Role of Cited2 in Hematopoiesis

描述(申请人提供)：Cited2[CBP/p300-与谷氨酸(E)和富含天冬氨酸(D)的尾部相互作用的反式激活因子2]是一个新的转录调节因子家族的创始成员之一。我的实验室克隆了Cited2，结果表明，Cited2受多种细胞因子的诱导，在过度表达时是一个转化基因。我们产生了Cited2基因缺失的小鼠，发现Cited2基因的缺失会导致胚胎在怀孕中后期死亡，并伴有几个发育缺陷。由于Cited2的表达与LTR-HSCs活性呈正相关，并诱导Bmi-1的表达，Bmi-1是胚胎和成人干细胞维持和更新所必需的多梳基因，因此我们研究了Cited2在胎肝造血中的作用。Cited2/-胎肝显示不同谱系的造血细胞数量显著减少。体外CFU和体内重建研究表明，Cited2在胎肝造血中起重要作用。微阵列分析和实时RT-PCR分析一致地确定了Cited2/-胎肝中与HSC和祖细胞功能障碍相对应的几个差异表达基因。我们已经证明，在发育过程中，Cited2是某些组织中HIF-1的负调控因子。最近，其他研究表明，FOXO_3在低氧条件下被激活，并通过Cited2抑制HIF-1诱导的细胞凋亡，并且在FoxO三基因敲除小鼠中发现Cited2的表达减少，从而使Cited2成为FoxO转录因子的下游靶点。由于骨髓局部缺氧在调节干细胞功能中起着重要作用，而Foxos是HSC抵抗氧化应激和维持HSC库的关键介质，我们拟采用条件基因敲除方法研究Cited2、HIF-1、Foxos和Bmi-1在成人造血中的相互作用，以验证FoxO/Cited2/HIF-1途径和Bmi-1在调节HSC功能中起重要作用的假设。该应用的总体策略是首先从功能上表征成人造血组织中的Cited2基因敲除表型(目标1)。接下来将进行两项机械论研究，以了解Cited2的行动。除了专注于Bmi-1(Aim 2)、FOX03/HIF-1(Aim 3)等特定分子及其相关途径外，还将同时采用更全基因组的方法来揭示新的机制。因此，这些研究将促进我们对调节正常和异常HSC生物学的分子机制的了解，从而为针对HSC用于再生医学和治疗不同血液系统恶性肿瘤的治疗开发提供新的见解。公共卫生相关性：这一建议不仅将提供对我们理解正常造血和干细胞生物学至关重要的信息，还将阐明体内转录因子/辅因子和信号分子之间的复杂相互作用，这可能为白血病发生的分子发病机制提供新的线索，并有助于确定特定治疗的靶点。

DESCRIPTION (provided by applicant): Cited2 [CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is one of the founding members of a new family of transcriptional modulators. My laboratory cloned Cited2 and showed that Cited2 is induced by many cytokines and it is a transforming gene when overexpressed. We generated Cited2 null mice and found that deletion of Cited2 causes embryos to die at mid- to late gestation with several developmental defects. Since Cited2 expression correlates positively with LTR-HSCs activity and induces the expression of Bmi-1, a polycomb group gene necessary for the maintenance and renewal of embryonic and adult stem cells, we studied the role of Cited2 in fetal liver hematopoiesis. Cited2-/- fetal liver displayed significant reduction in the numbers of hematopoietic cells in different lineages. In vitro CFU and in vivo reconstitution studies demonstrate that Cited2 plays an important role in fetal liver hematopoiesis. Consistently, microarray analysis and real-time RT-PCR analyses identified several differentially expressed genes corresponding to HSC and progenitor dysfunction in Cited2-/- fetal liver. We have shown that Cited2 is a negative regulator of HIF-1 in certain tissues during development. More recently, others showed that FoxO3 is activated under hypoxia and inhibits HIF-1-induced apoptosis through Cited2 and decreased expression of Cited2 was found in FoxO triple knockout mice, establishing Cited2 as a downstream target of FoxO transcription factors. Since regional hypoxia in the bone marrow plays an important role in regulating stem cell function and FoxOs are critical mediators of HSC resistance to oxidative stress and maintenance of the HSC pool, we would like to study the crosstalk between Cited2, HIF-1, FoxOs and Bmi-1 in adult hematopoiesis using conditional knockout approach to test the hypothesis that the FoxO/Cited2/HIF-1 pathway and Bmi-1 play an important role in regulating HSC functions. The overall strategy of the application is to first functionally characterize Cited2 knockout phenotypes in adult hematopoietic tissue (Aim 1). This will be followed by two mechanistic studies to understand Cited2 actions. In addition to focusing on specific molecules such as Bmi-1 (Aim 2), FoxO3/HIF-1 (Aim 3) and their associated pathways, a more genome-wide approach will also be undertaken in parallel to uncover novel mechanisms. The proposed studies therefore will advance our knowledge of molecular mechanisms regulating normal and abnormal HSC biology, and thus may offer new insights into the therapeutic development targeting HSC for regenerative medicine and for treatment of different hematological malignancies. PUBLIC HEALTH RELEVANCE: This proposal will not only provide information critical to our understanding of normal hematopoiesis and stem cell biology but also elucidate the complex interplay between transcription factors/cofactors and signaling molecules in vivo, which may provide new clues to the molecular pathogenesis of leukemogenesis and help identify targets for specific therapies.