Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae [II]

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. For the following datasets, we created a fusion protein between the heterochromatin protein Sir3 and the non-site-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. We also used a temperature-sensitive allele of SIR3 (sir3-8) fused to M.ECOGII to induce m6A methylation over time. Time courses involved a switch from restrictive temperature (37°C) to permissive temperature (25°C).酿酒酵母（Saccharomyces cerevisiae）中HML、HMR位点与端粒（telomeres）处的异染色质（heterochromatin）形成主要包含两个核心步骤：将Sir蛋白（Sir proteins）招募至沉默子（silencers），并使其在沉默结构域（silenced domain）内扩散。针对本数据集，我们将异染色质蛋白Sir3与非位点特异性细菌腺嘌呤甲基转移酶M.EcoGII构建为融合蛋白。我们采用长读长纳米孔测序（long-read Nanopore sequencing）对该融合蛋白介导的染色质互作位点进行全基因组定位，以检测被该融合蛋白甲基化的腺嘌呤位点。我们还将携带温度敏感型等位基因（temperature-sensitive allele）的SIR3突变体sir3-8与M.ECOGII融合，以实现随时间诱导的m6A甲基化。时间进程实验设置为将培养物从限制性温度（37℃）切换至允许性温度（25℃）。