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.

摘要 随着年龄的增长，干细胞自我更新和分化以维持组织完整性的内在能力急剧下降。 因此，了解随年龄增长导致干细胞功能障碍的过程对于未来的发展是至关重要的。 新的、有效的干细胞疗法，用于治疗与衰老相关的疾病。因此，我的长期目标是 阐明干细胞老化的表观遗传学机制，操纵它们使衰老的组织恢复活力，促进健康 衰老。更具体地说，这项建议提供的洞察力将被用来制定恢复肌肉和 造血干细胞功能，从而促进骨骼肌恢复，减少与年龄相关的全身性低血糖 慢性炎症分级。为了实现这一目标，我们将利用小鼠遗传模型，骨骼肌模型 退行性损伤和适度运动(自愿车轮跑步；VWR)，细胞培养系统，成像分析，小 分子抑制物、流式细胞术分析、恢复的生理指标、基因组学和表观基因组学(裂解 在目标和标记下；切割和标记下)。在老龄小鼠中，肌肉干细胞(MUSC)和造血干细胞以及 祖细胞(HSPC)的静止被打乱，导致再生能力降低。最近的研究使用VWR来 使衰老小鼠的MUSC和HSPC功能恢复安静和恢复活力。这两种干细胞的表观遗传格局 人口发生了戏剧性的变化，然而这些事件背后的机制以及它们对年龄相关的贡献 功能障碍仍未得到充分研究。赖氨酸甲基转移酶5a(Kmt5a)是唯一催化单甲基化的酶。 组蛋白H4(H4K20me1)上的赖氨酸20，Kmt5b和Kmt5c随后的二甲基化和三甲基化所需的， 分别进行了分析。H4K20的甲基化是染色质组织和转录调控的关键，但它在成人中的作用 干细胞是完全未知的，特别是在衰老的背景下。我们的初步数据显示，Kmt5a和H4K20me1 老年骨髓间充质干细胞的减少。MUSCs中Kmt5a的特异性缺失通过减少 干细胞，表明池子的静止被打乱，自我保护能力受损。使用最新开发的 表观基因组学技术切割和标记，我们评估了H4K20me1在成人和老年静止的MSC中，发现H4K20me1 大部分位于基因的转录起始点，并随着年龄的增长而显著减少。进一步的分析显示， 与年龄相关的H4K20me1的缺失使包括Rbpj在内的许多Notch基因沉默，Rbpj是维持MUSC静止的关键。 值得注意的是，Kmt5a抑制和随后在MuSCs中H4K20me1的丢失导致RNA聚合酶II丝氨酸2减少 磷酸化，暗示了启动子-近端停顿的受损释放，因此有效的基因沉默。因此， 我们建议研究老化的MSC中Kmt5a的丢失，以及随后的H4K20me1的丢失是否有助于破坏 它们的静止状态。此外，我们还将确定Kmt5a在调节RNA聚合酶II启动子近端的作用。 暂停，以及这一拟议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模型的适度运动可以通过恢复H4K20来恢复MUSC和HSPC表观基因组 甲基化。这项建议的具体目的是：1)确定Kmt5a在MUSC静止调节中的作用 2)确定VWR对老年MUSC和老年HSPC Kmt5a介导的表观遗传学重塑的影响。