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）的双重能力平衡自我更新和增殖，同时补充祖细胞以在一生中满足人体的需求。感染，贫血或有毒化疗等疾病带来的压力会破坏这种关键平衡，从而导致骨髓衰竭并最终导致死亡。了解控制自我更新和增殖的途径将为保护重要的HSC功能提供药理机会。 OTT1是T（1; 22）急性巨核细胞性白血病中分离为融合伴侣的基因，对于在增生胁迫期间保持HSC的自我更新至关重要。 OTT1是一种剪接组成分，具有转录激活/抑制域，RNA识别基序，并具有组蛋白脱乙酰基酶（HDAC），Notch-Effector RBPJ和组蛋白甲基转移酶Set1DB的结合位点。尽管已经发现OTT1与病毒RNA相互作用，但尚未确定生理靶标。初步数据显示，OTT1与参与替代剪接的C-MPL基因的区域相互作用。 C-MPL是血小板蛋白（THPO）的受体，这对于巨核细胞发育至关重要，在压力期间HSC中保持静态和增殖功能。低水平的C-MPL响应支持HSC静止和高信号反应允许扩散，但调节机制尚不清楚。对条件骨骼OTT1小鼠的HSC分析显示，MPL-TR同工型与MPL-FL（全长）的比率显着增加。 MPL-TR在体外描述了主要的负功能，并损害了体内的HSC植入。 OTT1在复合物中与C-MPL RNA上的剪接区域结合。此外，OTT1依赖性的表观遗传学修饰了C-MPL的剪接区域，包括组蛋白脱乙酰基化和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的激活还有利于MPL-TR的产生，通过与RBPJ相互作用，可以解释骨髓壁细分如何调节压力期间HSC静止和增殖。将研究分子机制OTT1介导的基础C-MPL替代剪接，并确定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响应的一种机制。最后，将确定MPL-TR对HSC增殖和静止的影响。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替代剪接的途径的综合策略可能会确定用于解决HSC功能受损的人类疾病的新型药理方法，例如从细胞毒性化学疗法中恢复，茎细胞失败，骨骼骨骼术后的植入术和骨骼较合子恢复。