Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae.
Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae.
复制标题
区分酿酒酵母中无声染色质结构的募集和传播。
作者：
Brothers M;Rine J
在酿酒酵母的HML、HMR和端粒处异染色质的形成涉及两个主要步骤：将Sir蛋白募集到沉默子中以及它们在整个沉默结构域中的扩散。我们开发了一种方法来研究这两个过程在单碱基对分辨率。使用异染色质蛋白Sir 3和非位点特异性细菌腺嘌呤甲基转移酶M.EcoGII之间的融合蛋白，我们使用长读Nanopore测序来检测融合蛋白甲基化的腺嘌呤，并通过ChIP-seq来绘制Sir 3-M. EcoGII的分布，从而在全基因组范围内绘制Sir 3-染色质相互作用的位点。一个沉默缺陷的突变体Sir 3缺乏其溴邻近同源（BAH）结构域，sir 3-bah，仍然招募到HML，HMR，和端粒。然而，在BAH结构域缺失的情况下，它无法从这些募集位点扩散出去。Sir 3的过表达没有导致HML、HMR和大多数端粒的进一步扩散。一些特殊的端粒，如6 R，表现出少量的Sir 3扩散，这表明端粒的边界响应于Sir 3-M。EcoGII过表达。最后，通过使用与M.ECOGII融合的SIR 3的温度敏感等位基因，我们追踪了诱导后第一次甲基化的位置，并发现在Sir 3占据整个基因座之前，HML和HMR处的基因开始受到抑制。
The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: the recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single basepair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the nonsite-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3–chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein and by ChIP-seq to map the distribution of Sir3–M.EcoGII. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah∆, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3-M.EcoGII overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.