Oncogene (2008) 27, 3384–3392 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE SIRT1 negatively regulates HDAC1-dependent transcriptional repression by the RBP1 family of

O Binda1, C Nassif1 and PE Branton1,2,3

1Department of Biochemistry, McGill University, Montre´al, Que´bec, Canada; 2Department of Oncology, McGill University, Montre´al, Que´bec, Canada and 3McGill Cancer Center, McGill University, Montre´al, Que´bec, Canada

Both RBP1 and the highly related BCAA play a considerable effort is being focused on the targeting of role in the induction of growth arrest and cellular deacetylase (HDAC) activity as part of cancer senescence via mechanisms involving transcriptional therapy (Baylin et al., 2001; Rountree et al., 2001; repression. While investigating the transcriptional repres- Minucci and Pelicci, 2006). sion activities of RBP1, we observed a genetic link Three classes of HDACs have been characterized thus between RBP1 and SIR2. Further work uncovered an far (Khochbin et al., 2001; Thiagalingam et al., 2003). interaction between RBP1 family proteins and the The zinc-binding class I and II HDACs share a similar mammalian homologue of SIR2, SIRT1. Interestingly, mechanism of action and the common property of being the HDAC-dependent transcriptional repression domain sensitive to the inhibitor trichostatin of RBP1 proteins, termed R2, is necessary and sufficient A (Finnin et al., 1999). The class I histone deacetylases for the interaction with SIRT1. In vitro and in vivo are related to the yeast Rpd3 protein while the class II binding studies indicated that the p33ING1b and p33ING2 are related to HDA1. HDAC1 is a class I deacetylase subunits of the mSIN3A/HDAC1 complex are responsible that is found in association with the mSIN3A complex for the recruitment of SIRT1 to the R2 domain. To (Hassig et al., 1997; Laherty et al., 1997). The class III investigate the biological relevance of this interaction, we HDACs share homology within their catalytic core used the activator resveratrol and the sirtuin domain with the yeast NAD þ -dependent histone inhibitor sirtinol in transcriptional repression assays and deacetylase Silent Information Regulator 2 (SIR2) and demonstrated that SIRT1 activity negatively regulates generally referred to as (reviewed in Blander and R2-mediated transcriptional repression activity. We there- Guarente, 2004). fore propose a novel mechanism of class I HDAC In a number of model organisms including yeast regulation by a class III HDAC. Explicitly, SIRT1 is (Kaeberlein et al., 1999), nematode (Tissenbaum and recruited by ING proteins and inhibits R2-associated Guarente, 2001) and Drosophila (Rogina and Helfand, mSIN3A/HDAC1 transcriptional repression activity. 2004), SIR2 activity is required for the extended lifespan Oncogene (2008) 27, 3384–3392; doi:10.1038/sj.onc.1211014; induced by calorie restriction (reviewed in published online 14 January 2008 Guarente, 2005; Guarente and Picard, 2005; Longo and Kennedy, 2006). In mammals, the SIR2 homologue Keywords: ; RBP1; BCAA; SIRT1; HDAC1; SIRT1 is the best characterized sirtuin. Its activities mSIN3A range from histone deacetylation (Braunstein et al., 1993; Imai et al., 2000; Vaquero et al., 2004; North et al., 2005) and transcriptional silencing (Shore et al., 1984; Parsons et al., 2003; Vaquero et al., 2004) to regulation Introduction of the transcriptional activities of (Luo et al., 2001; Vaziri et al., 2001; Langley et al., 2002) and p300 Histone deacetylation has a pivotal role in the regula- (Bouras et al., 2005) via deacetylation. tion of epigeneticevents leading to transcriptional The retinoblastoma-binding protein 1 (RBP1, also silencing. A number of processes linked to cell cycle known as ARID4A) and the breast carcinoma-asso- control and tumourigenesis appear to be regulated by ciated antigen (BCAA, also known as ARID4B) are histone deacetylases, including cell