体内的老和年轻的肌肉，使用AMPK和电 刺激以增加TET 2活化。最后，我将确定年龄相关的肌肉功能障碍是否是 在体内使用表观遗传重编程可逆。我们的初步结果表明，OSK表观遗传 重编程显著增强终末分化的体细胞（神经元）的再生， 以TET依赖性方式改善老年小鼠的视力。因为表观遗传衰老广泛存在于 在所有组织中，我预测OSK重编程的TET依赖机制并不局限于单一组织 型，并可能有利于老年骨骼肌。我将治疗年轻和年老的肌肉细胞在文化和肌肉 组织与OSK-AAV和测量肌原性，急性损伤后的修复，肌肉力量和其他标志， 肌肉老化如果我的目标得以实现，该领域将受益于我新开发的方法和综合 基于单核的转录组学和表观基因组学数据的分析。此外，拟议的实验 将定义TET 2去甲基化动力学在衰老肌肉中的作用，并确定TET 2失调是否是一种 肌肉减少症的司机最后，研究在衰老过程中驱动肌肉功能障碍的表观遗传机制 可能为抗衰老治疗提供新的靶点，如OSK-表观遗传重编程。

ABSTRACT The “relocalization of chromatin modifiers” theory of aging postulates that the degenerative process of aging is the result of a lifetime accumulation of epigenetic noise, which transcriptionally dysregulates cellular signaling pathways, triggering symptoms of tissue dysfunction and health deterioration. Evidence suggests that DNA methylation, in particular, may set the pace of the aging clock in several mammalian tissues, yet it is unclear whether these changes are reversible. Skeletal muscle is a particular tissue of interest because of its high metabolic load, biomass, and susceptibility to age-related dysfunction, termed sarcopenia. My approach is to first survey of the epigenomic landscape of individual nuclei isolated from young and old mouse muscle using snATAC-seq and integrate snRNA-seq data to test whether regions of chromatin accessibility in each discreet cell type correlate with gene expression from those regions. I will integrate whole genome bisulfite sequencing with 5hmC capture data, to find CpG sites that are hydroxymethylated in young muscle but remain methylated in old and see if these correlate with gene expression. To test the hypothesis that age-associated muscle dysfunction is the result of changes in the epigenetic landscape that lead to a TET2-driven loss of cellular identity, I will use the single nucleus and 5hmC data to determine if age- associated changes to the epigenome are correlated with a loss of cellular identity, as measured by significant changes to the transcriptomes of old muscle cells. Next, I will focus on the role of TET2 dioxygenase in adult skeletal muscle from young and old animals. I will perform CHiP-seq to locate TET2 in healthy young muscle and then determine if this localization is altered in old skeletal muscle. I will also test TET2 activity, including 5mC oxidation, and stability in culture and in vivo in old and young muscle, using AMPK and electrical stimulation to increase TET2 activation. Finally, I will determine if age-associated muscle dysfunction is reversible in vivo using epigenetic reprogramming. Our preliminary results show that OSK epigenetic reprogramming dramatically enhances regeneration of terminally differentiated somatic cells (neurons) and improves visual acuity in aged mice in a TET-dependent manner. Because epigenetic aging is seen broadly in all tissues, I predict that the TET-dependent mechanism of OSK reprogramming is not limited to a single tissue type, and that it may benefit aged skeletal muscle. I will treat young and old muscle cells in culture and muscle tissue with OSK-AAV and measure myogenicity, repair after acute injury, muscle force and other hallmarks of muscle aging. If my aims are achieved the field will benefit from my newly developed methods and integrative analyses of single nucleus-based transcriptomic and epigenomic data. Moreover, the proposed experiments will define the role of TET2 demethylation dynamics in aging muscle and determine if TET2 dysregulation is a driver of sarcopenia. Finally, investigating the epigenetic mechanisms that drive muscle dysfunction in aging may provide novel targets for anti-aging therapies, such as OSK-epigenetic reprogramming.