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 inflammat ory protein 1alpha/hematopoietic stem cell inhibitor. Promoters of the LD78alpha and LD78beta genes showed similar inducible activities in t wo leukemic cell lines, K562 and Jurkat, but the induction mechanisms differed between the two cell lines. Further characterization of the L D78alpha promoter indicated that multiple positive and negative regula tory elements are present, some of which are differentially required f or induction and repression of the promoter activity in different cell s. 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 facto rs. This ICK-1 element contained a direct repeat, and similar repeats were also found in the negative regulatory elements of hematopoietic g rowth factor interleukin-3 (IL-3) and granulocyte-macrophage colony-st imulating factor (GM-CSF) gene promoters. Nuclear extracts from K562 a nd Jurkat cells formed several protein-DNA complexes with the LD78alph a ICK-1 element, one of which was also observed with the IL-3 and GM-C SF ICK-1 elements. Results from in vivo and in vitro analyses suggeste d 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 temp erature and the salt concentration in the binding buffer. ICK-1B, anot her protein bound specifically by the LD78alpha ICK-1 element, was sho wn to be a positive factor important for induction of the promoter. Th ese 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.