Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Oncogene. 2010 May 6;29(18):2739-45. doi: 10.1038/onc.2010.21. Epub 2010 Feb 15.
Almost all neuroblastoma tumors express excess levels of Cyclin D1 (CCND1) compared to normal tissues and other tumor types. Only a small percentage of these neuroblastoma tumors have high-level amplification of the Cyclin D1 gene. The other neuroblastoma tumors have equally high Cyclin D1 expression without amplification. Silencing of Cyclin D1 expression was previously found to trigger differentiation of neuroblastoma cells. Overexpression of Cyclin D1 is therefore one of the most frequent mechanisms with a postulated function in neuroblastoma pathogenesis. The cause for the Cyclin D1 overexpression is unknown. Here we show that Cyclin D1 overexpression results from transcriptional upregulation. To identify upstream regulators, we searched in mRNA profiles of neuroblastoma tumor series for transcription factors with expression patterns correlating to Cyclin D1. GATA3 most consistently correlated to Cyclin D1 in four independent data sets. We identified a highly conserved GATA3 binding site 27 bp upstream of the Cyclin D1 transcriptional start. Chromatin immune precipitation confirmed binding of GATA3 to the Cyclin D1 promoter. Overexpression of GATA3 induced Cyclin D1 promoter activity, which decreased after site-directed mutagenesis of the GATA3 binding site in the Cyclin D1 promoter. Silencing of GATA3 resulted in reduced Cyclin D1 promoter activity and reduced Cyclin D1 mRNA and protein levels. Moreover, GATA3 silencing caused differentiation that was similar to that caused by Cyclin D1 inhibition. These finding implicate GATA3 in Cyclin D1 overexpression in neuroblastoma.
与正常组织和其他肿瘤类型相比，几乎所有神经母细胞瘤肿瘤都过度表达细胞周期蛋白 D1（CCND1）。只有一小部分神经母细胞瘤肿瘤存在细胞周期蛋白 D1 基因的高水平扩增。其他神经母细胞瘤肿瘤具有同等高水平的细胞周期蛋白 D1 表达而没有扩增。先前发现沉默细胞周期蛋白 D1 的表达会引发神经母细胞瘤细胞的分化。因此，细胞周期蛋白 D1 的过表达是神经母细胞瘤发病机制中最常见的机制之一，其假定功能是上调Cyclin D1 的表达。Cyclin D1 过表达的原因尚不清楚。在这里，我们表明 Cyclin D1 的过表达是转录上调的结果。为了鉴定上游调节剂，我们在神经母细胞瘤肿瘤系列的 mRNA 图谱中搜索与 Cyclin D1 表达模式相关的转录因子。在四个独立的数据集中，GATA3 与 Cyclin D1 最一致地相关。我们确定了一个高度保守的 GATA3 结合位点，位于 Cyclin D1 转录起始点上游 27 个碱基对。染色质免疫沉淀证实 GATA3 结合到 Cyclin D1 启动子上。GATA3 的过表达诱导 Cyclin D1 启动子活性增加，而 Cyclin D1 启动子中 GATA3 结合位点的定向突变后则降低。GATA3 的沉默导致 Cyclin D1 启动子活性降低以及 Cyclin D1 mRNA 和蛋白水平降低。此外，GATA3 的沉默导致分化，类似于 Cyclin D1 抑制所引起的分化。这些发现表明 GATA3 参与了神经母细胞瘤中 Cyclin D1 的过表达。
