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(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身体终生需要。感染、贫血或毒性化疗等疾病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基因是在t(1；22)急性巨核细胞白血病中分离的一个融合伴侣基因，在增殖应激过程中对维持HSCs的自我更新是必不可少的。Ott1是一个剪接体成分，具有转录激活/抑制结构域和RNA识别基序，并与组蛋白脱乙酰酶(HDAC)、Notch效应分子Rbpj和组蛋白甲基转移酶Set1db结合。虽然已经发现Ott1与病毒RNA相互作用，但还没有确定生理靶点。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，它对巨核细胞的发育以及在应激状态下维持HSCs的静止和增殖功能至关重要。低水平的c-MPL反应支持HSC的静止，而高水平的信号反应允许HSC增殖，但其调控机制尚不清楚。对条件缺失OTT1小鼠的HSCs的分析表明，MPL-TR亚型与MPL-FL(全长)的比率显著增加。MPL-tr在体外具有显性的负性功能，并在体内损害HSC的植入。Ott1与c-MPL RNA上的选择性剪接区以复合体结合。此外，对c-MPL选择性剪接区的OTT1依赖的表观遗传修饰，包括组蛋白去乙酰化和H3K4me3标记，提供了一种潜在的机制，能够调节MPL-TR：MPL-FL比率，从而调节对Thpo的反应。Notch的激活也有利于MPL-tr的产生，这可能是通过Ott1与Rbpj的相互作用来解释骨髓生态位如何在应激状态下调节HSC的静止和增殖。OTT1介导的c-MPL选择性剪接的分子机制将被研究，并建立相关的表观遗传修饰物的作用。将研究Ott1、Rbpj和c-mpl之间的联系，作为Notch控制HSC Thpo反应的机制。最后，将确定MPL-tr对HSC增殖和静止的影响，这与耐受造血应激有关。在这项建议中，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选择性剪接的途径的综合策略可能会找到新的药理学方法来解决因HSC功能受损而引起的疾病，如细胞毒性化疗后的恢复、干细胞移植后的移植物衰竭以及再生障碍性贫血等骨髓衰竭综合征。