Role of Cited2 in Hematopoiesis

描述（由申请人提供）：Cited2 [CBP/p300-与谷氨酸(E)和天冬氨酸(D)-富尾2相互作用的transactivators]是一个新的转录调节剂家族的创始成员之一。我的实验室克隆了Cited2，发现Cited2可被多种细胞因子诱导，过表达时为转化基因。我们产生了Cited2缺失的小鼠，发现Cited2缺失会导致胚胎在妊娠中后期死亡，并伴有几种发育缺陷。由于Cited2的表达与ltr - hsc活性呈正相关，并诱导Bmi-1的表达，而Bmi-1是胚胎和成体干细胞维持和更新所必需的多巢群基因，因此我们研究了Cited2在胎儿肝脏造血中的作用。在不同谱系中，Cited2-/-胎儿肝脏显示出造血细胞数量的显著减少。体外CFU和体内重构研究表明，Cited2在胎儿肝脏造血中起重要作用。一致地，微阵列分析和实时RT-PCR分析在Cited2-/-胎儿肝脏中发现了几个与HSC和祖细胞功能障碍相关的差异表达基因。我们已经证明，在发育过程中的某些组织中，Cited2是HIF-1的负调节因子。最近，其他研究表明FoxO3在缺氧条件下被激活，并通过Cited2抑制hif -1诱导的细胞凋亡，并且在FoxO三敲除小鼠中发现Cited2的表达减少，这表明Cited2是FoxO转录因子的下游靶点。由于骨髓中的局部缺氧在调节干细胞功能中起着重要作用，而FoxOs是HSC抗氧化应激和维持HSC池的关键介质，我们想通过条件敲除的方法研究成人造血过程中Cited2、HIF-1、FoxOs和Bmi-1之间的串音，来验证FoxO/Cited2/HIF-1通路和Bmi-1在调节HSC功能中发挥重要作用的假设。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（目的1）。接下来将进行两项机制研究，以了解Cited2的作用。除了关注特定的分子，如Bmi-1 (Aim 2), FoxO3/HIF-1 （Aim 3）及其相关途径外，还将采用更全基因组的方法来发现新的机制。因此，所提出的研究将促进我们对正常和异常HSC生物学调节的分子机制的认识，从而可能为针对HSC的治疗开发提供新的见解，用于再生医学和治疗不同的血液系统恶性肿瘤。公共卫生相关性：这一建议不仅将为我们理解正常造血和干细胞生物学提供关键信息，而且还将阐明体内转录因子/辅助因子与信号分子之间复杂的相互作用，这可能为白血病发生的分子发病机制提供新的线索，并有助于确定特异性治疗的靶点。

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.