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GENE REGULATION OF THE CYTOKINES IL3 AND GM CSF

细胞因子 IL3 和 GM CSF 的基因调控

基本信息

批准号：
2141906
负责人：
BERNARD MATHEY-PREVOT
金额：
$ 20.87万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
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

项目摘要

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、祖细胞的增殖和成熟在很大程度上受到可获得和供应的造血生长的影响因子，特别是白细胞介素3(IL3)和粒-巨噬细胞集落模拟因子(GM-CSF)。因此，IL-3和GM-CSF基因的调控表达将在造血过程中发挥重要作用差异化。以获得对潜在的分子事件的一些见解他们的调节，IL-3和GM-CSF的基因表达将首先被检测在一个模型细胞系中，MLA 144长臂猿T白血病细胞。随后，它将扩增至人T淋巴细胞、巨噬细胞和成纤维细胞。两个宽问题将得到解决。首先，描述了识别dna的实验。 IL-3和GM-CSF基因转录调控区激活，并鉴定和克隆相互作用的核因子有这样的地区。瞬时转染和核糖核酸酶保护分析将被用来识别与细胞因子基因的转录激活。这些DNA序列将用于确定凝胶滞留分析中的核因素，以及各种标准将被应用于描述核蛋白-DNA复合体，包括DNA序列特异性、DNA足迹和甲基化干扰。DNA与结合蛋白接触点的致突变作用因子及其对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对序列进行评估。已确认的核因素参与调控的两种细胞因子的表达将被利用寡核苷酸筛选cDNA表达文库进行克隆由结合的DNA序列的串联重复组成，或通过直接在COS细胞中的表达及凝胶滞留实验。另外，大自然和 Jun和Fos(或其相关蛋白)的作用将在激活IL-3基因。第二，我们将从分子的角度进行研究与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核蛋白将在使用前面列出的相同技术的不同细胞类型。分子如果确定了新的核因子，也将进行克隆。在……里面除了这些话题，人类表达IL-3的可能性神经细胞将通过常规的RNA分析进行研究。