Functions of the TG-Interacting Transcription Factor (TGIF) in Hematopoiesis

描述（由申请人提供）：我们已经发现，干细胞表达的转录抑制因子转化生长干扰因子（TGIF）的定量表达是急性髓性白血病（AML）患者生存的最具鉴别性和最强大的预测因子。较低水平的TGIF与较短的生存期相关，而较高水平的TGIF预测较长的生存期（p=0.00001）。为了了解我们临床发现的生物学基础，我们研究了TGIF在骨髓祖细胞系和Tg/'f敲除小鼠模型中的功能。在髓系祖细胞系（如HL60）中，TGIF敲低会导致显著的生长抑制、相对分化阻断和髓系转录程序的中断。对Tg/7缺失小鼠造血干细胞（HSC）功能的分析表明，在竞争性移植研究中，与野生型HSC相比，HSC产生的集落形成单位更少，具有生长优势。这些数据表明，敲除Tg/f可能会改变HSC静止、分化和自我更新等关键功能之间的平衡。因此，我们假设Tgif在HSC功能中起着基本的调节作用，并将通过以下方式来检验这一假设：1)更彻底地定义Tgif在HSC和祖细胞功能中的作用；2)确定Tgif对hsc作用的细胞基础；3)确定Tgif对造血干细胞作用的分子基础。这些目标将在使用各种标准和最先进的分子技术的一系列实验中进行，包括限制稀释竞争再种群分析，系列移植，体内细胞周期分析，以及使用自然发生的TGIF突变和体外HSC转导的结构-功能表征。除了目前使用的Tgif敲除模型外，我们还将采用一种新的条件Tgif敲除等位基因，这将为我们的实验设计提供很大的灵活性。该提案还概述了一项计划，旨在帮助候选人在学术医学领域取得成功。该候选人拥有范德比尔特大学微生物学和免疫学博士学位，并完成了儿科住院医师培训和遗传学奖学金培训。候选人将在博士的指导下扩展其造血和白血病发生的知识。斯蒂芬·勃兰特和斯科特·希伯特。指导委员会将定期提供额外的指导。相关性（见说明书）：在急性白血病中，包括AML，造血干细胞的静止、自我更新和分化之间的正常平衡被破坏。我们的初步数据表明，Tgif可能调节这种平衡，并且它在AML中也具有作为预后指标的临床作用。因此，我们对Tgif如何调节关键HSC功能的研究将为HSC生物学提供新的见解，并增加我们对AML发病机制的理解，从而更好地诊断和治疗这种毁灭性疾病。

DESCRIPTION (provided by applicant): We have discovered that quantitative expression of the stem cell expressed transcriptional represser Transforming-growth-interfering factor (TGIF) is the most discriminating and powerful predictor of patient survival identified in acute myelogenous leukemia (AML). Lower levels of TGIF correlated with shorter survival, whereas higher levels predicted longer survival (p=0.00001). To understand the biological basis for our clinical finding, we investigated TGIF's function in myeloid progenitor cell lines and in a Tg/'f knockout mouse model. TGIF knockdown in myeloid progenitor cell lines, such as HL60, resulted in significant growth inhibition, a relative differentiation block, and disruption of the myeloid transcription program. Analysis of hematopoietic stem cell (HSC) function in Tg/7-null mice showed that the HSCs produced fewer colony- forming units and had a growth advantage compared to wild-type HSCs in competitive transplant studies. These data suggest that Tg/f knockout may alter the balance between the key HSC functions of quiescence, differentiation and self-renewal. We thus hypothesize that Tgif plays a fundamental regulatory role in HSC function and will examine this hypothesis by 1) defining more thoroughly the role of Tgif'm HSC and progenitor cell function; 2) determining the cellular basis of Tgif's effects on HSCs; and 3) determining the molecular basis of Tgif's effects on HSCs. These aims will be carried out in a series of experiments using a variety of standard and state-of-the-art molecular techniques, including limiting dilution competitive repopulation analysis, serial transplantation, in vivo cell cycle analysis, and structure-function characterizations using naturally occurring TGIF mutations and ex vivo HSC transduction. In addition to the Tgif knockout model currently in use, we will also employ a new conditional Tgif knockout allele, which will offer us great flexibility in our experimental design. This proposal also outlines a plan designed to help the candidate develop a successful career in academic medicine. The candidate has a Ph.D. in Microbiology and Immunology from Vanderbilt University and has completed residency training in Pediatrics and fellowship training in Genetics. The candidate will expandhis knowledge of hematopoiesis and leukemogenesis under the mentorship of Drs. Stephen Brandt and Scott Hiebert. A mentoring committee will provide added guidance at regular intervals. RELEVANCE (See instructions): In acute leukemias, including AML, the normal balance between quiescence, self-renewal and differentiation of HSCs is disrupted. Our preliminary data suggest that Tgif may regulate this balance and that it also has a clinical role as a prognostic indicator in AML. Thus, our investigations of how Tgif regulates key HSC functions will provide new insights into HSC biology as well as increase our understanding of AML pathogenesis, leading to better diagnosis and treatment of this devastating disease.