The Role of Zinc Finger Genes in Leukemogenesis

项目总结 这项研究的目的是为了了解造血细胞的恶性转化。 通过鉴定Ikaros蛋白的生物学功能。伊卡洛斯对造血发育和 作为一种肿瘤抑制因子。Ikaros基因交替拼接以产生多个锌指蛋白 参与基因调控和染色质重塑。 我们的初步数据显示：1)Ikaros在进化上保守的多个位置被磷酸化，通过 细胞周期G1期和S期的CK2和其他激酶2)Ikaros在特定氨基上的磷酸化 酸调节其DNA结合能力和亚细胞定位。3)Ikaros与Bclxl基因启动子结合 在体内，Ikaros活性的失调与Bclxl基因的表达上调有关。 我们建议的具体目标是：具体目标#1：确定特定的磷酸化位点 负责Ikaros在转录、细胞增殖和分化的调节功能。我们 假设Ikaros的磷酸化干扰其转录调控功能和 染色质重塑并影响细胞增殖。我们将定义关键的磷酸化位点 Ikaros在DNA结合、亚细胞定位和蛋白质-蛋白质相互作用中的作用。伊卡洛斯的角色 将使用小鼠白血病细胞系统研究控制细胞周期进展的磷酸化 来源于伊卡洛斯缺陷小鼠。这些细胞将用野生型或Ikaros模拟磷酸盐进行转导 突变体来定义对Ikaros在细胞周期控制中的功能至关重要的位点。伊卡洛斯的角色 磷酸化在调节T细胞分化和T细胞增殖中的作用将在体内研究。小鼠干细胞 靶向破坏Ikaros的小鼠细胞将被含有野生型的逆转录病毒载体感染 伊卡洛斯或仿磷伊卡洛斯突变体，并移植到亚致死剂量的伊卡洛斯基因敲除小鼠体内。 拟磷化的伊卡洛斯突变体恢复正常T细胞分化的能力将与 野生型伊卡洛斯。具体目的#2：剖析Ikaros调控Bclxl表达的机制。 以往的研究表明，Ikaros活性降低会导致Bclxl基因的过度表达。我们 假设Ikaros通过负向调节Bclxl的表达而发挥其肿瘤抑制活性。为了测试 这一假设，我们将确定增加的Ikaros表达是否下调了Bclxl的转录 我们将绘制出Bcl-xl上游调控元件(URE)的区域，这些区域是 对Ikaros调控的Bclxl表达至关重要。 这些研究将首次提供对信号转导途径的详细功能分析， 控制Ikaros的肿瘤抑制功能。我们的研究将提供新的重要信息 控制造血细胞增殖的机制及其对病理生理学的影响 以及白血病的治疗。

PROJECT SUMMARY The objective of the proposed study is to understand the malignant transformation of hematopoietic cells by identifying the biological functions of Ikaros proteins. Ikaros is essential for hematopoietic development and acts as a tumor suppressor. The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Our preliminary data show that: 1) Ikaros is phosphorylated at multiple evolutionarily conserved sites by CK2 and other kinases during G1 and S phase of the cell cycle 2) Phosphorylation of Ikaros at specific amino acids regulates its DNA-binding ability and subcellular localization. 3) Ikaros binds to the Bcl-xL gene promoter in vivo and disregulation of Ikaros activity is associated with upregulation of Bcl-xL gene expression. The specific aims of our proposals are: Specific Aim #1: To identify the specific phosphorylation sites responsible for Ikaros function in the regulation of transcription, cellular proliferation and differentiation. We hypothesize that phosphorylation of Ikaros interferes with its function in transcriptional regulation and chromatin remodeling and influences cellular proliferation. We will define phosphorylation sites that are critical for Ikaros function in DNA-binding, subcellular localization, and protein-protein interaction. The role of Ikaros phosphorylation in controlling cell cycle progression will be studied using a murine leukemia cellular system derived from Ikaros deficient mice. These cells will be transduced with wild type or Ikaros phosphomimetic mutants to define sites that are critical for Ikaros' function in cell cycle control. The role of Ikaros' phosphorylation in regulating T cell differentiation and T cell proliferation will be studied in vivo. Murine stem cells from mice with targeted disruption of Ikaros will be infected with retroviral vectors containing wild type Ikaros or phosphomimetic Ikaros mutants and transplanted into sublethally irradiated Ikaros knockout mice. The ability of phosphomimetic Ikaros mutants to restore normal T cell differentiation will be compared to that of wild type Ikaros. Specific aim #2: To dissect the mechanism by which Ikaros regulates Bcl-xL expression. Previous studies suggest that decreased Ikaros activity leads to overexpression of the Bcl-xL gene. We hypothesize that Ikaros exerts its tumor suppressor activity by negatively regulating Bcl-xL expression. To test this hypothesis we will determine whether increased Ikaros expression downregulates Bcl-xL transcription in human lymphoma cells and we will map the regions of the Bcl-xL upstream regulatory element (URE) that are critical for Ikaros-modulated control of Bcl-xL expression. These studies will provide the first detailed functional analysis of the signal transduction pathways that control the tumor suppressor function of Ikaros. Our research will provide new and important information on the mechanisms controlling the proliferation of hematopoietic cells and will yield insights into the pathophysiology and treatment of leukemia.