Department of Medicine/Cancer Prevention, Stony Brook University, Stony Brook, New York, United States of America.
PLoS One. 2012;7(9):e44661. doi: 10.1371/journal.pone.0044661. Epub 2012 Sep 6.
Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5'-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5'-primer extension study with 5'RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.
新出现的证据表明，人类癌症中 KIAA1199 的表达上调预示着生存不良。控制癌症中 KIAA1199 表达的调节机制仍有待描述。在本研究中，我们已经从调节 KIAA1199 基因表达的角度分离和表征了人 KIAA1199 启动子。包含 KIAA1199 基因 5'-侧翼序列的人基因组 DNA 的 3.3kb 片段具有抑制和激活元件。通过缺失诱变方法，确定了 1.4kb 的近端区域作为包含靠近转录起始位点的 TATA 盒的基本 KIAA1199 启动子。5'-引物延伸研究与 5'RACE DNA 测序分析相结合表明，人类 KIAA1199 基因使用一个主要的转录起始位点。生物信息学分析表明，1.4kb 的 KIAA1199 启动子包含推定的激活调节元件，包括激活蛋白-1(AP-1)、Twist-1 和 NF-κB 位点。顺序缺失和定点突变分析表明，AP-1 和远端 NF-κB 位点是 KIAA1199 基因表达所必需的。使用电泳迁移率变动分析和染色质免疫沉淀的进一步分析证实了这些顺式和反式作用元件在控制 KIAA1199 基因表达中的必要性。最后，我们发现人类乳腺癌标本中上调的 KIAA1199 表达与调节区的低甲基化相关。在人乳腺癌细胞系中，DNA 甲基化在调节 KIAA1199 表达中的作用得到了重现。总之，我们的研究揭示了控制人类癌症中 KIAA1199 基因表达的调节机制。
