FUNCTIONAL-CHARACTERIZATION OF THE PROMOTER REGION OF THE PLATELET-ACTIVATING-FACTOR RECEPTOR GENE - IDENTIFICATION OF AN INITIATOR ELEMENTESSENTIAL FOR GENE-EXPRESSION IN MYELOID CELLS

To understand the molecular mechanisms that direct the expression of t he gene encoding the platelet-activating factor (PAF) receptor, the 5' -flanking region of the human PAF receptor gene was cloned, and its pr omoter activity in myeloid cell lines was characterized By the 5'-rapi d amplification of cDNA ends method and primer extension, the transcri ption initiation site was mapped to an adenosine residue 137 bases ups tream of the ATG translation initiation codon, The promoter region lac ks a typical TATA or CCAAT box. However, the sequence encompassing the transcription initiation site shows high homology to the initiator (I nr) sequence. Transfection of promonocytic U937 cells with recombinant plasmids containing a series of truncated segments of the 5'-flanking region linked to the luciferase reporter gene revealed that the seque nce from nucleotides -44 to +27 relative to the transcription initiati on site was sufficient to promote a high level of gene expression, The promoter activity was much lower in nonexpressing HeLa cells and prom yeIocytic HL-60 cells, which express relatively low levels of the PAF receptor, Gel mobility shift analysis demonstrated the binding of nucl ear factors extracted from myelocytic cells to the -16/+18 sequence co ntaining the Inr element, No binding activity was detected using the n uclear extracts from the non-myelocytic HeLa cells, The DNA-protein co mplexes were sequence-specific since the binding was not significantly affected by the mutated Inr sequences or the Inr sequence of the term inal deoxynucleotidyltransferase gene, Furthermore, point mutations in the Inr element significantly reduced promoter activity in both U937 and THP-1 cell lines. When Me(2)SO or retinoic acid was used to induce granulocytic differentiation of HL-60 cells, a distinct Inr-protein c omplex was induced concurrently, but the complex was not observed in 1 2-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiated H L-60 cells or Me(2)SO-induced differentiated U937 cells, indicating th at the inducible Inr binding activity is granulocyte-specific. These r esults suggest that distinct nuclear factors interact with the unique Inr element and play a role in the transcriptional regulation of the P AF receptor in various myeloid cells.