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一生的需求。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提供保护重要HSC功能的药理学机会。在t（1;22）急性巨核细胞白血病中作为融合伴侣分离的HSCs 1基因对于在增殖应激期间维持HSC的自我更新是必不可少的。Notch 1是一种剪接体成分，具有转录激活/抑制结构域、RNA识别基序，并具有组蛋白脱乙酰酶（Hisp）、Notch效应子Rbpj和组蛋白甲基转移酶Set 1db的结合位点。尽管已经发现BMP 1与病毒RNA相互作用，但尚未鉴定出生理靶点。初步数据显示，c-Mpl 1与参与选择性剪接的c-Mpl基因的一个区域相互作用。C-Mpl是血小板生成素（Thpo）的受体，其对于巨核细胞发育以及在应激期间维持HSC中的静止和增殖功能至关重要。低水平的c-Mpl应答支持HSC静止，而高水平的信号传导应答允许HSC增殖，但调节机制尚不清楚。对来自条件性缺失的MPL 1小鼠的HSC的分析显示Mpl-TR同种型与Mpl-FL（全长）的比率显著增加。Mpl-TR已经描述了在体外显性负功能并且在体内损害HSC植入。C-Mpl 1与c-Mpl RNA上的可变剪接区复合结合。此外，对c-Mpl的可变剪接区的Mp 11依赖性表观遗传修饰，包括组蛋白脱乙酰化和H3 K4 me 3标记，提供了能够调节Mpl-TR：Mpl-FL比率并由此调节对Thpo的应答的潜在机制。Notch的激活也有利于Mpl-TR的产生，其通过Notch 1与Rbpj的相互作用，可以解释骨髓小生境如何调节应激期间HSC的静止和增殖。将研究c-Mpl选择性剪接的分子机制，并建立相关的表观遗传修饰剂的作用。将检查Notch 1、Rbpj和c-Mpl之间的联系作为Notch控制HSC Thpo应答的机制。最后，将确定Mpl-TR对与承受造血应激相关的HSC增殖和静止的影响。综合策略，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，在这个建议可能会发现新的药理学方法，以解决人类疾病所产生的损害HSC功能，如细胞毒性化疗，移植失败后干细胞移植和骨髓衰竭综合征，如再生障碍性贫血的恢复。