Department of Neuro-oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
Oncogene. 2013 Jun 27;32(26):3119-29. doi: 10.1038/onc.2012.331. Epub 2012 Sep 3.
Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.
肿瘤干细胞被认为是多种癌症恶性肿瘤的起源细胞，其可以通过细胞表面标志物（如 CD133，一种五跨膜蛋白）的表达来鉴定。CD133+细胞可形成神经球，具有自我更新和分化能力，并具有致瘤性。然而，尽管 CD133 与干细胞有关，但 CD133 与肿瘤发生之间的因果关系仍有待确定。我们假设，在驱动干细胞状态中起作用的特定表观遗传和转录因子可能同时调控干细胞中 CD133 的表达，因此分析了胶质瘤干细胞和胶质瘤细胞系中 CD133 启动子的结构和调控。最初，通过分析转录起始位点上游的 CD133 启动子驱动的荧光素酶表达 5'和 3'缺失构建体的活性，确定了一个最小启动子区域。该区域包含一个 CpG 岛，在 CD133-ve 胶质瘤干细胞（GSC）和胶质瘤细胞中高度甲基化，但在 CD133+ve 细胞中未甲基化。在该区域的几个预测 TF 结合位点中，研究了串联 Sp1（-242 和-221）和两个 Myc（-541 和-25）结合位点的作用。Sp1 或 Myc 的过表达增加了 GSC 和胶质瘤细胞系中 CD133 最小启动子驱动的荧光素酶活性和 CD133 水平。Sp1 抑制剂米托蒽醌降低了 GSC 中最小启动子活性和下调 CD133 水平。凝胶阻滞实验证明了 Sp1 与其预测位点的直接结合，该结合可被寡核苷酸结合位点序列竞争性抑制，并通过抗 Sp1 超迁移证实了这种相互作用。在 GSC 中进行的 Sp1 和 Myc 抗体染色质免疫沉淀（ChIP）分析显示，Sp1 和 Myc 结合位点富集的区域。在 CD133-ve 细胞中，ChIP 分析显示甲基-DNA 结合蛋白 MBD1、MBD2 和 MeCP2 与甲基化 CpG 岛结合，并抑制转录。这些结果表明，Sp1 和 Myc 调节 GSC 中 CD133 的转录，启动子甲基化和甲基-DNA 结合蛋白通过排除转录因子结合来抑制 CD133 的转录。
