Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription.
Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription.
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功能蛋白质组学建立了 SIRT7 与染色质重塑复合物的相互作用，并扩展了其在 RNA 聚合酶 I 转录调节中的作用。
DOI：
10.1074/mcp.m111.015156
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Cristea,IleanaM
中科院分区：
文献类型：
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作者：
Tsai,Yuan-Chin;Greco,ToddM;Boonmee,Apaporn;Miteva,Yana;Cristea,IleanaM
在哺乳动物中，SIRT7是唯一存在于核仁中转录核糖体DNA的酶。最近的报道证实，SIRT7与RNA Pol I机制有关，是rDNA转录所必需的。尽管SIRT7与酵母组蛋白去乙酰基酶Sir2同源，但目前的研究表明，SIRT7本身几乎没有去乙酰基酶活性。由于到目前为止只描述了两个SIRT7相互作用：RNA Pol I和上游结合因子，因此鉴定与SIRT7相关的蛋白质和复合体对于了解其功能至关重要。在这里，我们提出了SIRT7交互网络的第一个特征。我们系统地研究了三个EGFP标记的SIRT7结构的蛋白质相互作用：野生型，影响rDNA转录的点突变，以及缺失预测的螺旋卷曲结构域的缺失突变体。采用蛋白质组学和生物信息学相结合的方法整合基因本体论分类、功能蛋白质网络和与SIRT7共分离的蛋白质的标准化丰度。由此得到的精炼蛋白质组数据证实了SIRT7与RNA Pol I和上游结合因子的相互作用，并突出了与RNAPol I和II依赖的转录过程中涉及的因子以及几个核仁定位的染色质重塑复合体的关联。尤其丰富的是B-wich复合体的成员，如Mybbp1a、WSTF和Snf2h。显著的相互作用通过选择的反应监测类方法得到验证，该方法使用稳定同位素的代谢标记、共聚焦显微镜、相互免疫亲和沉淀以及与内源SIRT7的共分离。为了扩大目前对SIRT7依赖的rDNA转录调控机制的了解，我们发现小干扰RNA介导的SIRT7敲除导致RNA Pol I蛋白水平降低，但不能导致信使RNA水平下降，这在不同类型的细胞中得到证实。在SIRT7相互作用网络的背景下，RNA Pol I蛋白水平的下调导致我们认为SIRT7在转录过程中将染色质重塑复合体的功能与RNA Pol I机制联系起来起着至关重要的作用。
Among mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of RNA Pol I protein levels placed in the context of SIRT7 interaction networks led us to propose that SIRT7 plays a crucial role in connecting the function of chromatin remodeling complexes to RNA Pol I machinery during transcription.