Many genes expressed by multicellular organisms are expressed in one cell type but not another. Two cis-acting elements, promoters and enhancers have been shown to be responsible for tissue specificity. However, the biochemical basis for this cell type specificity is not clearly understood. We are using the K562 leukemic cell line as a model system to increase our understanding of events associated with tissue specific and developmental expression in normal marrow progenitor cells. The human globin genes exhibit a high degree of sequence conservation not only in their coding region but also in their 5'- flanking regions. Despite the considerable degree of sequence homology, the globin genes are expressed in a distinct developmental manner. Therefore, this is an interesting system for studying the co-evolution of cis- and trans-acting elements in addition to investigating the molecular mechanisms which control tissue and developmental specific gene expression. To this end we are using techniques which will enable us to identify and characterize trans-acting factors which interact with the (epsilon) embryonic globin gene, which is the predominate hemoglobin message in K562 cells. We have used a labelled DNA fragment containing the epsilon promoter in a DNA-protein binding assay. We have been able to detect a DNA-protein complex using a 0.3M KC1 nuclear extract and the epsilon promoter fragment. We have determined that the complex is specific by competition assays. At present we are attempting to footprint the DNA-protein complex and identification of the protein is being attempted by UV cross-linking experiments.
