Abstract
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.
Highlights
The correlation between histone acetylation status and transcriptional regulation is well established in that hyperacetylation is commonly associated with transcriptional activation, whereas hypoacetylation is associated with transcriptional repression [1,2,3]
We propose a model in which sirtuin 7 (SIRT7) regulates RNA Pol I transcription through interaction with chromatin remodeling complexes
We have not ruled out the possibility that other Pol I subunits or associated proteins might be downregulated at the mRNA level, our results demonstrate for the first time that SIRT7 knockdown does not affect the mRNA level of RPA194 but instead modulates its protein level, pointing toward a post-transcriptional or post-translational role in maintaining the stability of Pol I complexes
Summary
The correlation between histone acetylation status and transcriptional regulation is well established in that hyperacetylation is commonly associated with transcriptional activation, whereas hypoacetylation is associated with transcriptional repression [1,2,3]. Based on homology of the sirtuin core domain, there are seven sirtuins (SIRT1–7) in human cells with functions not limited to histone deacetylation, because many additional cellular proteins have been identified as substrates [4, 6]. Candidate interactions that showed significant enrichment in SIRT7 isolations relative to EGFP control isolations were subjected to gene ontology classification and functional network analysis, revealing a subset of nucleolar-enriched proteins, chromatin remodeling and modification factors, and transcriptional regulators. By correlating normalized immunoisolated protein abundance with these prominent functional categories, we refined the proteomic data set into targets of significant biological interest These included Pol I- and II-dependent transcriptional processes as well as several nucleolar chromatin remodeling complexes, in particular the B-WICH complex. We propose a model in which SIRT7 regulates RNA Pol I transcription through interaction with chromatin remodeling complexes
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