1. Volume 52, Issue 3, Pages 303-313 (November 2013)
Repression of RNA Polymerase I upon Stress Is Caused by Inhibition of RNA- Dependent Deacetylation of PAF53 by SIRT7 
Sifan Chen, Jeanette Seiler, Magaly Santiago-Reichelt, Kerstin Felbel, Ingrid Grummt, Renate Voit 
Molecular Cell 
Volume 52, Issue 3, Pages (November 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1 PAF53 Is Acetylated by CBP
(A) PAF53 is acetylated in vivo. HEK293T cells expressing Flag-PAF53 were treated for 5 hr with TSA (500 nM), NAM (10 mM), or both inhibitors. After lysis in the presence of HDAC inhibitors, Flag-PAF53 was bound to and eluted from the anti-Flag beads and analyzed on immunoblots with anti-ac-K and anti-PAF53 antibodies. The relative level of PAF53 acetylation (rel. acet.) is indicated below.
(B) CBP acetylates PAF53. GFP-PAF53 was coexpressed with CBP-HA, p300-HA, or Flag-PCAF in HEK293T cells. Cells were treated with NAM (5 mM) and TSA (500 nM) 5 hr before harvesting, and expression of the HATs was probed on immunoblots with anti-HA and anti-Flag antibodies. Acetylation of purified GFP-PAF53 was monitored with anti-ac-K and anti-PAF53 antibodies.
(C) CBP acetylates PAF53 at K373. GFP-tagged wild-type (WT) or mutant (K373R) PAF53 was expressed in HEK293T cells alone or together with CBP-HA. After treatment with HDAC inhibitors and affinity purification, the acetylation state of GFP-PAF53 was monitored on immunoblots.
(D) Wild-type and mutant PAF53 localize to nucleoli. Live-cell imaging of U2OS cells expressing GFP-PAF53 or GFP-PAF53/K373R. The images show GFP fluorescence and Hoechst staining. The scheme above depicts the amino acid sequence flanking K373 of PAF53. Scale bar, 10 μm.
(E) Increased rDNA occupancy of the acetylation-deficient mutant PAF53/K373R. ChIP shows the association of PAF53 and UBF in U2OS cells stably expressing GFP-PAF53 or PAF53/K373R. Precipitated DNA was assayed by qPCR using primers that amplify different regions of rDNA shown in the scheme above. The amount of immunoprecipitated DNA is calculated to input DNA. Bars denote the mean value (±SD) from three biological replicates.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 SIRT7 Interacts with and Deacetylates PAF53
(A) PAF53 interacts with SIRT7. GST-tagged SIRT7, SIRT1, SIRT6 immobilized on glutathione Sepharose were incubated with lysates containing in vitro-translated 35S-labeled PAF53. Bound PAF53 was visualized by PhosphorImaging. Ten percent of input and 75% of bead-bound PAF53 are shown.
(B) PAF53 is associated with SIRT7 in vivo. Pol I was immunoprecipitated from HeLa nuclear extracts with antibodies against RPA194. Coprecipitation of PAF53 and SIRT7 was analyzed on immunoblots with antibodies specific to PAF53 and SIRT7.
(C) SIRT7 deacetylates PAF53 in vitro. GST-PAF53 was preacetylated with CBP, incubated with baculovirus-expressed His-tagged SIRT2, SIRT6, or SIRT7 in the absence or presence of 1 mM NAD+, and analyzed on western blots with anti-ac-K and anti-GST antibodies. Coomassie-stained His-tagged SIRT2, SIRT6, and SIRT 7 are shown on the left.
(D) PAF53 is hyperacetylated in SIRT7-deficient cells. Flag-PAF53 was overexpressed in HEK293T cells that were transfected with control- (sh-ctrl) or SIRT7-specific shRNA (sh-SIRT7). The western blots show depletion of SIRT7 in cell lysates as well as the level of immunoprecipitated PAF53 probed with anti-ac-K and anti-PAF53 antibodies.
(E) Depletion of CBP and SIRT7 impairs pre-rRNA synthesis. Northern blot monitoring pre-rRNA levels in U2OS cells transfected with nontargeting siRNA (si-ctrl), or CBP- and SIRT7-specific siRNAs. Blotted RNA was hybridized to a 32P-labeled riboprobe comprising antisense sequences from +150 to +1 of human pre-rRNA. Quantification of pre-rRNA as well as ethidium bromide-stained 28S and 18S rRNA are shown below. The bars on the right show CBP- and SIRT7-mRNA levels normalized to β-actin mRNA. Bars denote means ± SD from three independent biological replicates.
