Regulated c-myc Destabilization during Differentiation

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DESCRIPTION (provided by applicant): Aberrant c-myc expression favors cell growth rather than differentiation, a hallmark of the neoplastic phenotype. Thus, our major goal is to understand how c-myc expression is regulated during differentiation. Leukemia cell lines are undifferentiated cells that proliferate continuously in culture. However, activation of the appropriate signal transduction pathways can induce a program of gene expression leading to their differentiation into cells of erythroid, myeloid, or lymphoid lineage. An essential requirement for differentiation of many hematopoietic cell types is the down-regulation of the c-myc proto-oncogene, since its constitutive expression blocks differentiation. Activation of the differentiation program leads to a 20- to 50-fold decrease in c-myc mRNA within 2-3 hours. Both transcriptional and posttranscriptional mechanisms can contribute to rapid down-regulation. The major posttranscriptional mechanism involves activation of a pathway that destabilizes c-myc mRNA 4-fold compared to dividing cells. A cell-free mRNA decay system reconstitutes the differentiation-induced destabilization of c-myc mRNA. However, it requires at least two components for reconstitution. One component fractionates to the 130,000 x g, post-ribosomal supematant (S 130) from either dividing or differentiating cells, and is thus constitutively active. This component contains both RNA and protein subunits. The other component is polysome-associated and is induced or activated by differentiation signals. The coding region of c-myc mRNA appears necessary and sufficient for down-regulation of the mRNA during differentiation. Moreover, the last 249 nt of the coding region, known as the c-myc coding region determinant or CRD, can confer regulation upon a heterologous mRNA. A family of RNA-binding proteins, known as CRDbinding proteins, or CRD-BPs, bind the CRD and appear to control access of a polysome-associated endoribonuclease to the CRD. We plan to utilize a combination of biochemical and genetic experiments to dissect the roles of the soluble and polysome-associated factors, the CRD, and the CRD-BPs in the destabilization of c-myc mRNA during cellular differentiation. These studies should have a major impact on our understanding of the posttranscriptional mechanisms that contribute to the silencing of this important gene to permit differentiation. By extension, our findings are likely to apply to many genes controlled during development, cell growth, and neoplastic transformation.