We plan a comprehensive study of the role of specific methylation of DNA in the control of globin gene expression during avian erythroid differentiation. By using recombinant DNA technology, molecular biological methods, and chicken globin gene clones isolated and characterized by the principal investigator, we plan to investigate the role of DNA methylation both in vitro and in vivo. The avian model system was chosen because of our previous experience with it, and its technical advantages for conducting investigations of DNA methylation. Our in vivo studies will examine the differences in site-specific DNA methylation in active versus inactive globin genes 1) during the normal avian developmental globin gene switch, 2) during stress induced Alpha-globin gene switching in adult animals, and 3) during induction of avian erythroblastosis virus transformed cells to globin production. The latter system will also allow us to test the biologic effect which perturbation of DNA methylation can have upon differentiation and globin gene expression. In vitro studies will be aimed at elucidating physical and biochemical effects of methylation upon DNA structure and its ability to act as a transcriptional template. The ultimate goal of this research project is to shed light on the basic mechanism of control of cellular differentiation of the level of gene expression. Such knowledge would be applied directly to hereditary diseases such as the thalassemia syndromes, in which the defect lies at the level of globin gene expression and in which manipulation of globin expression could lead to correction of the disorder. Perhaps of greater impact on human disease would be the potential for understanding controls involved in normal differentiation and the abnormal differentiation mechanisms present in hereditary developmental defects and leukemia or other cancerous conditions.