The SCFDia2 ubiquitin E3 ligase ubiquitylates Sir4 and functions in transcriptional silencing.
The SCFDia2 ubiquitin E3 ligase ubiquitylates Sir4 and functions in transcriptional silencing.
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DOI：
10.1371/journal.pgen.1002846
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发表时间：
2012
期刊：
影响因子：
4.5
通讯作者：
Zhang Z
中科院分区：
文献类型：
--
作者：
Burgess RJ;Zhou H;Han J;Li Q;Zhang Z
在芽殖酵母中，转录沉默对于调节基因表达和维持基因组完整性是重要的，需要沉默信息调节因子（Sir）蛋白。此外，Rtt106，一个参与核小体组装的组蛋白伴侣，在转录沉默中发挥作用。然而，在有丝分裂细胞分裂过程中转录沉默是如何调节的还不清楚。我们发现，细胞缺乏Dia2，参与DNA复制的SCFDia2 E3泛素连接酶的一个组成部分，在端粒和HMR基因座的沉默显示缺陷和Dia2的F-盒和C-末端区域，Dia2的泛素化活性的两个重要区域，需要适当的转录沉默在这些基因座。此外，我们发现Sir蛋白在dia2 Δ突变细胞中存在错误定位。Dia2和Rtt106突变导致HMR基因座沉默的协同丧失和HMR基因座Sir4蛋白的显著升高，表明dia2 Δ突变细胞中的沉默缺陷至少部分是由于沉默染色质中Sir4水平的改变。支持这一想法，我们表明，SCFDia2泛素化Sir4在体外和体内。此外，Sir4与沉默染色质的结合在细胞周期中受到动态调节，并且这种调节在dia2 Δ突变细胞中丢失。这些结果表明，SCFDia2复合物参与转录沉默，泛素化Sir4，并在细胞周期中调节转录沉默。异染色质对于维持基因组稳定性和调节基因表达是重要的。异染色质蛋白1（HP1）是一种与哺乳动物细胞异染色质基因座上赖氨酸9甲基化的组蛋白H3（H3K9me3）结合的蛋白质，在细胞周期中受组蛋白H3丝氨酸10（H3S10ph）磷酸化的动态调节。与哺乳动物细胞相比，芽殖酵母沉默染色质的转录沉默需要沉默信息调节因子（Sir）蛋白，而H3K9me3和H3S10ph在芽殖酵母中不存在。因此，目前尚不清楚芽殖酵母中的沉默染色质是否以及如何在细胞周期中受到调节。在这里，我们表明，SCFDia2泛素E3连接酶复合物调节转录沉默。我们发现，SCFDia2泛素化Sir4，酵母沉默染色质的结构组成部分，和Sir4水平下降，在细胞周期中的Dia2依赖性的方式。伴随着在细胞周期中端粒沉默染色质上Sir4的减少，端粒相关基因的表达增加。因此，我们提出，在芽殖酵母沉默染色质的转录沉默调节在细胞周期中，部分由SCFDia2介导的Sir4泛素化染色质。
In budding yeast, transcriptional silencing, which is important to regulate gene expression and maintain genome integrity, requires silent information regulator (Sir) proteins. In addition, Rtt106, a histone chaperone involved in nucleosome assembly, functions in transcriptional silencing. However, how transcriptional silencing is regulated during mitotic cell division is not well understood. We show that cells lacking Dia2, a component of the SCFDia2 E3 ubiquitin ligase involved in DNA replication, display defects in silencing at the telomere and HMR locus and that the F-box and C-terminal regions of Dia2, two regions important for Dia2's ubiquitylation activity, are required for proper transcriptional silencing at these loci. In addition, we show that Sir proteins are mislocalized in dia2Δ mutant cells. Mutations in Dia2 and Rtt106 result in a synergistic loss of silencing at the HMR locus and significant elevation of Sir4 proteins at the HMR locus, suggesting that silencing defects in dia2Δ mutant cells are due, at least in part, to the altered levels of Sir4 at silent chromatin. Supporting this idea, we show that SCFDia2 ubiquitylates Sir4 in vitro and in vivo. Furthermore, Sir4 binding to silent chromatin is dynamically regulated during the cell cycle, and this regulation is lost in dia2Δ mutant cells. These results demonstrate that the SCFDia2 complex is involved in transcriptional silencing, ubiquitylates Sir4, and regulates transcriptional silencing during the cell cycle. Heterochromatin is important for the maintenance of genome stability and regulation of gene expression. Heterochromatin protein 1 (HP1), a protein that binds to histone H3 methylated at lysine 9 (H3K9me3) at heterochromatin loci in mammalian cells, is dynamically regulated during the cell cycle by phosphorylation of histone H3 serine 10 (H3S10ph). Compared to mammalian cells, transcriptional silencing at budding yeast silent chromatin requires silent information regulator (Sir) proteins, and H3K9me3 and H3S10ph are not present in budding yeast. Therefore, it is not known whether and how silent chromatin in budding yeast is regulated during the cell cycle. Here, we show that the SCFDia2 ubiquitin E3 ligase complex regulates transcriptional silencing. We show that SCFDia2 ubiquitylates Sir4, a structural component of yeast silent chromatin, and that Sir4 levels decrease during the cell cycle in a Dia2-dependent manner. Concomitant with the reduction of Sir4 at telomeric silent chromatin during the cell cycle, the expression of a telomere-linked gene increases. Therefore, we propose that transcriptional silencing at budding yeast silent chromatin is regulated during the cell cycle, in part by SCFDia2-mediated Sir4 ubiquitylation on chromatin.
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