GENE REGULATION OF THE CYTOKINES IL-3 AND GM-CSF

细胞因子 IL-3 和 GM-CSF 的基因调控

• 批准号: 3242635
• 负责人: BERNARD MATHEY-PREVOT
• 金额: $ 18.52万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3242635
• 关键词: DNA binding protein; T lymphocyte; cell growth regulation; cell type; chromatin; clone cells; colony stimulating factor; complementary DNA; cytokine; fibroblasts; gene expression; genetic regulation; genetic transcription; granulocyte; hematopoietic stem cells; immunogenetics; interleukin 3; methylation; molecular cloning; neurons; nucleic acid sequence; nucleoproteins; oligonucleotides; pancreatic ribonuclease; transfection

The proliferation and maturation of hematopoietic stem and progenitor cells is strongly affected by the availability and supply of hematopoietic growth factors, particularly interleukin-3 (IL3) and granulocyte-macrophage colony simulating factor (GM-CSF). As a result, regulation of IL-3 and GM-CSF gene expression will weigh heavily in the process of hematopoietic differentiation. To gain some insights into the molecular events underlying their regulation, IL-3 and GM-CSF gene expression will be first be examined in a model cell line, MLA 144 gibbon T leukemic cells. It will subsequently be broadened to human T lymphocytes, macrophages and fibroblasts. Two broad issues will be addressed. First, experiments are described to identify DNA regions of IL-3 and GM-CSF genes responsible for transcriptional activation, and to characterize and clone nuclear factors which interact with such regions. Transient transfection and RNase protection assays will be performed to identify the regulatory DNA regions which confer transcriptional activation of the cytokine genes. These DNA sequences will be used to identify nuclear factors in gel retardation assays, and various criteria will be applied to characterize the nuclear protein-DNA complexes, including DNA sequence specificity, DNA footprinting, and methylation interference. Mutagenesis of the contact points between DNA and the bound factors will be performed and its effect on DNA binding and transcriptional activation of reporter genes in which the mutated motif replaces wild-type sequences will be assessed. Nuclear factors which are confirmed to participate in the regulation of expression of the two cytokines will be cloned by screening cDNA expression libraries with oligonucleotides consisting of tandem repeats of the bound DNA sequence, or by direct expression in COS cells and gel retardation assay. Also, the nature and role of jun and fos (or their related proteins) will be evaluated in the activation of the IL-3 gene. Second, we will investigate in molecular terms how expression of IL-3 and GM-CSF, in contrast to T cells, is uncoupled in macrophage and fibroblast cells. Chromatin structure, promoter activities and DNA nuclear proteins will be compared and characterized in the different cell types using the same techniques listed earlier. Molecular cloning of new nuclear factors, if identified, will also be carried out. In addition to these topics, the possibility of IL-3 expression by human neuronal cells will be investigated by conventional RNA analyses.

造血干/祖细胞的增殖与成熟 受到造血生长的可用性和供应的强烈影响 因子，特别是白细胞介素-3（IL-3）和粒细胞-巨噬细胞集落 模拟因子（GM-CSF）。因此，IL-3和GM-CSF基因的调节 在造血过程中， 分化为了深入了解 它们的调节、IL-3和GM-CSF基因表达将首先被检查 在模型细胞系MLA 144巨噬细胞T白血病细胞中。随后， 扩大到人T淋巴细胞、巨噬细胞和成纤维细胞。两大 问题将得到解决。首先，描述了识别DNA的实验 IL-3和GM-CSF基因负责转录的区域 激活，并表征和克隆相互作用的核因子， 这些地区。瞬时转染和RNA酶保护测定将 进行鉴定的调控DNA区域，赋予 细胞因子基因的转录激活。这些DNA序列将 用于在凝胶阻滞测定中鉴定核因子，以及各种 将应用标准来表征核蛋白-DNA复合物， 包括DNA序列特异性、DNA足迹和甲基化 干扰DNA与结合物接触点的诱变 因子将进行及其对DNA结合和转录的影响 其中突变基序取代野生型的报告基因的激活 将评估序列。已证实的核因子 参与调节这两种细胞因子的表达， 通过用寡核苷酸筛选cDNA表达文库来克隆 由结合的DNA序列的串联重复序列组成，或通过直接 COS细胞中的表达和凝胶阻滞测定。此外，性质和 jun和fos（或其相关蛋白）的作用将在 IL-3基因的激活。其次，我们将从分子角度进行研究 与T细胞相比，IL-3和GM-CSF的表达如何解偶联， 巨噬细胞和成纤维细胞。染色质结构，启动子活性 和DNA核蛋白将进行比较和表征， 不同的细胞类型使用相同的技术之前列出的。分子 如果发现新的核因子，也将进行克隆。在 除了这些主题，IL-3表达的可能性，由人类 将通过常规RNA分析研究神经元细胞。