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

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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.