Regulated c-myc Destabilization during Differentiation

描述（由申请人提供）：异常c-myc表达有利于细胞生长而非分化，这是肿瘤表型的标志。因此，我们的主要目标是了解c-myc表达在分化过程中是如何调节的。白血病细胞系是在培养物中持续增殖的未分化细胞。然而，适当的信号转导途径的激活可以诱导基因表达程序，导致其分化为红系、髓系或淋巴系细胞。许多造血细胞类型分化的基本要求是下调c-myc原癌基因，因为其组成型表达阻断分化。分化程序的激活导致c-myc mRNA在2-3小时内减少20- 50倍。转录和转录后机制都有助于快速下调。主要的转录后机制涉及激活一条途径，使c-myc mRNA的不稳定性比分裂细胞高4倍。无细胞mRNA衰变系统重建分化诱导的c-myc mRNA不稳定。然而，它需要至少两种组分用于重构。一种组分从分裂或分化细胞中分离到130，000 x g的核糖体后上清液（S 130），因此具有组成型活性。该组分包含RNA和蛋白质亚基。另一种成分是多核糖体相关的，由分化信号诱导或激活。 c-myc mRNA的编码区似乎是必要的，足以在分化过程中下调的mRNA。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，结合CRD，并似乎控制多核糖体相关的核糖核酸内切酶进入CRD。我们计划利用生物化学和遗传学实验的组合来剖析可溶性和多核糖体相关因子，CRD，和CRD-BP在细胞分化过程中c-myc mRNA的不稳定的作用。这些研究应该有一个重大的影响，我们的理解的转录后机制，有助于沉默这个重要的基因，允许分化。通过扩展，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.