Chromatin Association of Gcn4 Is Limited by Post-translational Modifications Triggered by its DNA-Binding in Saccharomyces cerevisiae
Chromatin Association of Gcn4 Is Limited by Post-translational Modifications Triggered by its DNA-Binding in Saccharomyces cerevisiae
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DOI：
10.1534/genetics.116.194134
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发表时间：
2016-12-01
期刊：
影响因子：
3.3
通讯作者：
Rosonina, Emanuel
中科院分区：
文献类型：
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作者：
Akhter, Akhi;Rosonina, Emanuel
酿酒酵母转录因子Gcn4在氨基酸饥饿过程中表达，其丰度受泛素介导的蛋白分解控制。CDK8是RNA聚合酶II介体复合体的一种激酶成分，它使Gcn4磷酸化，从而触发其泛素化/蛋白分解，并被认为将Gcn4的降解与靶基因的转录联系起来。除了磷酸化和泛素化，我们以前发现Gcn4以DNA结合依赖的方式变得Sumoyl化，而非Sumoylate形式的Gcn4显示染色质占有率增加，但只有在CDK8存在的情况下。为了进一步研究Gcn4与染色质的关联是如何调节的，我们在这里研究了Gcn4总酰化的决定因素，以及它的翻译后修饰是如何协调的。值得注意的是，将缺乏DNA结合域的Gcn4人工靶向异源DNA位点可以恢复其自然修饰位点的总甲基化，表明DNA结合足以在体内进行修饰。事实上，我们发现Gcn4的SUMO化既不需要靶基因的转录，也不需要磷酸化，但阻断它的SUMO化会改变它的磷酸化和泛素化模式，将GCN4 SUMO化置于这些CDK8介导的修饰的上游。Gcn4是一种超糖化形式，它强烈支持和基化在限制其与染色质结合方面的作用，显示出极大地减少了DNA占有率和靶基因的表达。重要的是，我们发现CDK8至少部分负责从DNA中清除超糖基化的Gcn4，进一步暗示了苏莫化是CDK8介导的磷酸化和降解的刺激因素。这些结果支持相扑在标记转录因子的DNA结合形式方面的一种新功能，该转录因子触发下游过程，限制其与染色质的关联，从而防止目标基因的不受控制的表达。
The Saccharomyces cerevisiae transcription factor Gcn4 is expressed during amino acid starvation, and its abundance is controlled by ubiquitin-mediated proteolysis. Cdk8, a kinase component of the RNA polymerase II Mediator complex, phosphorylates Gcn4, which triggers its ubiquitination/proteolysis, and is thought to link Gcn4 degradation with transcription of target genes. In addition to phosphorylation and ubiquitination, we previously showed that Gcn4 becomes sumoylated in a DNA-binding dependent manner, while a nonsumoylatable form of Gcn4 showed increased chromatin occupancy, but only if Cdk8 was present. To further investigate how the association of Gcn4 with chromatin is regulated, here we examine determinants for Gcn4 sumoylation, and how its post-translational modifications are coordinated. Remarkably, artificially targeting Gcn4 that lacks its DNA binding domain to a heterologous DNA site restores sumoylation at its natural modification sites, indicating that DNA binding is sufficient for the modification to occur in vivo. Indeed, we find that neither transcription of target genes nor phosphorylation are required for Gcn4 sumoylation, but blocking its sumoylation alters its phosphorylation and ubiquitination patterns, placing Gcn4 sumoylation upstream of these Cdk8-mediated modifications. Strongly supporting a role for sumoylation in limiting its association with chromatin, a hyper-sumoylated form of Gcn4 shows dramatically reduced DNA occupancy and expression of target genes. Importantly, we find that Cdk8 is at least partly responsible for clearing hyper-sumoylated Gcn4 from DNA, further implicating sumoylation as a stimulus for Cdk8-mediated phosphorylation and degradation. These results support a novel function for SUMO in marking the DNA-bound form of a transcription factor, which triggers downstream processes that limit its association with chromatin, thus preventing uncontrolled expression of target genes.