cycle progression, transcriptional (Lai et al., 1999a, b; Fleischer , senescence and apoptosis. et al., 2003; Binda et al., 2006) found in association Indeed, since a number of tumour suppressors have with the mSIN3A/HDAC1 complex (Lai et al., 2001; been reported to be epigenetically silenced in cancer, Skowyra et al., 2001; Kuzmichev et al., 2002; Fleischer et al., 2003; Meehan et al., 2004; Nikolaev et al., 2004; Correspondence: Professor PE Branton, Department of Biochemistry, Doyon et al., 2006). RBP1 and BCAA repress transcrip- McGill University, McIntyre Medical Building, Room 702, 3655 tion in both HDAC-independent (R1) and HDAC- Promenade Sir William Osler, Montre´ al, Que´ bec, Canada H3G 1Y6. E-mail: [email protected] dependent (R2) manners (Lai et al., 1999a; Fleischer Received 4 September 2007; revised 29 November 2007; accepted 3 et al., 2003; Binda et al., 2006). The R1 and R2 domains December 2007; published online 14 January 2008 have been functionally characterized in both human SIRT1 regulates HDAC1-dependent repression O Binda et al 3385 (Lai et al., 1999a; Binda et al., 2006) and murine RBP1 (O Binda and PE Branton, unpublished) as well as human BCAA (Binda et al., 2006). Although R1 and R2 are unique to RBP1 and BCAA, the R1 domain encompasses an A/T-rich interacting domain (ARID; Herrscher et al., 1995), a domain phylogenically conserved in (Kortschak et al., 2000), which, in the case of RBP1, displays sequence nonspecific low DNA-binding affinity (Patsialou et al., 2005). The R1 domain of RBP1 and BCAA are the only instances in the literature of ARID-mediated transcriptional repres- sion activity (Binda et al., 2006). Interestingly, adjacent to the carboxy terminal end of the ARID, two SUMOylation sites mediate strong transcriptional repression in RBP1 and BCAA (Binda et al., 2006). Sequence analysis (O Binda, unpublished observation) of the other ARID proteins (Wilsker et al., 2005) reveal that ARID1A and ARID1B have a conserved putative CKxE (see Hay, 2005 for review) SUMOylation site (LKPP) about 50 residues downstream of their ARID; ARID3B has an IKKE sequence about 60 residues from its ARID; ARID5A has an LKGE motif adjacent to the ARID and an IKIE motif about 30 residues further away; ARID5A putative SUMOylation motif is con- Figure 1 BCAA and RBP1 associate with SIRT1. (a) Endogen- served in ARID5B IKGE motif; while JARID1B is the ously expressed RBP1 and SIRT1 were immunoprecipitated from an extract of mouse brain nuclear proteins using the LY11 only Jumonji/ARID protein with a potential SUMOylation (Upstate, Billerica, MA, USA) and 2G1/F7 (Upstate) mouse motif (IKIE) about 60 residues after its ARID. monoclonal antibodies, respectively. The mouse monoclonal anti- Therefore, SUMOylation of sequences adjacent to the body RK5C1 (Santa Cruz, Santa Cruz, CA, USA) was used as a ARID may be involved in regulating its function(s), negative control. The immunoprecipitates were analysed by presumably DNA-binding affinity or transcriptional immunoblotting using either LY11 or a-SIR2 (Upstate). (b) H1299 cells were co-transfected with plasmids expressing Flag repression. alone or Flag-SIRT1 and HA-RBP1 or HA-BCAA. Whole cell Both RBP1 and BCAA can associate with the extracts were immunoprecipitated using a-Flag M2-agarose and mSIN3A/HDAC1 complex via a direct interaction analysed by immunoblotting using either the a-HA or the a-Flag between the SAP30 subunit and the R2 region (Lai M2 antibodies. et al., 2001; Binda et al., 2006). Overexpression of either of these two RBP1 family members leads to cell growth specific antibodies. Figure 1a shows that the mouse inhibition and induction of senescence (Binda et al., monoclonal antibody a-Gal4 (RK5C1), used as a 2006), a cellular event also controlled by the mSIN3A/ negative control, did not co-immunoprecipitate RBP1 HDAC1 complex subunits p33ING1b (Garkavtsev et al., or SIRT1 (first lane), but