Curradi Michela, Izzo Annalisa, Badaracco Gianfranco, Landsberger Nicoletta
Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi dell'Insubria, 21052 Busto Arsizio (Va), Italy.
Mol Cell Biol. 2002 May;22(9):3157-73. doi: 10.1128/MCB.22.9.3157-3173.2002.
DNA methylation and chromatin modification operate along a common pathway to repress transcription; accordingly, several experiments demonstrate that the effects of DNA methylation can spread in cis and do not require promoter modification. In order to investigate the molecular details of the inhibitory effect of methylation, we microinjected into Xenopus oocytes a series of constructs containing a human CpG-rich sequence which has been differentially methylated and cloned at different positions relative to a specific promoter. The parameters influencing the diffusion of gene silencing and the importance of histone deacetylation in the spreading effect were analyzed. We demonstrate that a few methylated cytosines can inhibit a flanking promoter but a threshold of modified sites is required to organize a stable, diffusible chromatin structure. Histone deacetylation is the main cause of gene repression only when methylation does not reach levels sufficient to establish this particular structure. Moreover, contrary to the common thought, promoter modification does not lead to the greater repressive effect; the existence of a competition between transactivators and methyl-binding proteins for the establishment of an open conformation justifies the results obtained.
DNA甲基化和染色质修饰沿着共同途径发挥作用以抑制转录；因此，多项实验表明DNA甲基化的效应能够顺式传播，且不需要启动子修饰。为了研究甲基化抑制作用的分子细节，我们将一系列构建体显微注射到非洲爪蟾卵母细胞中，这些构建体包含一个富含CpG的人类序列，该序列已被差异甲基化，并相对于特定启动子在不同位置进行了克隆。分析了影响基因沉默扩散的参数以及组蛋白去乙酰化在扩散效应中的重要性。我们证明，少数甲基化的胞嘧啶能够抑制侧翼启动子，但需要一定数量的修饰位点来构建稳定、可扩散的染色质结构。仅当甲基化未达到足以建立这种特定结构的水平时，组蛋白去乙酰化才是基因抑制的主要原因。此外，与普遍观点相反，启动子修饰并不会导致更强的抑制作用；反式激活因子与甲基结合蛋白之间为建立开放构象而存在的竞争能够解释所获得的结果。
