Hematopoietic regulation through Ott1-dependent alternative splicing

通过 Ott1 依赖的选择性剪接进行造血调节

• 批准号: 8579535
• 负责人: Glen D Raffel
• 金额: $ 39.63万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8579535
• 关键词: Acute Megakaryocytic Leukemias; Address; Affect; Alternative Splicing; Anemia; Antisense Oligonucleotides; Aplastic Anemia; Binding; Binding Sites; Biological Assay; Blood; Bone Marrow; Cell Proliferation; Cell physiology; Cells; Cessation of life; Chemicals; Chromatin; Clinical; Complex; Critical Pathways; Cytotoxic Chemotherapy; DNA; Data; Development; Dominant-Negative Mutation; Drug Targeting; Engraftment; Epigenetic Process; Equilibrium; Exons; Genes; Genetic; Genetic Transcription; Goals; HDAC1 gene; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Histone Deacetylase; Histone Deacetylation; In Vitro; Infection; Injury; Knock-out; Length; Link; MPL gene; Mediating; Megakaryocytes; Messenger RNA; Modification; Molecular; Mus; Nature; Outcome; Pancytopenia; Pathway interactions; Pharmaceutical Preparations; Physiological; Process; Production; Protein Isoforms; RNA; RNA Recognition Motif; RNA Splicing; Recovery; Regulation; Repression; Role; Signal Transduction; Spliceosomes; Stem cell transplant; Stem cells; Stress; Syndrome; Testing; Thrombopoietin; Time; Transcriptional Activation; biological adaptation to stress; chemotherapy; chromatin immunoprecipitation; graft failure; histone methyltransferase; human HDAC1 protein; human MPL protein; human disease; improved; in vivo; inhibitor/antagonist; mRNA Precursor; meetings; notch protein; novel; progenitor; public health relevance; response; self-renewal; viral RNA

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.

描述（由申请人提供）：造血取决于造血干细胞（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身体一生的需求。感染、贫血或有毒化疗等疾病带来的压力可能会破坏这种关键的平衡，导致骨髓衰竭并最终死亡。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 t(1;22) 急性巨核细胞白血病中作为融合伴侣分离出来的基因，对于在增殖应激期间维持 HSC 的自我更新至关重要。 Ott1 是一个剪接体组件，具有转录激活/抑制结构域、RNA 识别基序，并拥有组蛋白脱乙酰酶 (Hdac)、Notch 效应器 Rbpj 和组蛋白甲基转移酶 Set1db 的结合位点。尽管已发现 Ott1 与病毒 RNA 相互作用，但尚未确定生理目标。初步数据显示 Ott1 与参与选择性剪接的 c-Mpl 基因区域相互作用。 C-Mpl 是血小板生成素 (Thpo) 的受体，对于巨核细胞发育以及应激期间维持 HSC 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。对条件性缺失 Ott1 小鼠 HSC 的分析显示，Mpl-TR 亚型与 Mpl-FL（全长）的比率显着增加。 Mpl-TR 已在体外描述了显性失活功能，并在体内损害 HSC 植入。 Ott1 与 c-Mpl RNA 上的选择性剪接区域形成复合物结合。此外，对 c-Mpl 选择性剪接区域的 Ott1 依赖性表观遗传修饰（包括组蛋白脱乙酰化和 H3K4me3 标记）提供了一种能够调节 Mpl-TR:Mpl-FL 比例并从而调节对 Thpo 反应的潜在机制。 Notch 的激活也有利于 Mpl-TR 的产生，通过 Ott1 与 Rbpj 的相互作用，可以解释骨髓生态位如何在应激期间调节 HSC 的静止和增殖。将研究 Ott1 介导的潜在 c-Mpl 选择性剪接的分子机制，并建立相关表观遗传修饰剂的作用。 Ott1、Rbpj 和 c-Mpl 之间的联系将作为 Notch 控制 HSC Thpo 反应的机制进行检查。最后，将确定 Mpl-TR 对 HSC 增殖和与承受造血应激相关的静止的影响。该提案中利用遗传互补、化学抑制剂和反义寡核苷酸来靶向调节 c-Mpl 选择性剪接途径的综合策略可能会确定新的药理学方法来解决因 HSC 功能受损而引起的人类疾病，例如细胞毒性化疗的恢复、移植后的移植失败。干细胞移植和骨髓衰竭综合征，例如再生障碍性贫血。