Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription - PubMed (original) (raw)

Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription

Ethan Ford et al. Genes Dev. 2006.

Abstract

We investigated the role of SIRT7, one of the seven members of the mammalian sirtuin family. We show that SIRT7 is a widely expressed nucleolar protein that is associated with active rRNA genes (rDNA), where it interacts with RNA polymerase I (Pol I) as well as with histones. Overexpression of SIRT7 increases Pol I-mediated transcription, whereas knockdown of SIRT7 or inhibition of the catalytic activity results in decreased association of Pol I with rDNA and a reduction of Pol I transcription. Depletion of SIRT7 stops cell proliferation and triggers apoptosis. Our findings suggest that SIRT7 is a positive regulator of Pol I transcription and is required for cell viability in mammals.

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Figures

Figure 1.

Figure 1.

SIRT7 is a broadly expressed nucleolar protein. (A) RNA was isolated from the indicated tissues (left panels) and embryos (right panels) in wild-type adult mice, resolved by electrophoresis, and subjected to Northern blotting. Blots were probed with 32P-labeled cDNA specific to SIRT7 (top panels) or actin (bottom panels). Muscle actin found in heart and skeletal muscle samples migrated as a distinct band. (B) Proteins extracted from the indicated wild-type mouse tissues were resolved by SDS-PAGE and analyzed by Western blotting using antibodies specific to SIRT7 (top panel) or actin (bottom panel). For each tissue type, samples from two different mice were included. (C) Nucleolar localization of GFP-SIRT7 in live human U2OS cells. (D) Endogenous SIRT7, Pol I, and UBF were visualized by immunofluorescence microscopy in U2OS cells using α-SIRT7 (red), α-Pol I (green), and α-UBF (green) antibodies. The DNA was counterstained with Hoechst 33342. (E) During mitosis GFP-SIRT7 is associated with condensed chromatin in live U2OS cells stained with Hoechst 33342.

Figure 2.

Figure 2.

SIRT7 is associated with the rDNA and RNA Pol I. (A) Results of ChIP analysis showing the localization of SIRT7 and Pol I within the rDNA gene repeats. Cross-linked chromatin from 293T cells was precipitated by the indicated antibodies and analyzed by PCR with the indicated primer pair. (B) Endogenous SIRT7 interacts with RNA Pol I. Partially purified human nuclear extract was immunoprecipitated with control nonspecific human antibodies (lanes 2,5) and α-Pol I antibodies (lanes 3,6). Before immunoprecipitation, the protein fraction used was treated with DNase (lanes 4–6) or left untreated (lanes 1–3). The precipitated proteins were analyzed by Western blotting with α-SIRT7 and α-RPA-116 antibodies as indicated. The input lanes contain 10% of the protein fraction used for IP. (C) Pol I is a component of the TAP-SIRT7 complex. U2OS cell lines harboring the empty vector or TAP-SIRT7 were established. TAP-tagged complexes were purified on IgG-Sepharose and Calmodulin-agarose resins, and the eluted protein complexes were tested for copurifying proteins by Western blot analysis. (Lane 1) Fifty micrograms of nuclear extract (10% of the Input). (Lane 2) Eluate from mock-transfected cells. (Lane 3) Eluate from a cell line expressing TAP-IRT7. The Western blot was probed with an antibody specific for RPA-116 (top panel) and α-SIRT7 (bottom panel). (D) SIRT7 interacts with histones. Histones were isolated from butyric acid-treated HeLa cells by ion exchange chromatography, incubated with GST-SIRT7-Sepharose and GST-Sepharose beads. Bound proteins were eluted and separated by SDS-PAGE and visualized by staining with Coomassie.

Figure 3.

Figure 3.

