Xu L, Meng Y, Wallen R, DePinho R A
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Oncogene. 1995 Nov 2;11(9):1865-72.
The modulation of RNA polymerase II processivity through the untranslated N-myc first exon represents an important mechanism governing N-myc mRNA levels during normal development. In this study, we employed the rat embryo fibroblast (REF) cooperation assay as a functional means to (i) map N-myc first exon sequences involved in the regulation of N-myc gene expression and specifically their contribution to controlling a block to transcriptional elongation and (ii) determine whether this transcriptional control mechanism plays a role in governing the oncogenic activity of N-myc. Transfection of N-myc expression constructs harboring various deletions within the untranslated first exon revealed that a region encoding a potential stem-loop structure followed by a thymine stretch (stem-loop/T region) was required for efficient transcriptional attenuation. The increase in transcriptional read-through associated with all deletions involving the stem-loop/T region also resulted in a more aggressive malignant phenotype as evidenced by a significant enhancement in the subcloning efficiency of N-myc/ras transformed foci. Most significantly, when cell lines generated from overexpression of the intact N-myc expression construct were selected for anchorage-independent growth, a strong block to transcriptional elongation was completely eliminated in all cases examined. Since the subcloning efficiency of transformed foci and the capacity of permanently established cell lines for anchorage-independent growth are direct correlates of more advanced stages of malignant transformation, our findings suggest that loss of transcriptional attenuation represents an important genetic event in the progression of N-myc-induced cellular transformation.
通过未翻译的N - myc第一外显子对RNA聚合酶II持续合成能力的调节，是正常发育过程中控制N - myc mRNA水平的重要机制。在本研究中，我们采用大鼠胚胎成纤维细胞（REF）协同试验作为一种功能手段，来（i）定位参与N - myc基因表达调控的N - myc第一外显子序列，特别是它们对控制转录延伸阻滞的贡献，以及（ii）确定这种转录控制机制是否在调控N - myc的致癌活性中发挥作用。转染在未翻译的第一外显子内含有各种缺失的N - myc表达构建体，结果显示，有效转录衰减需要一个编码潜在茎环结构并随后紧跟一段胸腺嘧啶序列的区域（茎环/T区域）。与涉及茎环/T区域的所有缺失相关的转录通读增加，也导致了更具侵袭性的恶性表型，N - myc/ras转化灶的亚克隆效率显著提高就证明了这一点。最重要的是，当选择由完整N - myc表达构建体过表达产生的细胞系进行不依赖贴壁生长筛选时，在所检测的所有情况下，对转录延伸的强烈阻滞都完全消除了。由于转化灶的亚克隆效率以及永久建立的细胞系不依赖贴壁生长的能力与恶性转化的更晚期阶段直接相关，我们的研究结果表明，转录衰减的丧失代表了N - myc诱导的细胞转化进程中的一个重要遗传事件。
