Hematopoietic regulation through Ott1-dependent alternative splicing

描述（由申请人提供）：造血依赖于造血干细胞（HSC）平衡自我更新和增殖的双重能力，同时补充祖细胞以满足人体一生的需求。感染、贫血或有毒化疗等情况带来的压力会破坏这种关键的平衡，导致骨髓衰竭，最终导致死亡。了解控制自我更新和增殖的途径将为保护重要的HSC功能提供药理学机会。Ott1是在t（1;22）急性巨核细胞白血病中作为融合伙伴分离出来的一个基因，对于在增殖应激下维持造血干细胞的自我更新至关重要。Ott1是一个剪接体成分，具有转录激活/抑制结构域、RNA识别基序，并具有组蛋白去乙酰化酶（Hdac）、缺口效应物Rbpj和组蛋白甲基转移酶Set1db的结合位点。虽然已经发现Ott1与病毒rna相互作用，但尚未发现生理靶点。初步数据显示，Ott1与c-Mpl基因中参与选择性剪接的区域相互作用。C-Mpl是血小板生成素（Thpo）的受体，Thpo对于巨核细胞的发育和维持hsc在应激状态下的静止和增殖功能至关重要。低水平的c- Mpl反应支持HSC静止，高水平的信号反应允许增殖，但调节机制尚不清楚。对条件缺失的Ott1小鼠hsc的分析显示，Mpl-TR亚型与Mpl-FL（全长）的比例显著增加。Mpl-TR已经在体外描述了显性负功能，并在体内损害HSC的植入。Ott1以复合物形式结合到c-Mpl RNA上的可选剪接区域。此外，对c-Mpl可选剪接区域的ott1依赖性表观遗传修饰，包括组蛋白去乙酰化和H3K4me3标记，提供了一种能够调节Mpl-TR:Mpl-FL比率的潜在机制，从而调节对Thpo的反应。Notch的激活也有利于Mpl-TR的产生，Mpl-TR通过Ott1与Rbpj的相互作用，可能解释了骨髓生态位如何调节应激期间HSC的静止和增殖。研究人员将研究ott1介导的c-Mpl选择性剪接的分子机制，并确定相关表观遗传修饰因子的作用。Ott1， Rbpj和c-Mpl之间的联系将作为Notch控制HSC Thpo反应的机制进行研究。最后，我们将确定Mpl-TR对HSC增殖和静止的影响与造血应激的抵抗有关。利用遗传互补、化学抑制剂和反义寡核苷酸靶向调节c-Mpl选择性剪接的途径的综合策略，可能会发现新的药理学方法，以解决由HSC功能受损引起的人类疾病，如细胞毒性化疗后的恢复、干细胞移植后的移植物衰竭和骨髓衰竭综合征，如再生障碍性贫血。

DESCRIPTION (provided by applicant): Hematopoiesis depends on the dual ability of the hematopoietic stem cell (HSC) to balance self-renewal and proliferation while replenishing progenitors to supply the body's needs over a lifetime. Stress brought on by conditions such as infection, anemia or toxic chemotherapy can disrupt this critical balance resulting in bone marrow failure and ultimately death. Understanding the pathways controlling self-renewal and proliferation will provide pharmacologic opportunities to protect vital HSC function. Ott1, a gene isolated as a fusion partner in t(1;22) acute megakaryocytic leukemia, is essential for maintaining self-renewal of HSCs during proliferative stress. Ott1 is a spliceosome component, has a transcriptional activation/repression domain, RNA Recognition Motifs and possesses binding sites for Histone Deacetylase (Hdac), Notch-effector Rbpj and the histone methyltransferase Set1db. Although Ott1 has been found to interact with viral RNAs, no physiologic targets have been identified. Preliminary data shows Ott1 interacts with a region of the c-Mpl gene involved in alternative splicing. C-Mpl is the receptor for Thrombopoietin (Thpo), which is critical for megakaryocyte development and maintaining both quiescence and proliferative function in HSCs during stress. Low level c- Mpl responses support HSC quiescence and high signaling responses allow proliferation, yet the modulating mechanism is not known. Analysis of HSCs from conditionally-deleted Ott1 mice show a dramatic increase in the ratio of Mpl-TR isoform to Mpl-FL (full length). Mpl-TR has described dominant negative function in vitro and impairs HSC engraftment in vivo. Ott1 binds in complex to the alternatively spliced region on c-Mpl RNA. In addition, Ott1-dependent epigenetic modifications to the alternatively spliced region of c-Mpl, including histone deacetylation and H3K4me3 marking provide a potential mechanism capable of regulating the Mpl-TR:Mpl-FL ratio and thereby modulating response to Thpo. Activation of Notch also favors Mpl-TR production which through Ott1 interaction with Rbpj, may explain how the bone marrow niche regulates HSC quiescence and proliferation during stress. The molecular mechanism Ott1-mediated underlying c-Mpl alternative splicing will be investigated and the role of associated epigenetic modifiers established. The link between Ott1, Rbpj, and c-Mpl will be examined as a mechanism for Notch to control HSC Thpo response. Finally, the effects of Mpl-TR on HSC proliferation and quiescence in relation to withstanding hematopoietic stress will be determined. The comprehensive strategy utilizing genetic complementation, chemical inhibitors, and antisense oligonucleotides to target the pathways regulating c-Mpl alternative splicing in this proposal may identify novel pharmacologic approaches to address human disease arising from impaired HSC function such as recovery from cytotoxic chemotherapy, graft failure after stem cell transplantation and bone marrow failure syndromes such as aplastic anemia.