CONTROL OF DIFFERENTIATION OF ERYTHROLEUKEMIC CELLS

The long-term goal of the proposed research is to understand the processes that control differentiation of erythroid cells. The approach is to analyze the properties of permanent mouse erythroleukemia (MEL) cell lines that can be induced to undergo terminal erythroid differentiation in cell culture. A major aim is to study the early events that control the transition of these cells from proliferating, nondifferentiating tumor cells to cells that are nontumorigenic and committed to terminal differentiation. A second aim is to learn how the amounts of globin mRNA and other specific mRNAs are controlled during terminal erythroid differentiation. A primary method to be used in these studies is cellular genetics, including DNA-mediated gene transfer of cloned genes and gene chimeras. The cellular oncogene c-myc undergoes dramatic changes in expression in the period before MEL cells become irreversibly committed to terminal differentiation. To test the role of c-myc in controlling differentiation, additional copies of an independently regulated c-myc gene will be introduced into MEL cells. Further tests of a role for c-myc will be accomplished by studying changes in c-myc expression when the probability and kinetics of differentiation are altered. C-myc gene transcription and mRNA stability will be measured to learn how rapid changes in expression occur. Differentiating MEL cells appear to utilize both transcriptional and post-transcriptional mechanisms to control the levels of specific mRNAs. To identify cis-acting DNA sequences and diffusible protein factors that regulate globin gene transcription, cloned globin genes will be introduced into MEL cells and their expression will be studied during induced differentiation. The isolation of mutant MEL cells with defects in transcriptional control factors also will be undertaken. To investigate post-transcriptional control of mRNA production, the base sequences in mRNA that control its turnover will be studied. The sequences will be identified by constructing and introducing into cells gene chimeras between two genes whose transcripts differ in stability. This method will then be applied to specific mRNAs that have different stabilities in terminally differentiating MEL cells. (M)