Magdinier F, Wolffe A P
Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Building 18T, Room 106, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4990-5. doi: 10.1073/pnas.101617298. Epub 2001 Apr 17.
DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2'-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase-PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.
肿瘤抑制基因的DNA甲基化是人类癌症的一个常见特征。细胞周期蛋白依赖性激酶抑制剂基因p16/Ink4A在多种恶性组织中发生高甲基化，位于9号染色体p21上游20 kb处的p14/ARF基因在癌组织中也发生甲基化。p14/ARF（ARF，可变阅读框）并不抑制细胞周期蛋白或细胞周期蛋白依赖性激酶复合物的活性；然而，这两种基因产物在癌症病因学中的重要性在于它们分别参与了两条主要的细胞周期调节途径：p53和视网膜母细胞瘤蛋白Rb。由不同启动子驱动的不同的第一个外显子被剪接到共同的外显子2和3上，产生的蛋白质在不同的阅读框中翻译。这两个基因在正常细胞中表达，但当它们的CpG岛发生高甲基化时，可以被选择性或协同沉默。在此，我们通过染色质免疫沉淀试验检测了与异常甲基化启动子相关的甲基-CpG结合蛋白的存在、乙酰化组蛋白H3和H4的分布，并通过定量逆转录聚合酶链反应检测了5-氮杂-2'-脱氧胞苷（5aza-dC）和曲古抑菌素A化学处理对结肠细胞系中基因诱导的影响。我们观察到甲基-CpG结合蛋白MBD2靶向甲基化调节区域并排除乙酰化组蛋白H3和H4，导致局部无活性染色质构型。当发生甲基化时，这些基因可被5aza-dC诱导，但5aza-dC和曲古抑菌素A的联合作用可导致强大的基因表达。因此，甲基-CpG结合蛋白和组蛋白脱乙酰酶似乎在体内协同作用，DNA甲基化对组蛋白乙酰化具有主导作用，并抑制癌症中高甲基化的肿瘤抑制基因的表达。