that SIRT1 was co-immuno- 1996; Garkavtsev and Riabowol, 1997) and p33ING2 precipitated with RBP1 (second lane; LY11). Likewise, (Pedeux et al., 2005). Induction of this phenotype by RBP1 was co-immunoprecipitated with a SIRT1-specific RBP1 and BCAA requires the presence of an intact R1 antibody (third lane; 2G1/F7), demonstrating an inter- region as well as the R2 HDAC-dependent transcrip- action in vivo between RBP1 and SIRT1. tional repression domain (Binda et al., 2006). To demonstrate an interaction between SIRT1 and Preliminary studies in yeast on transcriptional repres- the RBP1-related protein BCAA, H1299 cells were sion by RBP1 (A Lai, BK Kennedy and PE Branton, transfected with plasmid DNAs expressing full-length unpublished) suggested a geneticlink with SIR2. Here HA-tagged BCAA or RBP1 along with Flag-SIRT1 or we demonstrate for the first time that association of the Flag alone. Figure 1b shows that both RBP1 family class III HDAC, SIRT1, with the R2 region of RBP1 members are detected specifically in Flag-SIRT1 and BCAA proteins alleviates their HDAC-dependent immunoprecipitates (lanes 3 and 4), but not with the transcriptional repression activity. These findings sug- control immunoprecipitates (lanes 1 and 2). gest a novel mechanism of regulation of the class I In mammals, there are seven members in the sirtuin deacetylase HDAC1 transcriptional repression activity family of proteins (see Michan and Sinclair, 2007 for by the class III deacetylase SIRT1. review). SIRT1, SIRT6 and SIRT7 are nuclear whereas SIRT2, SIRT3, SIRT4 and SIRT5 are cytoplasmic, although a small pool of nuclear SIRT2 exists (North Results et al., 2003; Bae et al., 2004) that probably accumulates via regulation of its nuclear export (Wilson et al., 2006). BCAA and RBP1 associate with SIRT1 To determine if sirtuins other than SIRT1 associate with Endogenously expressed RBP1 and SIRT1 were im- RBP1 proteins, co-immunoprecipitation experiments munoprecipitated from a nuclear protein extract using were conducted on whole cell protein extracts from

Oncogene SIRT1 regulates HDAC1-dependent repression O Binda et al 3386 cells transiently expressing either HA-RBP1 or HA- complex and an hitherto uncharacterized class III- BCAA, and Flag-SIRT1-7 (see Supplementary Figure containing complex. S1). As expected, the cytoplasmic sirtuins did not co-immunoprecipitate with either BCAA or RBP1. However, SIRT4 expression being very low, as reported The R2 region of BCAA and RBP1 associates with elsewhere (North et al., 2005), a possible interaction SIRT1 could not be neglected. Furthermore, neither SIRT6 nor To determine which region of RBP1 proteins was SIRT7 nuclear sirtuins associated with RBP1 family required for SIRT1 association, a panel of amino- and members. These results suggested that SIRT1 may carboxy-terminal deletion mutant forms of RBP1 and perform a specialized function towards RBP1 and BCAA as well as the BCAAMCF-7 isoforms (Binda et al., BCAA that cannot be compensated by the other nuclear 2006) were tested (see Supplementary Figure S2 for an sirtuins. In addition, they indicated that the RBP1 illustration of RBP1/BCAA derivatives and a summary family members associate with two distinct classes of of the results). Co-immunoprecipitation experiments HDACs: the class I, present in the mSIN3A/HDAC1 showed that the association with SIRT1 was dependent

Figure 2 The R2 region of BCAA and RBP1 associates with SIRT1. (a) Illustration of RBP1 and BCAA most relevant features with their position in amino acid residues. Immunoprecipitation and immunoblotting studies were conducted as in Figure 1b except that truncated forms of HA-RBP1 and HA-BCAA were expressed. (b) Immunoprecipitation studies were conducted as in panel (a), but the R2 region was in vitro transcribed and labelled with biotinylated lysine-tRNA during in vitro translation, then detected using HRP-conjugated streptavidin.

