Targeting H4K20 methylation to rejuvenate aged stem cell epigenome and regenerative function.

靶向 H4K20 甲基化以恢复衰老干细胞表观基因组和再生功能。

• 批准号: 10644982
• 负责人: Roméo Sébastien Blanc
• 金额: $ 10.08万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10644982
• 关键词: ATAC-seq; Acute; Adult; Affect; Age; Age Months; Aging; Cell Count; Cell Culture System; Cells; Cellular biology; Chromatin; Chronic; DNA Polymerase II; Data; Data Collection; Development; Disease; Elderly; Environment; Enzymes; Epigenetic Process; Event; Exercise; Extramural Activities; Flow Cytometry; Functional disorder; Funding; Future; Gene Expression; Gene Silencing; Genes; Genetic Models; Genetic Transcription; Genomics; Goals; Health Benefit; Hematopoietic stem cells; Histone H4; Homeostasis; Human; Image; Impairment; Individual; Inflammation; Inflammatory; Injury; Knockout Mice; Ligands; Lysine; Maintenance; Mammals; Measures; Mediating; Mentors; Mentorship; Methylation; Methyltransferase; Modeling; Moderate Exercise; Modification; Molecular; Mus; Muscle; Muscle satellite cell; Natural regeneration; Phase; Phenotype; Phosphorylation; Physiological; Plasma; Positioning Attribute; Process; Productivity; Quality of life; RNA; RNA Polymerase II; Reaction; Recovery; Regenerative capacity; Regulation; Regulator Genes; Regulatory Element; Rejuvenation; Reporting; Repression; Research; Role; Running; Serine; Signal Transduction; Skeletal Muscle; Skeletal muscle injury; Sorting; Stimulus; TFF1 gene; Tamoxifen; Techniques; Testing; Tissues; Training; Transcription Initiation Site; Transcriptional Regulation; Work; adult stem cell; age related; aged; career; cell regeneration; comorbidity; cytokine; epigenetic regulation; epigenome; epigenomics; genome-wide; genome-wide analysis; healthy aging; improved; inducible Cre; insight; large datasets; mouse genetics; muscle aging; muscle regeneration; notch protein; novel; preservation; prevent; programs; promoter; regeneration function; response; restoration; sedentary; self-renewal; skills; small molecule inhibitor; stem cell aging; stem cell biology; stem cell fate; stem cell function; stem cell population; stem cell therapy; stem cells; tissue regeneration; transcriptome sequencing; translational medicine

ABSTRACT As we age, the intrinsic ability of stem cells to self-renew and differentiate to maintain tissue integrity dramatically declines. Therefore, understanding the processes leading to stem cell dysfunction with age is essential for the future development of novel, effective stem cell-based therapies to treat disorders associated with aging. Therefore, my long-term goal is to elucidate the epigenetic mechanisms of stem cell aging, manipulate them to rejuvenate aged tissue, and promote healthy aging. More specifically, the insight provided by this proposal would be used to devise strategies to rejuvenate muscle and hematopoietic stem cell function, and therefore promote skeletal muscle recovery and reduce age-associated systemic low- grade chronic inflammation. To accomplish this objective, we will utilize mouse genetic models, models of skeletal muscle degenerative injury and moderate exercise (voluntary wheel running; VWR), cell culture systems, imaging analysis, small molecule inhibitors, flow cytometry analysis, physiological measures of recovery, genomics, and epigenomics (Cleavage Under Targets and Tagmentation; CUT&Tag). In aged mice, both muscle stem cell (MuSC) and hematopoietic stem and progenitor cell (HSPC) quiescence is disrupted, leading to reduced regenerative capacity. Recent studies used VWR to restore quiescence and rejuvenate both MuSC and HSPC function in aged mice. The epigenetic landscape in both stem cell populations changes dramatically, yet the mechanisms underlying these events as well as their contribution to age-associated dysfunction remain understudied. The lysine methyltransferase 5a (Kmt5a) is the sole enzyme catalyzing monomethylation of lysine 20 on histone H4 (H4K20me1), which is required for subsequent di- and tri-methylation by Kmt5b and Kmt5c, respectively. Methylation of H4K20 is critical for chromatin organization and regulation of transcription, yet its role in adult stem cells is entirely unknown, especially in the context of aging. Our preliminary data show that Kmt5a and H4K20me1 decrease in aged MuSCs. Specific deletion of Kmt5a in MuSCs recapitulates aging phenotype by decreasing the pool of stem cells, suggesting disruption of quiescence and impaired self-preservation of the pool. Using the recently developed epigenomic technique CUT&Tag, we assessed H4K20me1 in adult and aged quiescent MuSCs and found that H4K20me1 is mostly located at the genes’ transcriptional start site and significantly decreases with age. Further analysis revealed that age-associated loss of H4K20me1 silenced numerous Notch genes including Rbpj, critical to maintaining MuSC quiescence. Significantly, Kmt5a inhibition and subsequent loss of H4K20me1 in MuSCs led to decreased RNA Polymerase II serine 2 phosphorylation, suggesting the impaired release of promoter-proximal pausing and therefore potent gene silencing. Thus, we propose to examine if the loss of Kmt5a, and consequently H4K20me1, in aging MuSCs contributes to the disruption of their quiescence state. Also, we will determine the role of Kmt5a in regulating RNA Polymerase II promoter-proximal pausing, and how this proposed mechanism contributes to controlling MuSC fate and function. Last, we will determine if moderate exercise using a VWR model can rejuvenate MuSC and HSPC epigenome through the restoration of H4K20 methylation. The specific aims of this proposal are: 1) Determine the role of Kmt5a in MuSC quiescence regulation during aging and 2) Determine the impact of VWR on Kmt5a-mediated epigenetic remodeling in aged MuSC and aged HSPC.

