This proposal is designed to test the hypothesis that trans-acting regulatory factors govern the expression of adult and embryonic globin genes in normal erythroid cells. Mutations (acquired or inborn) will be analyzed which appear to affect the expression of adult and embryonic globin genes in order to characterize the possible mechanisms by which developmentally specific patterns of globin gene expression can be changed in normal and abnormal cells. A transfection system, recently developed in our laboratory, which involves the introduction of the v-abl oncogene into mouse erythroleukemia cells to activate the mouse embryonic globin genes, will also be used to characterize the biochemical mechanisms governing adult and embryonic globin gene expression. Additional patients with altered adult and embryonic globin gene expression will be studied to define whether the mutations causing these unusual patterns of globin gene expression are syntenic or asyntenic to the globin genes. The adult and embryonic globin genes in mouse erythroleukemia cells before and after v-abl induced activation of embryonic globin genes will be characterized to test whether changes in methylation, DNAase I sensitivity, RNA processing, MRNA stability, and transcription have occurred. cDNA libraries will be made from the RNA of cells before and after transfection induced activation of embryonic globin genes expression, to isolate cDNA clones which are specific for cells in which oncogene induced activation of embryonic genes has occurred. The genes which encode such molecules will be studied with respect to their molecular properties and their capacity to activate embryonic globin gene expression. cDNA clones which are found to be specific for cells in which embryonic globin gene activation has occurred following v-abl oncogene transfection, will be used as hybridization probes to test whether any mRNA sequences in patients with altered alpha and embryonic globin gene expression are changing in a manner similar to or diametrically opposite to that observed in the transfection system. The goal of these experiments is to identify genes which are either coordinately regulated with the adult and embryonic globin genes in patients exhibiting altered states of expression of these genes or to identify molecules which are mediating these changes. By this means, we hope to clarify the mechanisms governing normal development, and the ways in which disruption of this program leads to disease in man.
