The development of the myeloid lineage from totipotent stem cells is the result of the expression of transcription factors that are either constitutively expressed or differentially induced at specific times during differentiation. A number of key transcription factors have been found that direct myeloid development. A number of specific genes affected by these transcription factors have been revealed and their expression contributes to the overall phenotype of myeloid cells. Understanding the process of normal myeloid development will require the identification of transcription factors and their co- factors that activate or silence lineage-specific genes. The restricted pattern of expression of the leukocyte integrin CD11d serves as an excellent model to identify some of these factors. CD11d is expressed on stem cells, myelomonocytic cells, and strongly on macrophage foam cells, and splenic red pulp and alveolar macrophages. Its expression pattern clearly differs from myeloid-specific leukocyte integrins CD11b and CD11c. We determined that CD11d is regulated during myeloid differentiation of stem cells, and during differentiation of monocytes to macrophages and foam cells. We propose that myeloid-specific transcription factors can be identified with a study of the CD11d promoter. Further, identification of cis elements and the interacting transcription factors that control CD11d expression could provide a means to direct expression of specific genes to myeloid cells in gene therapy approaches. The goals of this project are to identify myeloid-specific transcription factors that control CD11d expression and test their role in myeloid differentiation. We present evidence for 3 sites within a region in CD11d that confer myeloid-specificity, and that transcription factors that bind this region affect myeloid differentiation. One-hybrid analysis of this region led to the isolation of two myeloid-specific cDNAs. We also present evidence that CD11d expression and foam cell development may utilize shared transcription factors. Two silencers, one being cell-specific, are also in the CD11d locus. We propose to further characterize these and possibly other essential cis elements by site-directed mutagenesis, transfection analysis, DNase I hypersensitivity, mobility shift and DNase I footprint analyses. Yeast one-hybrid technology will be used to isolate novel myeloid-specific transcription factors. Model systems in which to study the CD11d promoter and myeloid-specific factors will include embryonic stem cells, peripheral blood monocytes, and myeloid cell lines. Identified factors will be up- or downregulated through forced overexpression or antisense approaches to determine their effect on myeloid differentiation. Transgenic analysis of the CD11d cis elements will be done to complement these studies. These studies will expand our understanding of normal myeloid development and leukemogenesis.