(F) rDNA occupancy of PAF53 is impaired in SIRT7-deficient cells. ChIP-qPCR shows rDNA occupancy of PAF53 and UBF in U2OS cells transfected with control si-RNA (si-ctrl), SIRT7-specific (si-SIRT7), or CBP-specific (si-CBP) siRNA. Precipitated DNA was assayed by qPCR using the indicated primer pairs that amplify the rDNA promoter (H0), the 18S and 28S rRNA coding region (H4, H8), or the 3′ end of the pre-rRNA coding region (H13). Bars denote means ± SD from three independent biological replicates.
(G) Depletion of SIRT7 does not alter chromatin compaction at rDNA. FAIRE analysis was performed on five regions of rDNA (H0, H4, H8, H13, and H18; see Figure 1E, top) in U2OS cells transfected with siRNAs specific to SIRT7 (si-SIRT7) or nontargeting siRNA (si-ctrl). The graphs depict the relative FAIRE signals after qPCR analysis. Bars denote means ± SD from three independent biological replicates. All p values were ≥0.05.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Hypoacetylation of PAF53 Promotes Pol I Transcription
(A) NAD+-dependent stimulation of Pol I transcription in vitro. Transcription was assayed in reactions containing linearized template DNA and 40 or 60 μg of nuclear extract (NE) from HeLa cells supplemented with 0.8 mM NAD+ or not. Run-off transcripts were analyzed by gel electrophoresis and PhosphorImaging. The relative amount of transcripts (rel. tx) is indicated below.
(B) SIRT7 is essential for Pol I transcription. Nuclear extracts were prepared from HEK293T cells expressing nontargeting shRNA (sh-ctrl) or SIRT7-specific shRNA (sh-SIRT7). Protein levels of SIRT7, tubulin, UBF, and RPA116 were monitored on western blots (left panel). In vitro transcription was performed with 20, 40, and 60 μg of nuclear extracts in the absence or presence of 0.8 mM NAD+. Run-off transcripts were analyzed and quantified as above (Figure 3A).
(C) PAF53 is hyperacetylated under cellular stress. HEK293T cells expressing Flag-PAF53 were cultured in normal conditions (untreated), cultured in glucose-free medium for 12 hr (−glucose), or treated with anisomycin (10 μM, 1.5 hr) or actinomycin D (AMD, 100 ng/ml, 2 hr). Immunoprecipitated Flag-PAF53 was analyzed on immunoblots with anti-PAF53 and anti-ac-K antibodies. The numbers below represent the relative level of acetylated PAF53.
(D) Energetic and transcriptional stress abrogate the association of PAF53 with SIRT7. (Left and middle panels) HEK293T cells expressing Flag-SIRT7 and GFP-PAF53 were cultured in glucose-rich or glucose-free medium (12 hr) or treated with actinomycin D (AMD). Flag-SIRT7 was immunoprecipitated, and coprecipitated GFP-PAF53 was monitored on immunoblots with anti-GFP antibodies. (Right) Pol I (RPA194) was immunoprecipitated from lysates of HEK293T cells grown in the presence or absence of glucose, and coprecipitation of PAF53 and Pol I was monitored on immunoblots using antibodies against PAF53 and RPA116.
(E) SIRT7 is released from nucleoli upon stress. Direct and indirect immunofluorescence of GFP-SIRT7 (green) and UBF (red) in NIH 3T3 cells cultured in normal conditions (+glucose) or in glucose-deficient medium (−glucose). The righthand panels show fluorescent images of AICAR-treated (2 mM, 12 hr) or untreated U2OS-GFP-SIRT7 cells. Nuclei were stained with Hoechst Scale bar, 10 μm.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Stress-Induced Hyperacetylation of PAF53 Reduces rDNA Occupancy of Pol I and Represses Transcription
(A) Stress-induced downregulation of pre-rRNA synthesis is compromised in cells expressing PAF53/K373R. Northern blots show pre-rRNA of parental U2OS and HEK293T cells and cells expressing wild-type (WT) or mutant (K373R) GFP-PAF53. Cells were grown in normal conditions, treated with AICAR (1 mM, 12 hr), or cultured in glucose-free medium (12 hr) as indicated. Ethidium bromide-stained 18S rRNA is shown below. The numbers indicate the relative level of pre-rRNA.
(B) Stress-induced loss of Pol I from rDNA is reduced in cells expressing mutant PAF53/K373R. ChIP showing rDNA occupancy of Pol I (RPA116) and UBF in U2OS cells expressing wild-type or mutant GFP-PAF53/K373R. The bar diagram shows the association of Pol I (RPA116) and UBF with rDNA (amplicon H1 and H8, see Figure 1E) in untreated or AICAR-treated (1 mM, 12 hr) cells. Bars denote means ± SD from three independent biological replicates.
