Regulated c-myc Destabilization during Differentiation

描述(申请人提供)：c-myc异常表达有利于细胞生长而不是分化，这是肿瘤表型的特征。因此，我们的主要目标是了解c-myc在分化过程中的表达是如何调节的。白血病细胞系是未分化的细胞，在培养中持续增殖。然而，激活适当的信号转导通路可以诱导一系列基因表达，使其分化为红系、髓系或淋巴系细胞。许多造血细胞类型分化的一个基本要求是c-myc原癌基因的下调，因为它的结构性表达阻碍了分化。分化程序的激活会导致c-myc mRNA在2-3小时内下降20-50倍。转录和转录后机制都可以促进快速下调。主要的转录后机制包括激活一条途径，使c-myc mRNA的稳定性比分裂细胞高4倍。一个无细胞的信使核糖核酸衰变系统重构了分化诱导的c-myc信使核糖核酸失稳。然而，它至少需要两个组成部分才能重组。一种组分从分裂或分化的细胞中分离到130,000×g，核糖体后上皮瘤(S 130)，因此具有结构性活性。该成分既含有RNA亚基，又含有蛋白质亚基。另一组分是多聚体相关的，由分化信号诱导或激活。 C-myc基因的编码区似乎是分化过程中下调该基因表达的必要条件和充分条件。此外，编码区的最后249个核苷酸，称为c-myc编码区决定簇或CRD，可以对异源mRNA进行调控。一个RNA结合蛋白家族，称为CRD结合蛋白，或CRD-BP，与CRD结合，似乎控制着与CRD相关的多聚体内切核酸酶对CRD的访问。我们计划利用生化和遗传实验的组合来剖析可溶性和多聚体相关因子CRD和CRD-BPS在细胞分化过程中c-myc mRNA失稳中的作用。这些研究应该对我们理解转录后机制有重大影响，转录后机制有助于沉默这一重要基因以允许分化。推而广之，我们的发现可能适用于许多在发育、细胞生长和肿瘤转化过程中受到控制的基因。

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.