Regulation of yeast chromosome III architecture and mating-type switching by a Sir2/condensin-bound region of the recombination enhancer [HiC-Seq]

Saccharomyces cerevisiae switches its mating type (MATa or MATalpha) through gene conversion using one of two possible donors, HMLalpha or HMRa, both of which are maintained by the SIR silencing complex as heterochromatic cassettes on opposing ends of chromosome III. In MATa cells, HMLalpha on the left arm is preferentially chosen as the donor through a mechanism requiring a cis-acting sequence called the recombination enhancer (RE). The left half of the RE is required for this donor preference activity, whereas the right half has been implicated in regulating chromosome III structure. In this study we have identified a MATa-specific Sir2 and condensin binding site within the right half of the RE that maintains chromosome III in a switching-competent conformation by preventing MATa from interacting with the default HMRa donor on the right arm. Within the RE, Sir2 strongly represses transcription of a small MATa-specific gene of unknown function (RDT1). Upon expression of HO endonuclease to induce the switching process, Sir2 redistributes from the RE to the double-stranded DNA break site at MATa, thus derepressing RDT1 transcription to coincide with the timing of mating-type switching. Condensin is also displaced from the RE in response to the HO-induced break, likely contributing to the transient change in chromosome III conformation required for effective switching without disruption of HML and HMR heterochromatin. Overall design: S. cerevisiae HiC data for WT, SIR2?, and a 100bp? of RDT1 promoter region strains.

酿酒酵母（Saccharomyces cerevisiae）可通过基因转换，利用HMLalpha或HMRa两种潜在供体切换其交配型（MATa或MATα）。上述两个供体均由SIR沉默复合物维持为异染色质盒，定位于三号染色体的相对两端。在MATa型细胞中，三号染色体左臂的HMLalpha会被优先选为供体，这一过程依赖于一类被称为重组增强子（recombination enhancer，RE）的顺式作用序列（cis-acting sequence）介导的机制。重组增强子的左半区域是该供体选择活性所必需的，而其右半区域则参与调控三号染色体的高级结构。本研究中，我们在重组增强子的右半区域鉴定出一个MATa特异性的Sir2与凝缩蛋白（condensin）结合位点；该位点可通过阻止MATa与右臂的默认供体HMRa发生相互作用，将三号染色体维持在具备交配型切换能力的构象状态。在重组增强子区域内，Sir2会强力抑制一个功能未知的小型MATa特异性基因RDT1的转录。当HO核酸内切酶表达以诱导交配型切换进程时，Sir2会从重组增强子重新分布至MATa位点的双链DNA断裂位点，从而解除对RDT1转录的抑制，使RDT1的转录时机与交配型切换的进程相契合。凝缩蛋白同样会响应HO诱导的DNA断裂而从重组增强子处解离，这可能有助于在不破坏HML与HMR异染色质的前提下，实现有效切换所需的三号染色体构象瞬时改变。整体实验设计：本研究获取了野生型（WT）、SIR2缺陷型（SIR2?）以及RDT1启动子区域100bp?菌株的高通量染色体构象捕获（Hi-C）数据。