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维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 新颖、有效的基于干细胞的疗法来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，操纵它们使衰老组织恢复活力，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肌肉活力的策略。 造血干细胞功能，从而促进骨骼肌恢复并减少与年龄相关的全身性低血压 级慢性炎症。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、小型 分子抑制剂、流式细胞术分析、恢复的生理测量、基因组学和表观基因组学（Cleavage 在“目标和标签”下；剪切并标记）。在老年小鼠中，肌肉干细胞（MuSC）和造血干细胞 祖细胞（HSPC）的静止状态被破坏，导致再生能力降低。最近的研究使用 VWR 恢复衰老小鼠的静止状态并恢复 MuSC 和 HSPC 功能。两种干细胞的表观遗传景观 人口发生了巨大变化，但这些事件背后的机制及其对与年龄相关的影响 功能障碍仍有待研究。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 是随后 Kmt5b 和 Kmt5c 进行二甲基化和三甲基化所必需的， 分别。 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 老化 MuSC 减少。 MuSC 中 Kmt5a 的特异性缺失通过减少衰老表型 干细胞，表明静止状态被破坏，池的自我保护能力受损。使用最近开发的 表观基因组技术 CUT&Tag，我们评估了成人和老年静态 MuSC 中的 H4K20me1，发现 H4K20me1 主要位于基因的转录起始位点，并随着年龄的增长而显着减少。进一步分析表明 与年龄相关的 H4K20me1 缺失使许多 Notch 基因沉默，包括 Rbpj，这对维持 MuSC 静止至关重要。 值得注意的是，MuSC 中 Kmt5a 抑制和随后 H4K20me1 的缺失导致 RNA 聚合酶 II 丝氨酸 2 减少 磷酸化，表明启动子近端暂停的释放受损，因此有效的基因沉默。因此， 我们建议检查老化 MuSC 中 Kmt5a 和 H4K20me1 的丢失是否会导致 他们的静止状态。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，以及该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 模型进行适度运动可以通过恢复 H4K20 来恢复 MuSC 和 HSPC 表观基因组的活力 甲基化。该提案的具体目标是：1）确定Kmt5a在MuSC静息调节中的作用 衰老；2) 确定 VWR 对衰老 MuSC 和衰老 HSPC 中 Kmt5a 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.