SIRT7 is an activator of Pol I transcription. (A) Overexpression of SIRT7 stimulates Pol I transcription from an rDNA minigene reporter. 293T cells were cotransfected with a Pol I reporter plasmid and increasing amounts of an expression plasmid encoding Flag-SIRT7. RNA was isolated from transfected cells. The amount of reporter transcripts was determined by Northern blot analysis and quantified using a PhosphorImager. (Bottom panel) Western blot of the transfected cells probed with α-Flag antibody. (B) Knockdown of SIRT7 by RNAi impairs rRNA synthesis. (Left panel) Western blot of extracts from U2OS cells transfected with control-dsRNA (lane 1) or SIRT7-specific dsRNA (lane 2). (Right panel) Pol I transcription was analyzed by RT–PCR. RNA was isolated from cells treated with the respective dsRNAs. Increasing amounts of cDNA were used for PCR with primers that amplify a region of the 5′ external transcribed spacer of the human pre-rRNA. Results from two independent experiments are shown (one in lanes 1,2,5,6 and the other in lanes 3,4,7,8). As a control, RT–PCR was performed to analyze expression of GAPDH. The PCR-products were quantified using a PhosphorImager. (C) Overexpression of SIRT7H188Y and SIRT7S112A does not augment cellular Pol I transcription. (Top panel) Northern blot analysis of pre-rRNA transcripts from 293T cells transfected with different amounts of plasmids expressing wild-type SIRT7 or the point mutants H188Y and S112A. Pre-rRNA levels were determined by PhosphorImager. (Bottom panel) Expression levels of Flag-SIRT7, Flag-SIRT7H188Y, and Flag-SIRT7S112A were monitored on Western blots using α-Flag antibodies. (D) The SIRT7 mutants H188Y and S112A were expressed as GFP-tagged proteins in U2OS cells and their cellular localization was examined in live cells. (E) SIRT7H188Y is associated with rDNA. Flag-SIRT7 wild type (WT) and Flag-SIRT7H188Y mutant (H188Y) were overexpressed in 293T cells and assayed for binding to rDNA by ChIP. Immunoprecipitations were performed with mouse IgGs (lanes 3,4) and α-Flag (M2) antibodies (lanes 5,6); 0.5 and 1.5 μL of whole-cell extract DNA (lanes 1,2) and precipitated DNA (lanes 3–6) were amplified with rDNA primers A (promoter, top panel) and C (28S coding region, bottom panel). (F) Nicotinamide represses rRNA synthesis. NIH3T3 cells were cultured for 6 h in medium containing 40 nM TSA or 5 mM nicotinamide, and pre-rRNA levels were measured by Northern blot analysis using a 32P-labeled riboprobe specific for the 5′-transcribed external spacer. The blot was subsequently reprobed for cytochrome c oxidase (cox 1) mRNA. (G) Treatment with actinomycin D, but not with nicotinamide, releases SIRT7-GFP from nucleoli. U2OS cells expressing SIRT7-GFP were cultured in the presence of either 50 ng/mL of actinomycin D for 2 h to inhibit Pol I activity or 5 mM nicotinamide for 6 h to inhibit NAD+-dependent deacetylase activity. Localization of SIRT7-GFP was examined in live cells.

Figure 4.

Figure 4.

SIRT7 stimulates the association of RNA Pol I with the rDNA. (A) Western blot showing overexpression levels of SIRT7 proteins. (B) Enrichment of Pol I at the transcribed region of the rDNA genes in the presence of ectopic SIRT7. Chromatin was prepared from cells harboring empty vector (pCMV) or plasmids expressing wild-type SIRT7 (SIRT7-WT) and mutant SIRT7 (SIRT7-S112A and SIRT7-H188Y). The chromatin was precipitated with α-RPA116 antibodies and analyzed by PCR with the primer pairs indicated on the right side of the figure. (C) Following siRNA transfections, whole-cell lysates were analyzed by immunoblot for SIRT7 and β-actin. (D) ChIP analysis of Pol I levels in cells transfected with siRNAs. Chromatin was prepared from cells transfected with nonspecific (ns) or α-SIRT7 siRNAs. The chromatin was precipitated with α-RPA116 antibodies and analyzed by PCR with the primer pairs indicated on the right side of the figure. (E) Western blot of SIRT7 and α-tubulin in 20 μg of whole-cell lysates from U2OS cells transfected with the pSUPER or pSUPER–shSIRT7 vectors. (F, right) U2OS cells transfected with the indicated vectors were fixed 5 d post-transfection and visualized by phase contrast. (Left) Apoptotic cells were detected by TUNEL staining.

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