To understand the regulation of SIRT1-mediated deacetylation in vivo
, biochemical purification was used to identify cellular factors that stably interact with SIRT1. We isolated physiologically formed protein complexes containing SIRT1 from cell extracts of native HeLa cells by conducting affinity chromatography with affinity-purified antisera raised against the carboxy (C) terminus (amino acids 480–737) of SIRT1 (Supplementary Fig. 1a
). As expected, we identified SIRT1 as the major component of the complexes, but several protein bands were also co-purified with SIRT1. Mass spectrometry of a prominent protein band of approximately 130 kilodaltons (kDa) from the SIRT1 complexes revealed peptide sequences corresponding to the DBC1 protein (Supplementary Fig. 1b
, Gi: 24432106). The DBC1
gene was initially identified as it is localized to a region of chromosome 8p21 that was homozygously deleted in human breast cancer; however, the molecular function of DBC1 is poorly understood18,19
To examine the interaction between endogenous DBC1 and SIRT1, cell extracts from human osteosarcoma U2OS cells were immunoprecipitated with the anti-SIRT1 antibody or with the control IgG. As expected, western blot analysis revealed that DBC1 was clearly detected in the immunoprecipitations obtained with the anti-SIRT1 antiserum (lane 3, ) but not with the control antibody (lane 2). Previous studies indicate that HIC1 can also interact with SIRT1 (ref. 11
); nevertheless, we failed to detect a strong interaction between HIC1 and SIRT1 in these cells under the same conditions. To prove the specificity of the SIRT1 antibody, we performed the co-immunoprecipitation in SIRT1-depleted U2OS cells treated with SIRT1
-specific RNAi (lanes 6 and 7). Indeed, we failed to detect any DBC1 from the anti-SIRT1 immunoprecipitates with these SIRT1-depleted cells (lane 7). We also performed a reciprocal co-immunoprecipitation assay. As shown in , endogenous SIRT1 was readily immunoprecipitated with the DBC1-specific antibody (lane 3), but not with a control antibody (lane 2).
DBC1 interacts with SIRT1 in vivo and in vitro
Next, we tested whether SIRT1 binds DBC1 in vitro
. As shown in , 35
-translated DBC1 bound the central core domain of SIRT1 (lane 3) but showed no affinity for either its amino (N)-terminal (lane 2) or C-terminal (lane 4) domains. Conversely, we identified the N terminus of DBC1 as the SIRT1-binding domain (Supplementary Fig. 3
). Because the enzymatic core sequence represents the most conserved region within the mammalian SIRT protein family, we examined whether DBC1 interacts with other members of this family. Thus, Flag-tagged derivatives of the seven human SIRT polypeptides (SIRT1–7) were each expressed in 293 cells and extracts of the transfected cells were immunoprecipitated with the anti-Flag antibody. Western blots revealed that endogenous DBC1 was clearly detected in the immunoprecipitates of Flag–SIRT1 (lane 2, ). Although similar expression levels for all seven Flag–SIRT polypeptides were detected, none of the other SIRT proteins (SIRT2–7) were able to co-immunoprecipitate DBC1 (lanes 3–8, ). These results demonstrate the specificity of the SIRT1 and DBC1 interaction.
Interestingly, when purified Flag–SIRT1 complexes from human cells were analysed by gel-filtration chromatography on a Superose 12 column, we observed that SIRT1 and DBC1 polypeptides co-eluted in fraction 15 with an apparent molecular mass of 440 kDa (lane 4, ). In contrast, a DBC1-free form of SIRT1 eluted in fractions 19–21 (lanes 6 and 7), suggesting that at least two distinct SIRT1 complexes exist in human cells. As expected, we found that SIRT1 from fraction 21 had a strong NAD-dependent deacetylase activity for p53 (lane 3, ). Surprisingly, however, no activity was detected with fraction 15 (lane 2), raising the notion that SIRT1-mediated deacetylation is inhibited by additional factors in the complexes, such as DBC1. To evaluate a role for DBC1 in regulating SIRT1 function, we examined whether DBC1 can inhibit the deacetylase activity of SIRT1 in a purified system. To this end, Flag-tagged forms of SIRT1 and DBC1 were purified under high-stringency conditions for in vitro
deacetylation assays. As indicated in , deacetylation of p53 was observed when the Flag–SIRT1 protein was incubated with acetylated p53 (lane 2). However, this activity was strongly repressed by Flag–DBC1 in a dose-dependent manner (lanes 3 and 4). DBC-mediated repression is apparently as potent as the effects obtained with 5 mM of nicotinamide (NIA) (lane 5), a known inhibitor of SIRT1-mediated deacetylation4
DBC1 inhibits SIRT1-mediated deacetylation of p53
Moreover, to prove the specificity of DBC-mediated inhibition of SIRT1 deacetylase activity, we examined the effect of DBC1 on SIRT2-mediated deacetylation of tubulin. As shown in , deacetylation of tubulin was observed when the purified SIRT2 protein was incubated with acetylated tubulin, as previously reported20
. This activity was also inhibited by nicotinamide (lane 5); however, tubulin deacetylation by SIRT2 was not affected by purified DBC1 polypeptides (lanes 3 and 4). Finally, p53 could also be deacetylated by purified HDAC1 complexes, as we have previously shown17
(lane 2, Supplementary Fig. 4
); nevertheless, this deacetylase activity was not repressed by DBC1 (lanes 3 and 4). Thus, these data demonstrate that DBC1-mediated inhibitory effects specifically act on SIRT1 deacetylase activity.
