Active conformation of the globin genes in uninduced and induced mouse erythroleukemia cells

Abstract

Mouse erythroleukemia (MEL) cells are hematopoietic cells transformed with Friend virus complex and therefore able to proliferate continuously in culture, but these cells may also be induced to undergo erythroid maturation and accumulate globin mRNA. Using pancreatic DNAase I as a probe for transcriptionally active genes, we have compared the conformation of the globin genes in nuclei from induced and uninduced MEL cells. Three MEL cell lines were chosen: C-19 had a moderate level of globin mRNA in the uninduced state and exhibited an 8 fold increase after incubation in Me 2SO; GM979 had a very low level of globin mRNA in the uninduced state and exhibited a 45 fold increase on incubation in Me 2SO; 4-3 was noninducible, having a very low level of globin mRNA both before and after incubation in Me 2SO. Digestion of 10–15% of the nuclear DNA by DNAase I in nuclei from induced and uninduced cells from each of these lines resulted in virtually complete digestion of globin gene sequences. Two additional cell lines were characterized with regard to the conformation of the globin genes. A cybrid line formed by fusion of MEL cells to enucleated mouse fibroblasts was partially inducible in that 40% of the cells became benzidine-positive in Me 2SO; 40–45% of the globin gene sequences were resistant to DNAase I. In contrast, the globin genes in nuclei from a lymphoblast line (L5178Y-2BF) were completely sensitive to DNAase I, implying that these noninducible cells nonetheless shared certain properties with erythroid progenitor cells. Two types of controls were performed. Incubation of nuclei from mouse adult liver cells and a mouse hepatoma cells with pancreatic DNAase I did not lead to digestion of the globin genes, while the globin genes were digested in nuclei from mouse fetal erythroid liver. The specificity for digestion of the globin genes in MEL cell nuclei was tested by annealing these DNA samples to cDNA prepared with mouse myeloma cell mRNA naturally rich in immunoglobulin light chain mRNA sequences. DNA from all nuclei which had been exposed to DNAase I annealed equally to this probe. We conclude that Me 2SO-induced accumulation of globin mRNA in MEL cells is not due to changes in the conformation of the globin gene in nuclei, but rather that the potential for transcription of the globin gene is acquired at a point early in erythroid maturation beyond which MEL cells have progressed. While the conformation of specific genes in nuclei as reflected by their DNAase I sensitivity is necessary to permit their transcription, the actual rate of transcription of these genes and the accumulation of their mRNAs must depend upon other factors.