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: 3242636
• 负责人: BERNARD MATHEY-PREVOT
• 金额: $ 19.23万
• 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/3242636
• 关键词: DNA binding protein; RNase protection assay; T lymphocyte; cell growth regulation; cell type; chromatin; clone cells; colony stimulating factor; complementary DNA; cytokine; fibroblasts; gel mobility shift assay; gene expression; genetic regulation; genetic transcription; granulocyte; hematopoietic stem cells; immunogenetics; interleukin 3; ion exchange chromatography; methylation; molecular cloning; neurons; nucleic acid sequence; nucleoproteins; oligonucleotides; pancreatic ribonuclease; transcription factor; 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.

造血干细胞和祖细胞的增殖和成熟 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 (IL3) 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 表达在造血过程中起着重要作用 差异化。深入了解潜在的分子事件 首先将检查它们的调节、IL-3 和 GM-CSF 基因表达 在模型细胞系 MLA 144 长臂猿 T 白血病细胞中。随后将 扩大到人 T 淋巴细胞、巨噬细胞和成纤维细胞。两宽 问题将得到解决。首先，描述鉴定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 与这些地区。瞬时转染和 RNase 保护测定将 进行以确定赋予的调控DNA区域 细胞因子基因的转录激活。这些DNA序列将 用于鉴定凝胶阻滞测定中的核因子，以及各种 标准将用于表征核蛋白-DNA 复合物， 包括 DNA 序列特异性、DNA 足迹和甲基化 干涉。 DNA 和结合物之间接触点的诱变 将执行的因素及其对 DNA 结合和转录的影响 报告基因的激活，其中突变基序取代野生型 将评估序列。已证实的核因素 参与这两种细胞因子表达的调节 通过用寡核苷酸筛选 cDNA 表达文库来克隆 由结合的 DNA 序列的串联重复组成，或通过直接 COS细胞中的表达和凝胶阻滞测定。此外，自然和 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 的表达如何在 巨噬细胞和成纤维细胞。染色质结构、启动子活性 和 DNA 核蛋白将在 不同的细胞类型使用前面列出的相同技术。分子 如果确定的话，还将进行新核因子的克隆。在 除了这些主题之外，人类表达 IL-3 的可能性 神经元细胞将通过常规 RNA 分析进行研究。