The role of Kmt2c/MLL3 in hematopoietic stem cell self-renewal, commitment and exhaustion

项目总结 该提案的目的是了解KMT2C/MLL3如何调节造血干细胞(HSC)自身。 更新以及为什么KMT2C突变传递了一种可能导致白血病的选择性优势。KMT2C编码 MLL3，一个COMPASS家族的组蛋白甲基转移酶，结合增强子元件并促进转录。 KMT2C在人类白血病中发生突变，这既是7q染色体大量缺失的一部分，也是在特定的 结构域，如PHD锌指结构域(与染色质结合)或SET甲基转移酶结构域 (它可以启动增强子元件以供激活)。先前的小鼠研究已经证实，Kmt2c缺失 促进HSC自我更新，促进白血病发生，但其机制尚不清楚。为了更好地理解 Kmt2c如何调节HSC的自我更新，我们产生了生殖系和条件性功能丧失小鼠。Kmt2c 缺失增强了HSC的自我更新，这与先前的观察一致，但突变不会改变细胞 循环动力学本身。取而代之的是，Kmt2c缺失允许进行连续移植或化疗 HSC在多个分区周期后保持自我更新能力。这使得突变的造血干细胞能够在竞争中胜出。 骨髓恢复过程中的野生型造血干细胞。在没有压力的情况下，Kmt2c的缺失并不传达选择性的 优势。总而言之，我们的数据表明，Kmt2c突变可以缓解一种名为HSC衰竭的现象， 在这种情况下，HSC在几次累积分裂后失去自我更新能力。我们的机械数据表明 ML3启动HSCs对IL-1和可能的其他炎性细胞因子的反应，通过 增强IL-1信号转导或促进IL-1靶基因表达。这项提议的目的是 旨在扩大这些观察结果。目标1将测试Kmt2c/Mll3缺陷是否传递选择性 通过降低对IL-1和其他炎性细胞因子的敏感性来分离HSC的优势。目标2将 HSCs中MLL3靶向增强子的结构、调控和IL-1反应性的研究 有丰富的部门历史。增强子启动的变化可能允许HSC存档他们的部门历史和 在经历多个部门周期后，更倾向于承诺，而不是自我更新。AIM 3将测试MLL3是否需要 用于限制HSC自我更新能力的功能集或PHD域。这种结构-功能分析将有助于 我们更好地理解特定的KMT2C突变如何传递自我更新优势。如果我们可以 了解HSC在经历累积的自我更新分裂时如何变化，以及Kmt2c如何缺失 传递选择性自我更新的优势，我们最终可能能够在一段时间内保持HSC的功能 在不增加白血病风险的情况下，缓解压力，如化疗后时期。

PROJECT SUMMARY The goal of this proposal is to understand how KMT2C/MLL3 regulates hematopoietic stem cell (HSC) self- renewal and why KMT2C mutations convey a selective advantage that can lead to leukemia. KMT2C encodes MLL3, a COMPASS family histone methyltransferase that binds enhancer elements and promotes transcription. KMT2C is mutated in human leukemias, both as part of large deletions of chromosome 7q and at specific domains such as the PHD zinc finger domains (which bind chromatin) or the SET methyltransferase domain (which can prime enhancer elements for activation). Prior murine studies have established that Kmt2c deletions enhance HSC self-renewal and promote leukemogenesis, but the mechanism is not clear. To better understand how Kmt2c regulates HSC self-renewal, we generated germline and conditional loss-of-function mice. Kmt2c deletions enhanced HSC self-renewal, consistent with prior observations, but the mutations did not alter cell cycle kinetics by themselves. Instead, Kmt2c deletions allowed serially transplanted or chemotherapy treated HSCs to retain self-renewal capacity after multiple division cycles. This allowed the mutant HSCs to outcompete wild type HSCs during marrow recovery. In the absence of stress, Kmt2c deletions did not convey a selective advantage. Altogether, our data suggest that Kmt2c mutations mitigate a phenomenon, called HSC exhaustion, in which HSCs lose self-renewal capacity after several cumulative divisions. Our mechanistic data suggest that MLL3 primes HSCs to differentiate in response to IL-1, and possibly other inflammatory cytokines, by either enhancing IL-1 signal transduction or by facilitating IL-1 target gene expression. The aims of this proposal are designed to extend these observations. Aim 1 will test whether Kmt2c/MLL3 deficiency conveys a selective advantage to dividing HSCs by reducing sensitivity to IL-1 and other inflammatory cytokines. Aim 2 will characterize the structure, regulation and IL-1 responsiveness of MLL3 target enhancers in HSCs with short and extensive division histories. Changes in enhancer priming may allow HSCs to archive their division histories and favor commitment, rather than self-renewal, after multiple division cycles. Aim 3 will test whether MLL3 requires functional SET or PHD domains to restrict HSC self-renewal capacity. This structure-function analysis will help us better understand how specific KMT2C mutations might convey a self-renewal advantage. If we can understand how HSCs change as they undergo cumulative self-renewing divisions, and how Kmt2c deletions convey a selective self-renewal advantage, we may ultimately be able to preserve HSC function through periods of stress, such as post-chemotherapy periods, without increasing leukemia risk.