Mechanisms that Regulate Self-renewal and Proliferation in Hematopoietic Stem Cells

造血干细胞(HSC)的分化率和自体成活率至关重要 更新受到仔细的管理并保持平衡，因为当这种情况发生时，就会出现严重的疾病状态 平衡被打破了。不幸的是，维持这种平衡的机制人们知之甚少， 这种缺乏理解是研究进展的主要障碍，同时也是严重的 限制了基于HSC的治疗干预的临床潜力。这项研究的目的 旨在阐明调节HSC静止、自我更新和 分化，重点是关键的HSC调节因子，阴阳1号(YY1)。YY1是一个无处不在的 锌指转录因子，对小鼠的肝星状细胞发育至关重要。我们最近出版的出版物 结果表明，YY1基因缺陷的HSC不能自我更新，不能保持静止状态。此外， 干细胞因子(SCF)/c-Kit信号通路是HSC发育过程中的关键调控途径。 在YY1缺陷的HSCs中显著下调。YY1占据远端增强子和启动子 在Kit基因座测序并促进Kit基因在造血干细胞中的表达。因此，我们令人信服的数据 提示SCF/c-Kit通路是YY1调节HSC自身的关键下游调节因子 更新和宁静。我们的初步数据也提供了强有力的证据表明，依赖于YY1 抑制凝集素的核心成分--染色体结构维持(SMC)1和3 对于其在造血干细胞中建立静止状态的能力来说，是至关重要的。重要的是，有缺陷的HSC YY1缺失小鼠的静止状态完全被Smc3的杂合性所拯救。总而言之，我们的结果 支持YY1通过以下机制调节HSC自我更新和静止的假说 包括通过控制Kit基因座的染色体结构变化来激活SCF/c-Kit信号， 以及对凝集素的抑制。为了验证这一假设，一个N端截短的YY1突变体，它 缺乏YY1的转录激活和共激活因子招募功能，但它保留了 YY1的转录抑制和DNA环功能将在小鼠的骨髓中表达 YY1-/-条件性基因敲除小鼠，允许用以下方法评估YY1的作用机制 在SCF/c-Kit信号、粘附素表达、HSC自我更新和增殖方面。此外，a 将使用独特的YY1-/-SMC3+/-条件性基因敲除小鼠品系来研究其机制(S) YY1-调节粘附素及其在决定HSC自我更新和分化中的重要性。 拟议的研究将阐明迄今尚未得到充分描述的机制和途径 YY1的参与及其对HSC细胞命运的影响是如何介导的。这些机制可能会 包括YY1依赖对染色质可及性、高阶染色质/染色体 靶基因的结构和/或激活/抑制，包括Kit和Smc3。

It is of critical importance that the rates of hematopoietic stem cell (HSC) differentiation and self- renewal are carefully regulated and kept in balance, because severe disease states arise when this balance is disrupted. Unfortunately, the mechanisms that maintain this balance are poorly understood, and this lack of understanding represents a major impediment to research progress, while also severely restricting the clinical potential of HSC-based therapeutic interventions. The goal of the research proposed here is to elucidate mechanisms that regulate HSC quiescence, self-renewal and differentiation, with a focus on the critical HSC regulatory factor, Yin Yang 1 (YY1). YY1 is a ubiquitous zinc finger transcription factor that is essential for HSC development in mice. Our recent publication showed that YY1-deficient HSCs fail to self-renew and fail to maintain a quiescent state. Furthermore, Stem Cell Factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in YY1-deficient HSCs. YY1 occupies the distal enhancer and promoter sequences at the Kit locus and promotes Kit gene expression in HSCs. Thus, our compelling data implicates the SCF/c-Kit pathway as a critical downstream mediator for YY1 in regulating HSC self- renewal and quiescence. Our preliminary data also provide strong evidence that YY1-dependent repression of Structural Maintenance of Chromosomes (SMC) 1 and 3, core components of the cohesin complex, are critical to its ability to establish quiescence in HSCs. Importantly, defective HSC quiescence in Yy1 null mice is completely rescued by heterozygosity at Smc3. Collectively, our results support the hypothesis that YY1 regulates HSC self-renewal and quiescence by mechanisms that include activation of SCF/c-Kit signaling by controlling chromosome structural change at the Kit locus, and repression of cohesin. To investigate the hypothesis, an N-terminally truncated YY1 mutant, which lacks the transcriptional activation and co-activator recruitment functions of YY1, but which retains transcriptional repression and DNA looping functions of YY1, will be expressed in the bone marrow of Yy1-/- conditional knockout mice, which allows the YY1 mechanism of action to be evaluated with respect to SCF/c-Kit signaling, cohesin expression, HSC self-renewal and proliferation. In addition, a unique Yy1-/- SMC3+/- conditional knockout mouse strain will be used to investigate the mechanism(s) by which YY1-regulates cohesin and its importance in determining HSC self-renewal and differentiation. The proposed studies will elucidate as yet poorly characterized mechanisms and pathways in which YY1 participates and how its effects on HSC cell fate are mediated. These mechanisms are likely to include YY1-dependent effects on chromatin accessibility, higher-order chromatin/chromosome structure and/or activation/repression of target genes including Kit and Smc3.