1. Volume 42, Issue 2, Pages 210-223 (April 2011)
Stabilization of Suv39H1 by SirT1 Is Part of Oxidative Stress Response and Ensures Genome Protection 
Laia Bosch-Presegué, Helena Raurell-Vila, Anna Marazuela-Duque, Noriko Kane-Goldsmith, Adamo Valle, Jordi Oliver, Lourdes Serrano, Alejandro Vaquero 
Molecular Cell 
Volume 42, Issue 2, Pages (April 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 SirT1 Overexpression Alters Suv39h1 Renewal in PCH
(A) Subnuclear distribution of DAPI staining, Suv39h1-EGFP, H3K9me3, and HP1α in NIH 3T3 cells transfected with Suv39h1-EGFP ± SirT1.
(B–D) FRAP assays on the PCH foci of NIH 3T3 cells.
(B) Fluorescence signals from FRAP representative experiments in Suv39h1-EGFP ± SirT1 transfected cells before and after photobleaching, as indicated. Red arrows indicate PCH foci selected for photobleaching.
(C) Relative fluorescence intensity of the FRAP experiments.
(D) FRAP assay (as in C) of Suv39h1-EGFP expressed in WT and SirT1−/− MEFs untreated or treated with 2 mM H2O2 for 1 hr. Inset: western blot of SirT1 and actin in WT cells.
(E) Quantification and statistical analysis of the FRAP experiments in (B) to (D) and in Figure 2I, such as mobile population fraction (Mobile [%]), and half-time of fluorescence recovery (t1/2). SirT1 expression led to a significant increase in Mobile (%) and a significant decrease in t1/2 (p < ).
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Upregulation of SirT1 Levels Correlate to Higher Suv39h1 Levels
(A) Western blot of 293 cells transfected with the indicated factors. Levels of SirT1 (FLAG), Suv39h1, and actin were monitored.
(B) Western blot (as in A) with Myc-Suv39h1 cotransfected with FLAG-SirT1, FLAG-SirT1m, FLAG-SirT2, or Gal4BD-SirT1.
(C) Western blot of two different FLAG-SirT1 tetracycline-inducible cell lines before and after induction of FLAG-SirT1, alone or with constitutive expression of Myc-Suv39h1.
(D) Western blot as in (A), but with two different extraction methods.
(E) Protein levels of endogenous SirT1, Suv39h1, and actin in extracts from WT or SirT1−/− MEFs.
(F) SirT1 knockdown by RNAi in 293 cells.
(G) RNA levels of SirT1, Suv39h1 and actin extracted from 293 cells shown in lanes 2 and 4 of (A) by RT-PCR.
(H) RNA levels as in (G), but with WT, SirT1−/−, or Suv39h1/2−/− MEFs.
(I) FRAP analysis (as in Figure 1) in PCH of NIH 3T3 cells transfected with either 0.5 μg (as in Figure 1) or 2 μg Suv39h1-EGFP expression plasmid. Quantifications are shown in Figure 1E.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 SirT1 Regulates Suv39h1 Stability
(A) Summary of the experiment shown in (B).
(B) Curve of [35S]-Myc-Suv39h1 in the presence or absence of FLAG-SirT1. The plot is based on quantification of multiple experiments, one of which is shown in (C). The levels of [35S] in Myc-Suv39h1 elutions were normalized to the total amount of Myc-Suv39h1 (mean ± standard deviation [SD]). The dashed blue lines indicate the half-time (t1/2) of Myc-Suv39h1 under these conditions, calculated as the time at which the initial [35S]-Myc-Suv39h1 signal (set as 100%) drops to 50%. The black arrows indicate t1/2 of Myc-Suv39h1; t1/2 of Myc-Suv39h1+FLAG-SirT1; and the ratio of [35S]-Myc-Suv39h1 signals at 11 hr after pulse.
(C) A representative experiment of the multiple (n = 4) used to generate the graph in (B).
(D) Schematic of the experiment shown in (E).
(E) Similar experiment as in (B), but without a radioactive pulse. Myc-Suv39h1 protein levels, represented as means ± SD (left scale), were quantified relative to their level at the time of SirT1 transfection (set as 100%). SirT1 levels (right scale) are represented differently: the maximum level reached was assigned a value of 100% (mean ± SD).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Regulation of Suv39h1 Levels by SirT1 Requires the N-Terminal Domain of SirT1 and the Chromodomain of Suv39h1
(A) Constructs used in (B).
(B) 293 cells transfected with indicated vectors. Myc-Suv39h1 levels represented as means ± SD are plotted relative to the levels of Suv39h1+G4BD, and assigned a value of 1. ∗p < 0.05.
(C) Schematics of the different Myc-tagged constructs of Suv39h1 used in (D). The ability of these constructs to bind to SirT1 (Vaquero et al., 2007) is summarized on the right side.
(D) Western blot of cells transfected with the constructs described in (C): FL (Suv39h1), ΔChr (ΔChromo) or ΔSET ± FLAG-SirT1.
(E) Quantification of several experiments (n = 5) as in (D), myc-Suv39h1 levels are represented as means ± SD. ∗∗∗p <
(F) GFP fusion proteins used in (G) and (H).
(G) Western blot of 293 cells transfected with GFP, Chromo-GFP, or Nt-GFP, ± FLAG-SirT1. The red asterisk marks the size of GFP. When using the GFP fusion proteins (Chromo- and Nt-), a proportion of GFP protein was found as a result of sporadic degradation.
