Zentrum für Medizinische Biotechnologie, Abteilung Genetik, Universität Duisburg-Essen, 45117 Essen, Germany.
Nucleic Acids Res. 2010 Dec;38(22):7991-8000. doi: 10.1093/nar/gkq689. Epub 2010 Aug 10.
The silent mating-type loci HML and HMR of Saccharomyces cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for the proteins Rap1, Abf1 and Sum1 as well as for the origin recognition complex (ORC). Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. In this study, we constructed a synthetic HML-E silencer (HML-SS ΔI) that lacked this redundancy. It consisted solely of Rap1 and ORC-binding sites and the D2 element, a Sum1-binding site. All three elements were crucial for minimal HML silencing, and mutations in these elements led to a loss of Sir3 recruitment. The silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Δ. Moreover, deletions of SIR1 and DOT1 lead to complete derepression of the HML-SS ΔI silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing.
酿酒酵母的沉默交配型基因座 HML 和 HMR 含有永久被抑制的交配型信息。这种沉默是由侧翼序列元件（E- 和 I-沉默子）介导的。它们包含 Rap1、Abf1 和 Sum1 蛋白结合位点的组合，以及起始识别复合物 (ORC)。它们共同募集其他沉默因子，最重要的是抑制性 Sir2/Sir3/Sir4 复合物，在 HM 基因座建立异染色质样结构。然而，HM 沉默子表现出相当大的功能冗余，这阻碍了进一步沉默因子的鉴定。在这项研究中，我们构建了一个合成的 HML-E 沉默子（HML-SS ΔI），它缺乏这种冗余。它仅由 Rap1 和 ORC 结合位点以及 D2 元件（Sum1 结合位点）组成。这三个元件对于最小 HML 沉默都是至关重要的，这些元件的突变导致 Sir3 的募集丧失。沉默子对 rap1-12 的突变敏感，但对 orc 突变或 sum1Δ 的敏感性较低。此外，SIR1 和 DOT1 的缺失导致 HML-SS ΔI 沉默子的完全去抑制。因此，这个功能齐全的最小 HML-E 沉默子将有助于鉴定参与 HML 沉默的新因子。