Oncogene SIRT1 regulates HDAC1-dependent repression O Binda et al 3387 on the presence of the R2 region (Figure 2a), suggesting The tumour suppressor inhibitor of growth protein that SIRT1 associates with RBP1 proteins through the p33ING1b associates with SIRT1 (Kataoka et al., 2003) R2 region. To determine whether the R2 domain alone and both p33ING1b and p33ING2 are found in association can bind SIRT1, a fragment of BCAA corresponding to with the mSIN3A/HDAC1 complex (Skowyra et al., the R2 domain was in vitro translated and incubated 2001; Kuzmichev et al., 2002; Vieyra et al., 2002; Doyon with lysates from cells expressing either Flag-SIRT1 or et al., 2006). It is therefore plausible that these two ING Flag alone. Upon a-Flag immunoprecipitation, R2 was proteins could bridge SIRT1 to the R2 region of RBP1 detected only in precipitates containing Flag-SIRT1 proteins. To confirm that ING proteins associate with (Figure 2b). Thus R2 is necessary and sufficient for the RBP1 proteins, cells were transfected with cDNAs mediating association with SIRT1. expressing Flag-tagged ING proteins and their ability to interact with RBP1 proteins was examined by co- immunoprecipitation. Figure 4a and Supplementary SIRT1 is recruited to the RBP1 proteins via the ING Figure S3 show that indeed BCAA and RBP1 subunits of the mSIN3A/HDAC1 complex co-immunoprecipitated with both p33ING1b and p33ING2 SIRT1 could interact with the R2 region of RBP1 whereas, the isoform p24ING1c, which has a distinct, proteins either directly or indirectly via an R2-associated shorter amino terminal region, did not. This is in protein such as a subunit of the mSIN3A/HDAC1 agreement with previous studies, which demonstrated complex. We have shown previously that the mSIN3A/ that both p33ING1b (Skowyra et al., 2001; Kuzmichev HDAC1 complex is recruited to R2 through a direct et al., 2002) and p33ING2 (Doyon et al., 2006) are interaction with the SAP30 subunit (Lai et al., 2001; inherent components of the mSIN3A/HDAC1 complex, Binda et al., 2006). Thus, if the SIRT1 interaction with whereas the p24ING1c isoform is not incorporated in the R2 occurs via the mSIN3A/HDAC1 complex, depletion complex (Skowyra et al., 2001). of SAP30 might reduce the amount of R2-bound To address whether ING proteins could directly SIRT1. To determine if SIRT1 association with the R2 associate with SIRT1, in vitro binding assays were region requires the presence of SAP30, the latter was conducted with GST, GST-HDAC1 and GST-SIRT1 knocked down by RNA interference methods. Figure 3 affinity purified recombinant proteins. Figure 4b con- shows that treatment with SAP30-specific siRNA firms that in vitro translated SAP30 could bind directly significantly reduced the amount of SAP30 protein in to HDAC1 (Zhang et al., 1998). Interestingly, all ING the cells. In addition, RBP1 lost the capacity to associate proteins tested were found to bind directly to both with SIRT1 with decreased levels of SAP30. These HDAC1 and SIRT1. These results were then confirmed observations suggested that the interaction between R2 in vivo in co-immunoprecipitation studies. Figure 4c and SIRT1 occurs via the mSIN3A/HDAC1 complex, shows that both ING1 isoforms p24ING1c and p33ING1b as SAP30 is the sole subunit known to make direct associated in vivo with HDAC1 and SIRT1. In addition, contact with the R2 region. although p33ING2 expression was lower than that of ING1 isoforms, p33ING2 was also seen to associate with both HDAC1 and SIRT1 (data not shown). These findings imply that p33ING1b and p33ING2 are capable of bridging SIRT1 to the mSIN3A/HDAC1 complex by associating directly with the SAP30 subunit via their conserved amino terminal region (p33ING1b residues 1– 125; Kuzmichev et al., 2002) and to deacetylases via the PHD-containing carboxy terminal three-quarter (Figures 4b and c). Interestingly, p24ING1c, which lacks the first 69 amino terminal residues, fails to associate with either RBP1 or BCAA (Figure 4a), but retains the capacity to bind HDACs (Figures 4b and c). This suggests that the conserved region 1 (Doyon et al., 2006) may mediate important protein-protein interactions for the assembly of the mSIN3A/HDAC1 complex.