We then tested whether DBC1 expression rescues p53 from SIRT1-mediated deacetylation and repression in human cells. As expected, co-expression of SIRT1 induced p53 deacetylation (lane 4, ); however, the steady-state levels of acetylated p53 were restored by DBC1 expression in a dose-dependent manner (lanes 5–7). To elucidate the mechanism of DBC-mediated effects on SIRT1, we conducted a co-immunoprecipitation assay to test whether the interaction between SIRT1 and p53 is regulated by DBC1. As shown in , p53 was co-immunoprecipitated with SIRT1 (lane 2). Notably, the p53–SIRT1 interaction was significantly abrogated by DBC1 expression in a dose-dependent fashion (lanes 3–5). These results suggest that DBC1 represses SIRT1 activity in human cells and that these effects may act, in part, through blocking the interactions between SIRT1 and substrates (p53).
DBC1 acts as an inhibitor of SIRT1 in human cells
To explore the functional consequences of these interactions further, we tested whether DBC1 can influence SIRT1-mediated repression of p53 transcriptional activation. As shown in , SIRT1 strongly suppressed p53-mediated transactivation of the PUMA reporter in a luciferase assay. Again, this SIRT1-mediated suppression was abrogated by DBC1 expression in a dose-dependent manner. These data indicate that DBC1 can enhance p53-dependent transactivation of PUMA by inhibiting SIRT1. Because homozygous deletion of the DBC1
gene was reported in human cancers18,21
, inactivation of DBC1
may enhance the deacetylase activity of SIRT1 and thereby lead to inhibition of p53 function ().
To test the above hypothesis, we first examined whether short interfering RNA (siRNA)-mediated knockdown of endogenous DBC1 has any effect on p53 function. To avoid possible off-target effects caused by the DBC1 RNAi, we used two different RNAi sequences that target different regions of the DBC1 messenger RNA (mRNA). Thus, human osteosarcoma U2OS cells were transfected with the DBC1-specific siRNA#1 (DBC1-RNAi#1), DBC1-specific siRNA#2 (DBC1-RNAi#2) or a control siRNA (Control-RNAi). As shown in , RNAi-mediated knockdown of DBC1 expression had no obvious effect on p53 stability (lanes 2 and 3) but significantly reduced the expression levels of PUMA and BAX, two major transcriptional targets of p53. As expected, knockdown of p53 expression by p53-specific siRNA (p53-RNAi) completely abolished the expression of both PUMA and BAX (lane 4), validating that expression of these two targets is indeed p53-dependent. These experiments demonstrate that inactivation of endogenous DBC1 leads to down-regulation of p53 activity.
siRNA-mediated knockdown of DBC1 reduces p53 acetylation and its transcriptional and apoptotic activities
Moreover, to demonstrate that DBC1 acts on p53 by repressing SIRT1 deacetylase activity, we tested whether inactivation of DBC1 indeed reduces acetylation levels of endogenous p53 by SIRT1 and, more importantly, whether these effects are reversed by inactivation of SIRT1 expression. Thus, these cells were transfected with the DBC1-specific siRNA#1 (DBC1-RNAi#1), SIRT1-specific siRNA (SIRT1-RNAi) or a control siRNA (Control-RNAi). As shown in , RNAi-mediated knockdown of DBC1 expression significantly reduced the acetylation levels of endogenous p53 (Ac-p53, bottom panel, lane 3). Notably, the reduction of p53 acetylation was completely reversed by concomitant knockdown of SIRT1 (Ac-p53, bottom panel, lane 4). Similar results were also observed with DBC1-mediated effects on PUMA and BAX by concomitant knockdown of SIRT1 (PUMA and BAX, middle panel, lanes 3 and 4). Thus our data demonstrate that DBC1-mediated effects on p53 activation act mainly through SIRT1 in vivo.
To investigate the role of DBC1 in the stress response, we tested whether inactivation of DBC1 leads to inhibition of p53-dependent apoptosis on DNA damage. For this, U2OS cells were first transfected with either control or DBC1
-specific siRNAs and then exposed to etoposide. Thirty hours later, the cells were stained with DAPI and apoptosis was examined by TUNEL staining. The DBC1-depleted cells were highly resistant to apoptosis, displaying only 8.8% apoptotic cells compared with 20.5% of cells transfected with the control siRNA ( and Supplementary Fig. 11
). To confirm further the role of DBC1 in regulating p53-mediated apoptosis, we performed an apoptosis assay by using Annexin V staining followed by flow-cytometry analysis. Again, p53-mediated apoptosis was repressed in DBC1
knockdown cells ( and Supplementary Fig. 12
). As expected, inactivation of p53 in these cells completely abolished the apoptotic response by DNA damage. Notably, concomitant knockdown of SIRT1
reversed the inhibitory effects on p53-dependent apoptosis by DBC inactivation. These data demonstrate that DBC1 is critically involved in regulating the p53-mediated apoptotic response by repressing SIRT1 function.
Our findings may have significant implications in the treatment of both metabolic-related disorders and cancer. Small molecule inhibitors of the SIRT1 deacetylase have been proposed as novel anticancer agents22–24
, presumably through activating the apoptotic response in cancer cells. On the other hand, activation of SIRT1 in mice also protects them against diet-induced obesity and insulin resistance, mainly through regulating metabolic pathways25,26
. Although a role for mammalian SIRT1 in the regulation of lifespan has not been directly determined, the ability of resveratrol, a chemical found in red wine and other foods, to enhance SIRT1 activity and increase lifespan in lower organisms supports the feasibility of this approach in mammals3,27–30
. Therefore, both inhibitors and activators of SIRT1 could be therapeutically beneficial by affecting different SIRT1-mediated regulatory pathways. It will be intriguing to know whether DBC1 has differential effects in regulating apoptotic responses versus metabolic pathways and whether manipulations of the DBC1–SIRT1 interaction will help to find more potent activators and/or inhibitors for SIRT1 activity.