Control mechanisms regulating gene expression during normal differentiation of myeloid leukemic cells: Differentiation defective mutants blocked in mRNA production and mRNA translation

Regulation of gene expression during myeloid cell differentiation has been analyzed using clones of myeloid leukemic cells that differ in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein MGI. Changes in the relative rate of synthesis for specific proteins were compared to changes in the relative amounts of corresponding translatable poly(A)⁺ mRNAs, assayed in the reticulocyte cell-free translation system, using two-dimensional gel electrophoresis. Of the 217 proteins which changed during MGI-induced differentiation of normally differentiating MGI⁺D⁺ leukemic cells, 136 could be identified as products of cell-free translation. Eighty-four percent of the 70 decreases in synthesis, most of which occurred early during differentiation, were not accompanied by a parallel decrease in the amount of translatable mRNA, but were accompanied by a parallel shift of the corresponding mRNAs from the polysomal to the monosomal and free mRNA fractions. These results indicate that most of the early decreases in the synthesis of proteins were translationally regulated. In contrast, 81% of the proteins which increased in synthesis and 71% of the proteins that were induced de novo were regulated at the level of mRNA production. Experiments with differentiation defective mutants have shown that they were blocked both at the level of mRNA production and mRNA translation. The data with these mutants have suggested that there were different subsets of translationally regulated proteins which were separately regulated. The translational blocks for several proteins in these mutant clones have also made it possible to identify additional translational sites of regulation for protein changes that were controlled at the level of mRNA production during normal differentiation. The results indicate that translational regulation may predominantly have a different function in cell differentiation than regulation by mRNA production, and that differentiation-defective mutants can be blocked at either level.