The repression activity of the R2 region is negatively regulated by SIRT1 To decipher the functional role of SIRT1 interaction with the R2 transcriptional repression domain, luciferase reporter assays were conducted in the presence or Figure 3 SIRT1 is recruited to RBP1 via the mSIN3A/HDAC1 absence of resveratrol, a sirtuin activator (Howitz et al., complex. H1299 cells were co-transfected with the indicated 2003), using a Gal4 DNA binding domain fused to R2 siRNAs and a DNA vector expressing HA-RBP1. The a-HA immunoprecipitates from whole cell extracts were resolved by and the Gal4-responsive reporter G5TKluc. Figure 5a SDS–PAGE and analysed by immunoblotting using a-HA, shows that increasing amounts of resveratrol (0, 10, 50, a-SIRT1, or a-SAP30 antibodies. 100 mM) resulted in proportional relief of repression

Oncogene SIRT1 regulates HDAC1-dependent repression O Binda et al 3388

Figure 5 SIRT1 activity negatively regulates R2-mediated repres- sion. (a) HeLa cells were co-transfected with pG5TKluc, phRL RSVluc, and Gal4 or Gal4-R2. The cells were either treated with dimethyl sulfoxide or increasing amount of resveratrol, as indicated on the graphic, for a period of 24 h. Luciferase activity was arbitrarily set at 100% for the Gal4-alone control. (b) This experiment was conducted as in panel (a) except that the cells were treated with sirtinol, instead of resveratrol. (c) This experiment was conducted as in panel (a) except that a Gal4-HDAC1 expressing plasmid was included. The results are shown as ‘Fold Repression’ on the graphicfor easier comparisonbetween R2 and HDAC1 ING1b ING2 Figure 4 p33 and p33 allow SIRT1 recruitment to R2. repression. The experiments for this figure were conducted at least (a) DNA vectors expressing Flag-tagged INGs were co-transfected thrice in duplicate. with a plasmid expressing HA-BCAA in HeLa cells. Whole cell extracts were immunoprecipitated using a-Flag M2-agarose and analysed by immunoblotting using either the HA.11 or the a-Flag M2 antibodies, as indicated. (b) In vitro transcribed and translated SAP30 and ING proteins were incubated with GST, GST-HDAC1 mediated by R2, such that at a concentration of 100 mM, or GST-SIRT1. Complexes were pulled-down using glutathione the repression activity of R2 was completely alleviated. sepharose 4, washed extensively, resolved by SDS–PAGE, trans- On the contrary, using the sirtuin inhibitor sirtinol ferred to a PVDF membrane and analysed by immunoblotting et al M using HRP-conjugated streptavidin (Promega). (c) Flag-tagged (Grozinger ., 2001) at 100 m , R2-mediated repres- HDAC1 and SIRT1 were expressed in HeLa cells along with HA- sion was significantly enhanced (approximately three- tagged ING1 isoforms. Whole cell extracts were immunoprecipi- fold induction; Figure 5b). The Gal4 and Gal4-R2 tated using a-Flag M2-agarose and analysed by immunoblotting protein levels were not significantly affected by the drug using either the HA.11 or the a-Flag M2 antibodies. treatments (data not shown). These results suggested that SIRT1 activity inhibits the mSIN3A/HDAC1

Oncogene SIRT1 regulates HDAC1-dependent repression O Binda et al 3389 transcriptional repression activity associated with the Accordingly, chemical agonists and antagonists of R2 region. SIRT1 affected HDAC1-mediated transcriptional re- HDAC1 was recently shown to be acetylated (Qiu pression in a manner similar to R2 (Figure 5) without et al., 2006) and thus could be a target of the SIRT1 significantly affecting the expression level of HDAC1 deacetylase activity recruited to the mSIN3A/HDAC1 (data not shown). These results suggest that SIRT1 may complex. The highly acetylated form of HDAC1 is regulate not only the transcriptional repression activity found in repressed chromatin, while the deacetylase of R2, but also that of HDAC1 or HDAC1-associated found on transcriptionally active chromatin has a lower factors. level of (Qiu et al., 2006). Furthermore, this We are aware that studies on transcriptional repres- post-translational modification appears to regulate sion using the Gal4 reporter system may not accurately HDAC1 histone deacetylase activity in