ONCOGENES AND THE CONTROL OF GLOBIN GENE EXPRESSION

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Humans undergo two developmental switches in their hemoglobin phenotype, the embryonic to fetal switch occurring in early gestation and the fetal to adult switch occurring around the time of birth. Gene regulation has two main components, namely cis-acting DNA sequences and trans-acting molecules (presumably proteins that interact with DNA sequences in a specific manner to control gene expression. The K562 human leukemia cell line expresses all globin genes other than the adult beta-globin. Previous work from this laboratory showed that the beta-globin gene of K562 cells functions normally in a heterologous expression system. Elucidation of the mechanism of failure of beta-globin gene expression in K562 cells may provide an insight into globin gene expression and switching in normal erythroid cells. The human adenovirus E1a protein product acts in trans on the transcription of other viral genes as well as certain resident cellular genes such as hsp 70. An enhancer deficient human beta-globin gene is not transcribed in HeLa cells; however, cotransfection with the E1a gene results in easily detectable beta-globin mRNA. The c-myc protein nuclear matrix product has limited homology to adenovirus E1a protein and like E1a can induce expression from the hsp 70 promoter and can complement c-ras in transforming primary cells. We propose to transfect a hybrid myc plasmid with a dexamethasone responsive regulatory element (MMTV-Xba-myc) into K562 cells and study epsilon and beta-globin gene expression upon steroid induction. Hybrid epsilon- and beta-CAT plasmids will be cotransfected with MMTV-Xba-myc and the level of CAT activity assayed in heterologous systems. No suitable human erythroid cell lines expressing only beta-globin are available. Such line(s) will be established using MMTV-Xba-myc and a c-ras containing oncogene. The steroid inducible MMTV element should permit mimicry of the in vivo decrease in c-myc mRNA level seen with differentiation, as well as supply an adult hemoglobin forming cell line.