The Fission Yeast Ubiquitin-Conjugating Enzymes UbcP3, Ubc15, and Rhp6 Affect Transcriptional Silencing of the Mating-Type Region

Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.

由RNA聚合酶II（RNA polymerase II）转录的基因，当被插入到粟酒裂殖酵母（Schizosaccharomyces pombe）的mat2或mat3交配型位点附近时，会发生基因沉默。该沉默过程由多种基因产物和顺式作用元件（cis-acting elements）介导。本研究报道了通过筛选高拷贝数沉默干扰因子所鉴定出的新型反式作用因子（trans-acting factors）的相关发现。从强nmt1启动子（nmt1 promoter）出发，编码假定E2泛素缀合酶（E2 ubiquitin-conjugating enzymes）UbcP3、Ubc15（泛素缀合酶）或Rhp6（粟酒裂殖酵母Rad同源蛋白）的cDNA的表达，可解除沉默型交配型位点mat2、mat3以及插入其附近的报告基因的沉默状态。将ade6报告基因（ade6 reporter gene）插入交配型区域后，rhp6基因的缺失可轻微解除其沉默，而ubcP3或ubc15基因的敲除则对沉默过程无明显影响。Rhp18是酿酒酵母（Saccharomyces cerevisiae）Rad18p的粟酒裂殖酵母同源蛋白，而Rad18p是一种可与Rad6p发生物理相互作用的DNA结合蛋白。当UbcP3、Ubc15或Rhp6过表达时，Rhp18并非其解除沉默所必需的因子。过表达Rhp6活性位点突变体的实验结果表明，Rhp6的泛素缀合活性对于干扰沉默过程是必需的。然而，26S蛋白酶体（26S proteasome）的突变并不能抑制高剂量表达的UbcP3、Ubc15或Rhp6的作用，这提示沉默的丧失并非源于沉默因子的降解增强，而是源于泛素化（ubiquitination）介导的蛋白质翻译后修饰（posttranslational modification）。我们就UbcP3、Ubc15和Rhp6可能的作用模式对本研究结果的启示进行了讨论。