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

摘要 随着年龄的增长，干细胞自我更新和分化以保持组织完整性的内在能力急剧下降。 因此，了解随着年龄的增长导致干细胞功能障碍的过程对于未来的发展至关重要。 新的，有效的干细胞为基础的疗法，以治疗与衰老有关的疾病。因此，我的长期目标是 阐明干细胞衰老的表观遗传机制，操纵它们使衰老组织恢复活力，促进健康 衰老更具体地说，这一建议提供的见解将用于制定战略，以振兴肌肉， 造血干细胞功能，并因此促进骨骼肌恢复和降低年龄相关全身性低- 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（自愿轮跑; VWR），细胞培养系统，成像分析，小 分子抑制剂、流式细胞术分析、恢复的生理学测量、基因组学和表观基因组学（切割 在目标和标记下;剪切和标记）。在老年小鼠中，肌肉干细胞（MuSC）和造血干细胞以及 祖细胞（HSPC）的静止被破坏，导致再生能力降低。最近的研究使用VWR， 在老年小鼠中恢复安静并使MuSC和HSPC功能恢复活力。在干细胞和胚胎干细胞中， 人口发生了巨大的变化，但这些事件背后的机制以及它们对年龄相关疾病的贡献 功能障碍仍未得到充分研究。赖氨酸甲基转移酶5a（Kmt 5a）是催化单甲基化的唯一酶 - 组蛋白H4上的赖氨酸20（H4 K20 me 1），其是随后通过Kmt 5 b和Kmt 5c的二甲基化和三甲基化所需的， 分别H4 K20的甲基化对于染色质组织和转录调节是至关重要的，但其在成体细胞中的作用 干细胞是完全未知的，特别是在衰老的背景下。我们的初步数据表明，Kmt 5a和H4 K20 me 1 衰老的MuSC减少。MuSC中Kmt 5a的特异性缺失通过减少细胞周期表型库来重现衰老表型。 干细胞，这表明静止的破坏和池的自我保护受损。使用最近开发的 利用表观基因组技术CUT&Tag，我们评估了成年和老年静止MuSC中的H4 K20 me 1，发现H4 K20 me 1 主要位于基因的转录起始位点，并随年龄的增长而显著降低。进一步分析显示， 与年龄相关的H4 K20 me 1缺失沉默了许多Notch基因，包括Rbpj，这对维持MuSC静止至关重要。 值得注意的是，MuSC中的Kmt 5a抑制和随后的H4 K20 me 1丢失导致RNA聚合酶II丝氨酸2 这表明启动子近端暂停的释放受损，因此是有效的基因沉默。因此，在本发明中， 我们建议检查在老化的MuSC中Kmt 5a的丢失，以及随后的H4 K20 me 1的丢失是否有助于破坏 他们的平静状态。此外，我们将确定Kmt 5a在调节RNA聚合酶II启动子近端的作用， 暂停，以及这种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 使用VWR模型的适度运动可以通过H4 K20的恢复来恢复MuSC和HSPC表观基因组。 甲基化1）确定Kmt 5a在MuSC静止调节中的作用， 2）确定VWR对老年MuSC和老年HSPC中Kmt 5a介导的表观遗传重塑的影响。

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.