Achour M, Jacq X, Rondé P, Alhosin M, Charlot C, Chataigneau T, Jeanblanc M, Macaluso M, Giordano A, Hughes A D, Schini-Kerth V B, Bronner C
Institut Gilbert-Laustriat, UMR 7175 CNRS/Université Louis Pasteur (Strasbourg I), Départment de Pharmacologie et Physicochimie, Faculté de Pharmacie, Illkirch, France.
Oncogene. 2008 Apr 3;27(15):2187-97. doi: 10.1038/sj.onc.1210855. Epub 2007 Oct 15.
Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-S-transferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macro-molecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Co-immunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16(INK4A) gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16(INK4A). They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.
90千道尔顿反向CCAAT盒结合蛋白（ICBP90）在包括乳腺癌、前列腺癌和肺癌在内的多种癌症中过度表达。为了寻找与ICBP90的SET和环相关（SRA）结构域相互作用的蛋白质，我们利用双杂交系统筛选了胎盘cDNA文库。发现了几个编码DNMT1新结构域的克隆。通过谷胱甘肽-S-转移酶下拉实验，用纯化的蛋白质证实了ICBP90 SRA结构域与DNMT1结构域之间的相互作用。我们检测了Jurkat细胞和永生化人血管平滑肌细胞（HVTs-SM1）中ICBP90和DNMT1是否存在于同一大分子复合物中。免疫共沉淀实验表明ICBP90和DNMT1存在于同一分子复合物中，免疫细胞化学评估的共定位实验进一步证实了这一点。ICBP90和DNMT1的下调降低了主要促血管生成因子VEGF基因的表达，而p16（INK4A）基因和RB1基因的表达则显著增强。总之，这些结果表明DNMT1和ICBP90可能通过ICBP90的SRA结构域与DNMT1的一个新结构域的相互作用以及p16（INK4A）的上调参与VEGF基因表达。它们进一步表明ICBP90在肿瘤血管生成和肿瘤抑制基因沉默背景下的组蛋白泛素化与DNA甲基化关系中具有新作用。
