Rountree M R, Selker E U
Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
Genes Dev. 1997 Sep 15;11(18):2383-95. doi: 10.1101/gad.11.18.2383.
In plants, animals, and fungi, DNA methylation is frequently associated with gene silencing, yet little is known about the role of the methylation in silencing. In Neurospora crassa, repeated sequences are silenced by repeat-induced point mutation (RIP) and genes that have suffered numerous GC --> AT mutations by RIP are typically methylated at remaining cytosines. We investigated possible effects on transcription from methylation associated with RIP by taking advantage of 5-azacytidine, which prevents most methylation in Neurospora and a dim-2 mutation that abolishes all detectable methylation. Northern analyses revealed that methylation prevents the accumulation of transcripts from genes mutated by RIP. Measurements of transcription rates in vivo showed that methylation inhibits transcription severely but does not influence mRNA stability. Results of nuclear run-on experiments demonstrated that transcription initiation was not significantly inhibited by the dense methylation in the promoter sequences. In contrast, methylation blocked transcription elongation in vivo.
在植物、动物和真菌中，DNA甲基化常常与基因沉默相关，但对于甲基化在基因沉默中的作用却知之甚少。在粗糙脉孢菌中，重复序列通过重复序列诱导点突变（RIP）被沉默，并且因RIP而发生大量GC→AT突变的基因通常在剩余的胞嘧啶处被甲基化。我们利用5-氮杂胞苷（它可阻止粗糙脉孢菌中的大部分甲基化）和一个消除所有可检测到的甲基化的dim-2突变，研究了与RIP相关的甲基化对转录的可能影响。Northern分析表明，甲基化会阻止RIP突变基因转录本的积累。体内转录速率的测量结果显示，甲基化会严重抑制转录，但不影响mRNA的稳定性。核延伸实验结果表明，启动子序列中的密集甲基化不会显著抑制转录起始。相反，甲基化在体内会阻断转录延伸。
