Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.
Biochem Pharmacol. 2012 Dec 15;84(12):1581-91. doi: 10.1016/j.bcp.2012.09.014. Epub 2012 Sep 25.
Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the Match™ software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.
Sp1 是一种转录因子，通过其 DNA 结合结构域（含三个锌指）调控许多基因的表达。我们对鉴定 Sp1 调控的靶基因特别感兴趣，尤其是那些与增殖和癌症相关的基因。我们的方法是用 Sp1 mRNA 的 siRNA 处理 HeLa 细胞，降低 Sp1 的表达，从而降低该转录因子激活的基因的表达。全基因组 cDNA 微阵列分析表明，Sp1-siRNA 处理导致大量差异表达基因。由于这些基因代表 Sp1 激活的假定基因，我们将其分类为六个基因本体论类别，即增殖和癌症、mRNA 加工、脂质代谢、糖代谢、转录和翻译。使用 Match™软件在选定基因的启动子中找到了 Sp1 的假定结合位点。通过文献挖掘，选择了 11 个基因进行进一步验证。用 qRT-PCR 验证了 Sp1-siRNA 处理后 HeLa 细胞中 11 个基因和 Sp1 的下调表达。进行了 EMSA 和 ChIP 测定以检测 Sp1 与这些基因启动子的结合。我们观察到 Sp1 与 RAB20、FGF21、IHPK2、ARHGAP18、NPM3、SRSF7、CALM3、PGD 和 Sp1 自身启动子的结合。此外，在 HeLa 细胞中转染 Sp1 后，确定了与增殖和癌症相关的 RAB20、FGF21 和 IHPK2 基因的 mRNA 水平和这些三个基因的荧光素酶活性，以确认它们受 Sp1 调控。通过使用多聚嘌呤反向霍格斯坦发夹基因沉默，验证了这三个基因在增殖中的作用。