(H) Quantification from multiple experiments (n = 5) performed as in (G) and represented as in (E). ∗∗∗p <
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Suv39h1 Is Upregulated during Stress through a SirT1-Dependent Mechanism
(A) WT and SirT1−/− MEFs incubated with serum from rats fed ad libitum (AL) or under calorie restriction (CR) for 24 hr were analyzed to quantify endogenous SirT1, Suv39h1, and actin.
(B) Quantification of Suv39h1 levels normalized to actin from multiple experiments (mean ± SD, n = 3) as in (A); the CR levels are reported relative to the corresponding AL levels. ∗∗∗p <
(C) Experiment similar to that in (A), but instead of using AL and CR serums, incubating the MEFs in the presence or absence of 2mM H2O2 for 1 hr.
(D) Quantification from multiple experiments (n = 3) as in (C) and represented as in (B). ∗∗∗p <
(E) Protein levels of SirT1, Suv39h1, actin, histone H3, and H3K9me3 of livers from rats fed AL or under CR.
(F) Quantification for SirT1 and Suv39h1 (normalized to actin levels), and for H3K9me3 (normalized to histone H3 levels), from multiple experiments done as in (E) and represented as mean ± SD. ∗∗∗p <
(G) Immunohistochemistry experiments to detect SirT1 and H3K9me3 in AL and CR animals, with two of the livers from rats analyzed in (E) and (F).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
SirT1 Inhibits Degradation of Suv39h1 by Inhibition of Proteasome-Dependent Polyubiquitination of K87 in Suv39h1 Chromodomain
(A) Western blot of 293 cells transfected with Suv39h1 ± SirT1, alone or plus 50 μM lactacystin.
(B) Quantification of Myc-Suv39h1 under these conditions are represented as mean ± SD. ∗∗p < 0.02.
(C) Polyubiquitination levels of Suv39h1. Extracts from cells transfected in the indicated combinations were immunoprecipitated with Myc-tag antibodies (Suv39h1) and tested for FLAG (SirT1), Myc (Suv39h1), and HA (ubiquitination). Inputs and elutions are shown.
(D and E) Elutions of experiments as in (C), with the indicated factors.
(F) Levels of Suv39h1 in H1299 cells cotransfected with MDM2 ± SirT1. A quantification of these assays is shown in Figure S4C.
(G) Protein levels of the indicated factors in p53−/− or p53−/−/MDM2−/− MEFs.
(H) In vitro ubiquitination of Suv39h1 by recombinant MDM2 with the indicated proteins.
(I) Myc-Suv39h1 ubiquitination levels in WT and ΔChromo performed as in (C) (elutions shown).
(J) Primary and secondary structure of the Suv39h1 chromodomain (human, mouse, and Drosophila). The segment missing in the ΔChromo mutant is highlighted. The five candidate lysines are shown in red; K87 is highlighted in yellow.
(K) Suv39h1 WT and mutants K55A/K56A, K63A, K81A, and K87A were transfected ± FLAG-SirT1. The results of multiple experiments (n = 5) were plotted relative to WT (mean ± SD). ∗∗∗p < 0.002, ∗∗p < A representative experiment is shown in Figure S4I.
(L) Effects of SirT1 overexpression on Chromo-GFP WT or K87A. Quantification from multiple experiments (mean ± SD, n = 5) is shown related to WT. ∗∗∗p < 0.002, ∗∗p < A representative assay is shown in Figure S4J.
(M) Polyubiquitination levels of Suv39h1 WT or K87A in presence or absence of FLAG-SirT1 similarly as in (C).
(N) Polyubiquitination of Myc-Suv39h1 WT or K87A (α-HA signal) of Figure 6M, Figure S4H, and other experiments divided by the total amount of Suv39h1 protein obtained from the α-Myc signal. K87A polyubiquitination levels are represented as mean ± SD and relative to WT. ∗∗∗p <
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Mutation of K87 Increases Suv39h1 Turnover in PCH and Associates to Genome Protection
(A) FRAP analysis of Suv39h1-EGFP WT versus K87A ± SirT1 performed as in Figure 1C.
(B) Quantification and statistical analysis of the FRAP experiments described in (A). Note that K87A mutant led to a significant increase in Mobile (%) and a significant decrease in t1/2 (p < ).
(C) Experiment shown in (D) and (E).
(D) Immunofluorescence analysis for H3K9me3, γH2Ax, Suv39h1-EGFP, and DAPI (for micronuclei detection, indicated by yellow arrows). Some micronuclei were strongly stained with γH2AX (blue arrows).
(E) Genome stability was quantified by counting the γH2AX-foci-positive cells and the presence of micronuclei.
(F) FISH of PCH (main islet and islet a) on metaphase spread of Suv39h1WT-expressing NIH 3T3 cells. Note the presence of acentric chromosome fragments (green arrows and islet b) and Robertsonian translocations (red arrow and islet c).
(G) Real-time quantification of the levels of γ-satellite expression of the cells used in (D) together with Suv39h1/2 and SirT1 KO MEFs (mean ± SD). As shown all differences were statistical significant. ∗∗∗p < Both KO lines were compared to corresponding WT.
(H) Model of how SirT1 modulates Suv39h1 in PCH structure in response to stress.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions