Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription
| Autor: | Todd M. Greco, Yana Miteva, Yuan-Chin C Tsai, Apaporn Boonmee, Ileana M. Cristea |
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| Rok vydání: | 2012 |
| Předmět: |
Proteomics
RNA-induced transcriptional silencing Transcription Genetic Recombinant Fusion Proteins Blotting Western Green Fluorescent Proteins Down-Regulation RNA polymerase II Enhancer RNAs Biology Real-Time Polymerase Chain Reaction Biochemistry DNA Ribosomal Analytical Chemistry Cell Line Transcription (biology) RNA Polymerase I Tandem Mass Spectrometry Protein Interaction Mapping Transcriptional regulation RNA polymerase I Humans Sirtuins Protein Interaction Domains and Motifs RNA Messenger Protein Interaction Maps RNA Small Interfering Molecular Biology RNA polymerase II holoenzyme Cells Cultured Genetics Cell Nucleus General transcription factor Research Chromatin Assembly and Disassembly Cell biology Gene Expression Regulation Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Gene Knockdown Techniques Multiprotein Complexes biology.protein RNA Interference Pol1 Transcription Initiation Complex Proteins Chromatography Liquid Protein Binding |
| Zdroj: | Molecularcellular proteomics : MCP. 11(5) |
| ISSN: | 1535-9484 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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