Differentiation is intimately linked to tightly regulated temporal, quantitative,and tissue-specific gene expression. Knowledge of the molecular control of gene expression during myeloid differentiation is limited by a paucity of well defined models for this process. CD18 is the Beta chain of the leukocyte integrins, a family of antigens which mediate cell adhesive functions that are critical to the immune and inflammatory responses. In adequate expression of CD18 causes the severe immunodeficiency known as Leukocyte Adhesion Deficiency (LAD). Because CD18 transcription increases markedly during myeloid differentiation, this gene provides a relevant model for lineage-specific gene transcription during myeloid differentiation. The CD18 gene has been cloned, and the promoter and flanking regions isolated. The CD18 promoter contains potential AP-1, CREB, and Oct sites; a binding site for the leukocyte specific transcription factor PU.1; a putative myeloid specific element; and a potential retinoic acid response element. The CD18 promoter directs expression in a leukocyte-specific manner, and is responsive to phorbol ester and retinoic acid induced myeloid differentiation. By sequential deletion, a region encoding high level myeloid activity was localized. Binding to two sites in this region by PU.1 is required for high level myeloid activity of the CD18 promoter. Scanning mutagenesis of the promoter localized other positive and negative regulatory elements. Numerous nuclear proteins bind to the CD18 promoter in electrophoretic mobility shift assay (EMSA) and DNAse I footprinting. Some of these factors, including other ets family members and the Sp1 transcription factor, have now been identified. This proposal seeks to identify the cis DNA elements and the trans acting factors which regulate CD18 expression. Transient and stable transection will be used to define the functional DNA elements which control lineage- specific and inducible CD18 expression EMSA and DNAse I footprinting will be used to further characterize nuclear factors which bind to CD18 regulatory DNA elements. Greater understanding of the molecular control of CD18 expression may have an impact on the pathogenesis and therapy of LAD. Because chromosomal rearrangements in some forms of acute leukemia generate novel proteins which resemble transcription factors, myeloid trans acting factors may play an important etiologic role in leukemia.
