Identification of transcriptional targets of HOXA5
Identification of transcriptional targets of HOXA5
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DOI：
10.1074/jbc.m413528200
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发表时间：
2005-05-13
影响因子：
4.8
通讯作者：
Sukumar, S
中科院分区：
文献类型：
--
作者：
Chen, HX;Rubin, E;Sukumar, S
同源异型盒基因HOXA 5编码的转录因子已被证明在胚胎发生、造血和肿瘤发生中发挥重要作用。为了破译HOXA 5的下游信号通路，我们利用寡核苷酸微阵列分析来鉴定HOXA 5诱导的细胞与未诱导的细胞相比差异表达的基因。在Hs 578 T乳腺癌细胞中HOXA 5诱导9小时后的基因表达变化的比较分析鉴定了306个基因，其表达被调节至少2倍。这306个基因中的10个在诱导后6 h也上调至少2倍。所有这10个基因的表达通过半定量逆转录-PCR证实。在这10个基因中，它们最有可能是HOXA 5的直接靶基因，我们通过首先克隆其启动子开始了对多效因子基因的研究。瞬时转染实验表明HOXA 5可以特异性激活多效生长因子启动子。进行启动子缺失、染色质免疫沉淀测定和凝胶迁移测定以显示HOXA 5可以直接结合多效营养因子启动子上的一个结合位点。这些数据有力地表明，微阵列分析可以成功地识别HOXA 5的许多潜在的直接下游基因。对这些靶点的进一步功能分析将使我们能够更好地了解HOXA 5在胚胎发育和肿瘤发生中的多种功能。
The homeobox gene HOXA5 encodes a transcription factor that has been shown to play important roles in embryogenesis, hematopoiesis, and tumorigenesis. In order to decipher downstream signaling pathways of HOXA5, we utilized oligonucleotide microarray analysis to identify genes that are differentially expressed in HOXA5-induced cells compared with uninduced cells. Comparative analysis of gene expression changes after 9 h of HOXA5 induction in Hs578T breast cancer cells identified 306 genes whose expression was modulated at least 2-fold. Ten of these 306 genes were also up-regulated by at least 2-fold at 6 h post-induction. The expression of all of these 10 genes was confirmed by semi-quantitative reverse transcription-PCR. Among these 10 genes, which are most likely to be direct targets of HOXA5, we initiated an investigation into the pleiotrophin gene by first cloning its promoter. Transient transfection assays indicated that HOXA5 can specifically activate the pleiotrophin promoter. Promoter deletion, chromatin immunoprecipitation assay, and gel-shift assays were performed to show that HOXA5 can directly bind to one binding site on the pleiotrophin promoter. These data strongly suggest that microarray analysis can successfully identify many potential direct downstream genes of HOXA5. Further functional analysis of these targets will allow us to better understand the diverse functions of HOXA5 in embryonic development and tumorigenesis.