抽象的 随着年龄的增长，干细胞自我更新和分化以显着下降的固有能力。 因此，了解导致干细胞功能障碍随年龄的过程对于未来的发展至关重要 新型，有效的基于干细胞的疗法治疗与衰老相关的疾病。因此，我的长期目标是 阐明干细胞衰老的表观遗传机制，操纵它们以恢复衰老的组织并促进健康 老化。更具体地说，该提案提供的见解将用于制定恢复肌肉和的策略 造血干细胞功能，因此促进骨骼肌恢复并减少与年龄相关的全身低 - 等级慢性炎症。为了实现这一目标，我们将利用鼠标遗传模型，骨骼肌的模型 退化性损伤和中等运动（自愿行驶； VWR），细胞培养系统，成像分析，小 分子抑制剂，流式细胞仪分析，恢复物理测量，基因组学和表观基因组学（裂解 在目标和标签下；切割和标签）。在老年小鼠中，肌肉干细胞（MUSC）和造血干细胞和 祖细胞（HSPC）静止受到破坏，导致再生能力降低。最近的研究使用VWR 恢复静止并恢复年龄小鼠的MUSC和HSPC功能。两个干细胞的表观遗传景观 种群发生了巨大变化，但是这些事件的基础机制及其对年龄相关的贡献 功能障碍仍然被理解。赖氨酸甲基转移酶5A（KMT5A）是唯一的酶催化单甲基化 组蛋白H4上的赖氨酸20（H4K20ME1），这是KMT5B和KMT5C随后的DI-和三甲基化所必需的 H4K20的甲基化对于染色质组织和转录调节至关重要，但其在成人中的作用 干细胞完全未知，尤其是在衰老的背景下。我们的初步数据显示KMT5A和H4K20ME1 老化的MUSC减少。 MUSC中KMT5A的特定缺失通过减少池来概括衰老表型 干细胞，表明静止的破坏和池的自我保护受损。使用最近开发的 表观基因组技术剪切和标签，我们评估了成人和老化的MUSC中的H4K20me1，发现H4K20Me1 主要位于基因的转录起始位点，并且随着年龄的增长而显着降低。进一步的分析表明 与年龄相关的H4K20ME1损失沉默了许多Notch基因，包括RBPJ，对于保持MUSC静止至关重要。 值得注意的是，MUSC中的KMT5A抑制和随后的H4K20ME1丢失导致RNA聚合酶II序列2降低 磷酸化，表明启动子抗毒性暂停的释放受损，从而释放了潜在的基因沉默。那， 我们建议检查衰老的MUSC中KMT5A以及H4K20ME1的损失是否有助于破坏 他们的静止状态。另外，我们将确定KMT5A在调节RNA聚合酶II启动子促启动子中的作用 暂停，以及该提出的机制如何有助于控制MUSC的命运和功能。最后，我们将确定是否 使用VWR模型进行适度的运动可以通过修复H4K20恢复MUSC和HSPC表观基因组 甲基化。该提案的具体目的是：1）确定KMT5A在MUSC静止调节中的作用 衰老和2）确定VWR对老化MUSC和老年HSPC的KMT5A介导的表观遗传重塑的影响。