vitro (Qiu reflect chromatin regulation by RBP1 and BCAA. et al., 2006). We therefore overexpressed Gal4-tagged Unfortunately, there are no endogenous known HDAC1 in the context of the transcriptional repression to be regulated by either RBP1 or BCAA. In addition, experiments described above. Figure 5c shows that as neither RBP1 nor BCAA have been shown convincingly with R2, HDAC1-mediated transcriptional repression to be involved in the regulation of endogenous - activity can be alleviated by resveratrol whereas sirtinol dependent promoters. Furthermore, resveratrol inhibits significantly enhanced HDAC1 repression activity. cellular proliferation in numerous cell lines (Lu and Serrero, 1999; Sgambato et al., 2001) and sirtinol irreversibly affects the cell cycle by inducing senescence (G1-like cell cycle exit; Ota et al., 2006, 2007). Therefore, Discussion even if studies were performed on endogenous E2F- dependent genes it would be difficult to discern between We describe for the first time in this report the the cell cycle arrest effects and E2F-dependent tran- association of the class III deacetylase SIRT1 with scriptional regulation by RBP1/BCAA, if any. In fact, the mSIN3A/HDAC1 complex. In previous studies we did conduct such a study with the mSIN3A-regulated involving biochemically purified mSIN3A/HDAC1 cyclin B1 (Dannenberg et al., 2005), and found that complexes, SIRT1 has never been reported as an inherent its protein level was greatly reduced by sirtinol (data not subunit of the complex. The SIRT1 deacetylase has shown); however, as discussed above, we were con- previously been linked to other transcriptional regula- cerned that the sirtinol treatment may not have had an tors such as p53 and p300 and may therefore be involved RBP1/BCAA-specific effect on cyclin B1 expression, but in the regulation of the transcriptional repression rather induced an indirect effect due to the G1-arrest. activities of RBP1 proteins. Unlike the other nuclear sirtuins, SIRT6 and SIRT7, SIRT1 was found in association with both full-length SIRT1 is excluded from DAPI- and Hoechst 33342- RBP1 and BCAA (Figure 1). Interestingly, the interac- stained chromatin (heterochromatin; Michishita et al., tion with SIRT1 required the R2 region (Figure 2a), 2005; Bordone et al., 2006). Thus SIRT1 appears to which has already been shown to associate with class I associate with euchromatin despite its reported role in HDACs (Lai et al., 2001). Furthermore, SIRT1 was the the formation of heterochromatin (Vaquero et al., sole sirtuin found to associate with RBP1 proteins. 2004). These contradictory observations may reflect Because R2 is known to associate with the mSIN3A/ dual functions associated with SIRT1. Nonetheless, the HDAC1 complex via a direct interaction with the euchromatin localization of SIRT1 correlates with our SAP30 subunit (Lai et al., 2001; Binda et al., 2006) observation that SIRT1 activity negatively regulates and, as shown here, reduction of SAP30 levels by RNAi HDAC1-dependent transcriptional repression. considerably reduced the association of SIRT1 with It has been reported that SIRT1 activity can over- RBP1, it is likely that SIRT1 is recruited to RBP1 and come p53/PML-induced senescence (Langley et al., BCAA via the mSIN3A/HDAC1 complex. In vitro 2002). We have previously shown that the R2 region is binding assays demonstrated that the tumour suppres- necessary for induction of senescence by the RBP1 sors p33ING1b and p33ING2 provide a direct link between proteins (Binda et al., 2006) and since SIRT1 activity is SIRT1 and the mSIN3A/HDAC1 complex subunits deleterious to R2 repression activity, it may also HDAC1 and SAP30, suggesting that the association of negatively regulate RBP1-induced senescence. The ob- SIRT1 with the R2 region of RBP1 and BCAA occurs servation that inhibition of SIRT1 by either sirtinol or through the ING subunits of the mSIN3A/HDAC1 splitomicin induces senescence in H1299 (O Binda, complex. unpublished observation; Ota et al., 2006), HUVEC Recently, HDAC1 was shown to be acetylated and (Ota et al., 2007) and MCF-7 (Ota et al., 2006) cells this post-translational