CHARACTERIZATION OF CYTOKINE LD78 GENE PROMOTERS - POSITIVE AND NEGATIVE TRANSCRIPTIONAL FACTORS BIND TO A NEGATIVE REGULATORY ELEMENT COMMON TO LD78, INTERLEUKIN-3, AND GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE PROMOTERS

Cytokine LD78 is a human counterpart of the mouse macrophage inflammatory protein 1alpha/hematopoietic stem cell inhibitor. Promoters of the LD78alpha and LD78beta genes showed similar inducible activities in two leukemic cell lines, K562 and Jurkat, but the induction mechanisms differed between the two cell lines. Further characterization of the LD78alpha promoter indicated that multiple positive and negative regulatory elements are present, some of which are differentially required for induction and repression of the promoter activity in different cells. One of the negative regulatory elements, ICK-1, functioned in both cell lines in the absence and presence of stimulation and was shown to be a recognition site for positive and negative transcriptional factors. This ICK-1 element contained a direct repeat, and similar repeats were also found in the negative regulatory elements of hematopoietic growth factor interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoters. Nuclear extracts from K562 and Jurkat cells formed several protein-DNA complexes with the LD78alpha ICK-1 element, one of which was also observed with the IL-3 and GM-CSF ICK-1 elements. Results from in vivo and in vitro analyses suggested that the protein forming this complex functions as a negative factor. The binding affinity of this protein, ICK-1A, to the LD78alpha ICK-1 element was low and was significantly affected by the incubation temperature and the salt concentration in the binding buffer. ICK-1B, another protein bound specifically by the LD78alpha ICK-1 element, was shown to be a positive factor important for induction of the promoter. These results suggested that ICK-1A plays an important role in balanced expression of LD78, IL-3, and GM-CSF during hematopoietic cell growth and differentiation.