(C) SIRT7 rescues Pol I transcription in extracts from glucose-deprived cells. (Upper panel) Transcriptional activity of nuclear extract (35 μg) from HEK293T cells cultured in glucose-rich (+glu) or glucose-free (−glucose) medium supplemented with 0.8 mM NAD+ and Flag-SIRT7 (12.5 and 40 ng). (Lower panel) Nuclear extract from glucose-deprived HEK293T cells was supplemented with 0.8 mM NAD+, Flag-SIRT7 (25 and 50 ng), and immunopurified Flag-TIF-IA (20 ng) as indicated. Transcripts were analyzed by gel electrophoresis and PhosphorImaging. The numbers indicate the relative amount of run-off transcripts (rel. tx).
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Nucleolar Enrichment of SIRT7 Requires Nascent Pre-RNA
(A) The interaction of SIRT7 and PAF53 depends on RNA. GFP-PAF53 was precipitated from lysates of HEK293T cells coexpressing GFP-PAF53 and Flag-SIRT7 in the absence or presence of RNase A (20 μg/ml). Precipitation of GFP-PAF53 and coprecipitation of Flag-SIRT7 were monitored on immunoblots.
(B) RNase treatment abolishes nucleolar detention of SIRT7. Permeabilized U2OS-GFP-SIRT7 cells were incubated with RNase A (1 mg/ml, 10 min), and GFP-SIRT7 was visualized by direct immunofluorescence. Nuclei were counterstained with Hoechst 33342, and nucleoli were stained with anti-UBF antibodies (red). Scale bar, 10 μm.
(C) Northwestern blot showing SIRT7 binding to RNA. (Left) GST-tagged PAF53, Cdc14B, and SIRT7 were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with radiolabeled 5′ ETS RNA (from +10 to +389). Bound RNA was visualized by PhosphorImaging. The asterisk marks a proteolytic product of GST-Cdc14B. (Right) Northwestern blot comparing RNA binding of GFP-tagged SIRT7 with SIRT1, SIRT6, and TIF-IA. Coomassie-stained proteins are shown on the left.
(D) Pull-down assay showing binding of 32P-labeled 5′ ETS RNA (+10/+389) to SIRT7. Immobilized GFP-SIRT1, SIRT6, and SIRT7 were incubated with radiolabeled 5′ ETS RNA, and bound RNA was analyzed by gel electrophoresis and PhosphorImaging (right panel). The numbers indicate the relative amount of bound RNA. Coomassie-stained proteins are shown on the left.
(E) SIRT7 binds to RNA. Lysates from HEK293T cells expressing GFP-tagged SIRT1, SIRT6, or SIRT7 were incubated with streptavidin-coated Dynabeads (ctrl) or with Dynabeads containing 5′ ETS RNA (+10/+389). Bound proteins were analyzed on western blots using anti-GFP antibodies.
(F) SIRT7 is associated with pre-rRNA. Shown are RNA immunoprecipitation (RIP) assays from HEK293T cells expressing Flag-tagged SIRT1, SIRT6, or SIRT7. Cells were exposed to UV light (254 nm) and lysed, and RNA-protein complexes were bound to anti-Flag beads. After peptide elution and decrosslinking, coprecipitated RNA was purified and analyzed by RT-qPCR. The percentage of precipitated RNA relative to the respective input RNA is shown. Bars denote means ± SD from three independent biological replicates (∗∗∗p < 0.001). The positions of rDNA amplicons are shown above.
(G) SIRT7 binds to nascent pre-rRNA. HEK293T cells expressing Flag-tagged SIRT7 were treated with AICAR (2 mM, 12 hr) or left untreated (control). SIRT7-RNA complexes were isolated and analyzed as in Figure 5F. Bars denote means ± SD from two independent biological replicates (∗∗p = 0.003, ∗∗∗p < 0.001). The positions of amplicons are shown in Figure 5F.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Reversible Acetylation of PAF53 Regulates rDNA Transcription
Under normal growth conditions, elongating Pol I is associated with the histone acetyltransferase CBP and the deacetylase SIRT7. CBP acetylates the Pol I-associated factor PAF53 at lysine 373 (K373), whereas SIRT7 counteracts CBP-dependent acetylation, removing the acetyl group from K373. Under conditions of energetic stress, AMP-kinase (AMPK) phosphorylates TIF-IA at serine 635, which precludes transcription complex formation and inhibits transcription initiation (Hoppe et al., 2009). Binding to nascent pre-RNA (blue line) is essential for nucleolar retention of SIRT7, lack of nascent rRNA causing release of SIRT7 into the nucleoplasm. As a consequence, PAF53 remains acetylated at K373, which in turn reduces rDNA occupancy of Pol and reinforces transcription repression.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions