Our long term objective is to develop a detailed understanding of globin gene activation during erythroid cell differentiation. To model these events we are characterizing globin gene expression during the differentiation of murine erythroleukemia (MEL) cells. We have identified three proteins that bind specifically to the promoter of the alpha-globin gene DNase I footprinting and electrophoretic shift assays were used to monitor the enrichment of each protein through several chromatographic steps. DNA sequence affinity chromatography was then used to purify each protein. We propose to employ these purification protocols to accumulate a sufficient amount of each factor to obtain amino acid sequence data. These data will be used to prepare oligonucleotide probes that will be used to screen MEL cell cDNA libraries. In addition, direct screens of MEL cell cDNA expression libraries will be initiated. For these experiments, double-stranded oligonucleotide comprised of the binding site of each protein will be synthesized, ligated, nick-translated, and used to probe cDNA expression libraries. The identification of clone that encode alpha-globin specific DNA binding proteins will allow us to compare these proteins to other DNA binding factors, to characterize the functional domains of each protein, and to investigate their tissue distribution, synthesis, and turnover. Antisera could also be prepared against synthetic peptides derived from the deduced amino acid sequence of each protein. This analysis will help us to obtain a deeper understanding of how nuclear proteins regulate cell growth and differentiation. In this regard it is of interest to note that MEL cells are transformed erythroid precursors arrested in differentiation at the CFU-e state. We have shown, however, that MEL cell differentiation leads to characteristic changes in the activities of the identified alpha-specific binding proteins. It is not unreasonable to speculate that an understanding of how these factors function and how their activities are modified during differentiation might lead to the development of novel strategies for selectively altering factor activities. Because these factors appear to play important roles in mediating growth and differentiation, these insights could have important implications for approaches aimed at altering the uncontrolled growth and lack of differentiation characteristic of many tumor cells.
