Mac S M, D'Cunha C A, Farnham P J
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Mol Carcinog. 2000 Oct;29(2):76-86.
The N-myc gene is amplified in 20-25% of human neuroblastomas, and this amplification serves as a poor prognostic factor. However, few genes have been determined to be direct targets of N-myc. Our current studies focused on identifying N-myc target genes, especially those affected in cells such as neuroblastomas that have high levels of N-myc protein. To pursue this goal, we performed differential expression screens with cell-culture systems containing high versus low levels of N-myc. The design of our experiments was such that we should identify genes both upregulated and downregulated by N-myc. Accordingly, we identified 22 genes upregulated by N-myc and one gene downregulated by N-myc. However, only five of these genes responded to increased N-myc levels in more than one system. Further analysis of the regulation of these genes required determining whether they were direct or indirect targets of N-myc. Therefore, we used a formaldehyde crosslinking and immunoprecipitation procedure to determine whether N-myc was bound to the promoters of these putative target genes in living cells. We found that low levels of N-myc were bound to the promoters of the telomerase and prothymosin genes in neuroblastoma cells having low amounts of N-myc but that the amounts of N-myc bound to these promoters greatly increased with overexpression of N-myc. However, the amount of max bound to the promoters was high before and after induction of N-myc. Therefore, our studies suggest that N-myc competes with other max partners for binding to target promoters. Our use of the chromatin immunoprecipitation assay suggests a molecular explanation for the consequences of amplification of the N-myc gene in neuroblastomas.
N - myc基因在20% - 25%的人类神经母细胞瘤中发生扩增，这种扩增是一个不良预后因素。然而，很少有基因被确定为N - myc的直接靶点。我们目前的研究聚焦于鉴定N - myc的靶基因，尤其是那些在N - myc蛋白水平高的细胞（如神经母细胞瘤细胞）中受影响的基因。为实现这一目标，我们在含有高水平和低水平N - myc的细胞培养系统中进行了差异表达筛选。我们实验的设计旨在鉴定出被N - myc上调和下调的基因。相应地，我们鉴定出22个被N - myc上调的基因和1个被N - myc下调的基因。然而，这些基因中只有5个在不止一个系统中对N - myc水平的升高有反应。对这些基因调控的进一步分析需要确定它们是N - myc的直接还是间接靶点。因此，我们使用甲醛交联和免疫沉淀程序来确定N - myc在活细胞中是否与这些假定靶基因的启动子结合。我们发现，在N - myc含量低的神经母细胞瘤细胞中，低水平的N - myc与端粒酶和前胸腺素基因的启动子结合，但随着N - myc的过表达，与这些启动子结合的N - myc量大幅增加。然而，在诱导N - myc前后，与启动子结合的max量都很高。因此，我们的研究表明，N - myc与其他max伴侣竞争结合靶启动子。我们对染色质免疫沉淀分析的使用为神经母细胞瘤中N - myc基因扩增的后果提供了分子解释。
