Our long term objective is to develop a detailed understanding of erythroid cell differentiation. As such, the specific aims of the project have been to purify, and characterize nuclear factors that interact with the alpha- beta-globin genes. The purification of three identified factors has been published. A fourth factor, found only in nuclear extracts prepared from erythroid cells, has also been purified. Preliminary results show that all factors modulate transcription of globin gene promoters in vitro. Other factors have been identified, but not purified. We propose to extend our results by purifying these additional erythroid cell factors. We also propose to begin an extensive physical characterization of factors already purified. Physical characterization will include peptide mapping, amino acid sequence determination, characterization of factor subunit composition and structure, and determining if factors are phosphorylated or glycosylated. In addition, we intend to prepare polyclonal antisera against each purified factor. Antisera will be used to determine factor distribution, synthesis and turn-over rates, and potential associations with other cellular proteins. Lastly, we will continue to employ our erythroid cell derived in vitro transcription system to assess further the role each purified factor plays in mediating globin gene transcription. Our analysis will help us 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. It is not unreasonable to speculate that a detailed understanding of the physical structure and function of each identified factor might lead to a deeper appreciation of how nuclear factors transduce environmental signals that regulate growth and differentiation. These insights might provide novel strategies for selectively altering factor activities with important implications for approaches aimed at altering the uncontrolled growth and lack of differentiation characterization of many tumor cells.
