Interleukin-3 and GMCSF are hematopoietic growth factors (HGFs) important for the proliferation and maturation of hematopoietic progenitor cells. These HGFs are both coordinately and independently regulated. This laboratory has been principally interested in understanding at a molecular level how, where, and under what circumstances IL-3 is produced. This proposal extends previous work by further investigating the transcriptional regulation of IL-3 gene expression directed by the proximal IL-3 promoter and studying the mechanism of coordinate expression of IL-3 and GMCSF. First, the proximal IL-3 promoter appears to be sensitive to cyclosporin-A (CsA). A CsA-sensitive protein complex, distinct from NFAT, binds to the IL-3 proximal promoter at the Act-1 site. This complex will be characterized and isolated by expression cloning or purification. Second, the IL-3 promoter contains a CBF site that is necessary but not sufficient for gene activation. Function of this site appears to require the adjacent Act-1 region of the IL-3 promoter. The functional interdependence of the CBF and Act-1 """"""""unit"""""""" and its potential role on tissue-restricted expression of IL-3 will be explored. Expression mediated by IL-3 promoters containing various combinations of mutated CBF and/or Act-1 sites will be measured by a luciferase assay after transient transfection into both T- and non-T cells. In addition, the same assay and promoter constructs will be used to investigate the functional consequence of mutant CBF proteins created by the t(i,21) and inv(16) chromosomal rearrangements associated with M2- and M4Eo-acute myelogenous leukemia. The IL-3 promoter is uniquely suited for these studies as the function of the CBF site in this promoter is well known. Third, preliminary experiments identify a new, positive regulatory activity between -160 bp and -210 bp of the IL-3 promoter. There are two independent protein binding regions within this promoter segment. Mutational analysis will be used to identify the functionally relevant binding activity, and the protein(s) will be characterized and isolated as above. Finally, the coordinate regulation of IL-3 and GMCSF will be studied by testing the activity of an intergenic DNAse I hypersensitive region, containing an NFAT binding site, on both the IL-3 and GMCSF promoters. Similarities between the promoter elements of IL-3, GMCSF, and other genes important for normal hematopoiesis make the results obtained from their experiments applicable beyond these HGFs. The specific experiments proposed are designed to further the understanding of the interdependence between regulatory sites within a promoter with the explicit anticipation that this information will be relevant to the future construction of artificial promoter elements.
| Taylor, D S; Lee, Y; Sieff, C A et al. (1998) Phase I/II trial of PIXY321 (granulocyte-macrophage colony stimulating factor/interleukin-3 fusion protein) for treatment of inherited and acquired marrow failure syndromes. Br J Haematol 103:304-7 |
