Estève Pierre-Olivier, Chin Hang Gyeong, Pradhan Sriharsa
New England Biolabs, Ipswich, Massachusetts 01938-2723, USA.
J Biol Chem. 2007 Jan 26;282(4):2615-25. doi: 10.1074/jbc.M606203200. Epub 2006 Nov 22.
Human maintenance DNA cytosine methyltransferase (DNMT1) regulates gene expression in a methylation-dependent and -independent manner. Anti-apoptotic survivin gene down-regulation is mediated by p53 recruitment of DNMT1 to its promoter. Survivin inhibits programmed cell death, regulates cell division, and is expressed in cancer cells. The survivin gene promoter is CG-rich containing several Sp1 canonical, Sp1-like, cell cycle-dependent element/cell cycle gene homology region, and p53-binding sites. Here we demonstrate that Sp1 transcription factor(s) play a role in transcriptional activation of the survivin promoter in Drosophila and human cells. Sp1 inhibition in vivo by mithramycin A leads to down-regulation of a luciferase reporter driven by the human survivin promoter in transfected cells. Mithramycin A or Sp1-specific short interfering RNA down-regulated the endogenous survivin gene expression, confirming Sp1 as the primary determinant for transcriptional activation. Furthermore, immobilized DNMT1 ligand bound to seven consensus amino acids corresponding to the N-terminal region of the Sp class of transcription factors in a phage display analysis. In the co-immunoprecipitation assay, the endogenous Sp1 or Sp3 pulled down DNMT1 and methyltransferase activity. Similarly, a glutathione S-transferase pulldown assay between DNMT1 and Sp1 demonstrates a direct interaction between the two proteins. Fluorescent fusions of DNMT1 and Sp1 co-localized in the mammalian nucleus, thus supporting binary complex formation between both the proteins. The kinetics of survivin promoter occupancy via chromatin immunoprecipitation following doxorubicin treatment show the presence of Sp1 and gradual accumulation of transcriptional repressors p53, DNMT1, histone methyltransferase G9a, and HDAC1 onto the promoter along with histone H3K9me2. These data suggest that the Sp1 transcription factor acts as a platform for recruitment of transcriptional repressors.
人类维持性DNA胞嘧啶甲基转移酶（DNMT1）以甲基化依赖和非依赖的方式调节基因表达。抗凋亡存活素基因的下调是由p53将DNMT1募集至其启动子介导的。存活素抑制程序性细胞死亡，调节细胞分裂，并在癌细胞中表达。存活素基因启动子富含CG，包含多个Sp1典型、Sp1样、细胞周期依赖性元件/细胞周期基因同源区域以及p53结合位点。在此我们证明，Sp1转录因子在果蝇和人类细胞中存活素启动子的转录激活中发挥作用。在体内，丝裂霉素A抑制Sp1会导致转染细胞中由人类存活素启动子驱动的荧光素酶报告基因下调。丝裂霉素A或Sp1特异性小干扰RNA下调了内源性存活素基因表达，证实Sp1是转录激活的主要决定因素。此外，在噬菌体展示分析中，固定化的DNMT1配体与对应于Sp类转录因子N端区域的7个共有氨基酸结合。在免疫共沉淀试验中，内源性Sp1或Sp3下拉了DNMT1和甲基转移酶活性。同样，DNMT1和Sp1之间的谷胱甘肽S-转移酶下拉试验证明了这两种蛋白质之间的直接相互作用。DNMT1和Sp1的荧光融合蛋白在哺乳动物细胞核中共定位，从而支持了这两种蛋白质之间二元复合物的形成。阿霉素处理后通过染色质免疫沉淀对存活素启动子占据情况的动力学分析表明，启动子上存在Sp1，并且转录抑制因子p53、DNMT1、组蛋白甲基转移酶G9a和HDAC1以及组蛋白H3K9me2逐渐积累。这些数据表明，Sp1转录因子作为募集转录抑制因子的平台。
