The Role of Zinc Finger Genes in Leukemogenesis

锌指基因在白血病发生中的作用

• 批准号: 8296355
• 负责人: Sinisa Dovat
• 金额: $ 37.79万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8296355
• 关键词: Adolescent; Adult Lymphoma; Amino Acids; Binding; Biochemical; Biological; Biological Process; CD8B1 gene; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Cell physiology; Cells; Childhood; Childhood Acute Lymphocytic Leukemia; Chronic Lymphocytic Leukemia; DNA Binding; Data; Development; Down-Regulation; Exhibits; Functional disorder; G1 Phase; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hematopoietic; Human; Ikaros protein; Immune; In Vitro; Infant; Interleukin-2; Knockout Mice; Lymphoma; Malignant - descriptor; Malignant Neoplasms; Maps; Molecular; Mus; Nucleic Acid Regulatory Sequences; Patients; Peripheral; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Protein Dephosphorylation; Proteins; RNA Interference; RNA Splicing; Regulation; Regulatory Element; Research; Retroviral Vector; Role; S Phase; Signal Transduction; Signal Transduction Pathway; Site; Stem cells; System; T cell differentiation; T-Cell Proliferation; T-Lymphocyte; Testing; Transcriptional Regulation; Transplantation; Tumor Suppressor Proteins; Up-Regulation; Zinc Fingers; adult leukemia; bone marrow hyperplasia; chromatin remodeling; effective therapy; in vivo; insight; leukemia; leukemia/lymphoma; leukemogenesis; mouse model; mutant; novel; overexpression; promoter; protein protein interaction; reconstitution; research study; response; thymocyte

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.

项目概要 拟议研究的目的是了解造血细胞的恶性转化 通过鉴定 Ikaros 蛋白的生物学功能。 Ikaros 对于造血发育至关重要 作为肿瘤抑制剂。 Ikaros基因交替剪接生成多种锌指蛋白 参与基因调控和染色质重塑。 我们的初步数据表明：1) Ikaros 在多个进化保守位点被磷酸化 细胞周期 G1 和 S 期的 CK2 和其他激酶 2) Ikaros 在特定氨基处的磷酸化 酸调节其 DNA 结合能力和亚细胞定位。 3) Ikaros结合Bcl-xL基因启动子 Ikaros 活性的失调与 Bcl-xL 基因表达的上调有关。 我们提案的具体目标是： 具体目标#1：确定特定的磷酸化位点 负责 Ikaros 调节转录、细胞增殖和分化的功能。我们 假设 Ikaros 的磷酸化会干扰其转录调控功能 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-蛋白质相互作用中发挥作用。伊卡洛斯角色 将使用鼠白血病细胞系统研究控制细胞周期进程的磷酸化 源自 Ikaros 缺陷小鼠。这些细胞将用野生型或 Ikaros 磷模拟物转导 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 将在体内研究调节 T 细胞分化和 T 细胞增殖的磷酸化。鼠干 来自靶向破坏 Ikaros 的小鼠的细胞将被含有野生型的逆转录病毒载体感染 Ikaros 或拟磷 Ikaros 突变体并移植到亚致死照射的 Ikaros 基因敲除小鼠中。 拟磷化 Ikaros 突变体恢复正常 T 细胞分化的能力将与 野生型伊卡洛斯。具体目标#2：剖析 Ikaros 调节 Bcl-xL 表达的机制。 先前的研究表明，Ikaros 活性降低会导致 Bcl-xL 基因过度表达。我们 假设 Ikaros 通过负向调节 Bcl-xL 表达来发挥其肿瘤抑制活性。测试 根据这一假设，我们将确定 Ikaros 表达增加是否会下调 Bcl-xL 转录 人类淋巴瘤细胞，我们将绘制 Bcl-xL 上游调节元件 (URE) 的区域，这些区域是 对于 Ikaros 调节的 Bcl-xL 表达控制至关重要。 这些研究将对信号转导途径进行首次详细的功能分析， 控制Ikaros的抑癌功能。我们的研究将提供有关以下方面的新的重要信息 控制造血细胞增殖的机制并将深入了解病理生理学 和治疗白血病。