GENE REGULATION OF HUMAN INTERLEUKIN-9 (IL-9)

编码人IL-9的cDNA从HTLV-I转化的人 基于其的通过哺乳动物细胞表达克隆T细胞系的方法 刺激因子依赖性人类细胞增殖的能力 巨核细胞白血病细胞系 新分离的细胞因子随后 已显示支持体外人红系祖细胞的生长。 这项提案的长期目标是了解人类的作用， IL-9在分子和细胞水平上调节造血。 的 拟议工作的具体目标是：（1）确定监管机制 人IL-9基因在分子和细胞水平的表达。 这 将通过（i）分析IL-9基因所需的顺式元件 通过测量质粒构建体的启动子活性 含有IL-9 5 ′-侧翼区上游的不同部分， 荧光素酶报告基因在不同刺激下的各种细胞类型中， (ii)检查负责IL-9基因表达的反式作用因子 通过DNA酶I足迹、凝胶阻滞、甲基化干扰 测定和位点特异性诱变，和（iii）启动 反式作用因子及其分子的鉴定和纯化 克隆编码这些蛋白质的基因;（2）评估可能的 HTLV-I相关白血病中人IL-9表达的机制。 我们将 测试人IL-9在HTLV转化的细胞中的组成型表达是否 细胞是由于（i）由HTLV tax引起的反式激活机制 蛋白质或紫杉醇诱导的蛋白质或（ii）插入/激活机制 通过病毒整合。 这种新的基因调控的研究 细胞因子应该导致更好地理解调节机制 人IL-9在复杂的细胞因子网络中的产生， 人IL-9可能参与白血病的发生。

The cDNA encoding human IL-9 was isolated from a HTLV-I transformed human T cell line through mammalian cell expression cloning method based on its ability to stimulate the proliferation of a factor-dependent human megakaryocytic leukemic line. The newly isolated cytokine has subsequently been shown to support the growth of human erythroid progenitors in vitro. The long term objective of this proposal is to understand the role of human IL-9 in regulating hematopoiesis at the molecular and cellular level. The proposed work specifically aims to (1) determine the regulatory mechanism of human IL-9 gene expression at the molecular and cellular level. This will be achieved by (i) analyzing the cis-elements required for IL-9 gene expression through measuring the promoter activities of plasmid constructs containing various portions of IL-9 5'-flanking region upstream from a luciferase reporter gene in various cell types under different stimuli, (ii) examining the transacting factors responsible for IL-9 gene expression through DNase I footprinting, gel retardation , methylation interference assay and site-specific mutagenesis and (iii) initiating the characterization and purification of the transacting factors and molecular cloning for the genes encoding these proteins; (2) evaluate the possible mechanism of human IL-9 expression in HTLV-I associated leukemia. We will test whether the constitutive expression of human IL-9 in HTLV-transformed cells is due to (i) the transactivation mechanism elicited by HTLV tax protein or tax-induced proteins or (ii) the insertion/activation mechanism through viral integration. The study of gene regulation of this new cytokine should lead to a better understanding of the regulatory mechanism of human IL-9 production in the complicated cytokine network and the possible involvement of human IL-9 in leukemogenesis.