modification appeared to be correlates with increased R2-mediated repression and involved in the targeting of HDAC1 to heterochromatic RBP1-induced senescence, thereby providing indirect regions of the nucleus, thus suggesting a possible evidence suggesting that SIRT1 activity may antagonize regulatory mechanism via localization of histone deace- the growth arrest phenotype caused by RBP1 family tylase activity (Qiu et al., 2006). As a consequence, it protein overexpression. A recent study, has reported was postulated that SIRT1 may negatively regulate the that SIRT1 is gradually lost in aging cells (Sasaki et al., mSIN3A/HDAC1 repression activity associated with 2006). So, while SIRT1 levels fade away as the cells age, R2 via a mechanism involving deacetylation of HDAC1. HDAC1-dependent transcriptional repression could be

Oncogene SIRT1 regulates HDAC1-dependent repression O Binda et al 3390 relieved from SIRT1 inhibition and lead to senescence, (Novagen, Mississaga, ON, Canada) using SacI and XhoI for as observed with p53/PML (Langley et al., 2002). in vitro transcription/translation and in vitro binding assays. The NCOR and SMRT complexes, like mSIN3A/HDAC1, also rely on HDAC activity for Luciferase assays transcriptional silencing. They have been found in Transcriptional repression assays were essentially conducted as association with HDAC3 (Li et al., 2000; Yoon et al., described before (Binda et al., 2006). To assess SIRT1- 2003), HDAC4 (Huang et al., 2000; Fischle et al., 2002) dependent biological function on R2-mediated repression, the cells were treated with resveratrol (Sigma, Oakville, ON, and HDAC5 (Huang et al., 2000). However, many Canada), sirtinol (Sigma) or DMSO for 24 h prior to the subunits of the mSIN3A/HDAC1 complex, essentially luciferase assay measurements. HDAC1, SAP30 and mSIN3A, can be found in biochemically purified NCOR (Laherty et al., 1998) RNAi depletion of SAP30 and SMRT (Underhill et al., 2000) complexes. Further- The siRNA duplexes were obtained from Dharmacon (Lafayette, more, both NCOR and SMRT associate with SIRT1 CO, USA) and information on the targeted sequence can be through the repression domain RD1 (Picard et al., provided upon request. H1299 cells were seeded at a density of 2004), which is the main binding surface for mSIN3A 5 Â 105 cells per 60 mm-diameter plate the day prior to (Heinzel et al., 1997; Nagy et al., 1997), suggesting that transfection. Before transfection, the medium was replaced SIRT1 may be recruited to NCOR/SMRT via the with 2 ml of fresh DMEM containing 10% FBS. Then the cells mSIN3A complex. Importantly, SIRT1 was also shown were transfected with 8.0mg of HA expression vector DNA to negatively regulate the transcriptional activity of using 24 ml TransIT LT1 in 500 ml serum-free DMEM. In parallel, the siRNAs were transfected using 7.5 mlof20mM PPAR-g via its NCOR (Picard et al., 2004). It stock (50 nM final concentration) and 15 ml TransIT TKO therefore appears that SIRT1 activity can damper both (Mirus) in 500 ml. The medium was changed 24 h post- transcriptional activation (PPAR-g) and transcriptional transfection. The cells were rinsed with phosphate-buffered repression (RBP1/BCAA). saline (PBS), harvested and lysed in 450 ml nuclear lysis buffer The class I and III HDACs RPD3 and SIR2 are (Lai et al., 1999b) 48 h post-transfection. SAP30 knockdown known in lower organisms such as yeast and flies to have was detected by immunoblotting using the rabbit polyclonal opposite roles in the regulation of life span (Kim et al., a-SAP30 antibody (Abcam ab15034). 1999; Rogina et al., 2002; Rogina and Helfand, 2004). Our results suggest that not only do these two classes of Immunoprecipitation HDACs have opposite functions, but also that SIRT1 Co-immunoprecipitation experiments were conducted either in actually regulates HDAC1-mediated transcriptional H1299 or in HeLa cells. Briefly, 4.0 mg of HA plasmid and silencing. Furthermore, because of previous results from 4.0 mg of Flag plasmid DNAs were transfected into the cells. The cells were lysed 48 h post-transfection in nuclear lysis our lab and the implication of SIRT1 in preventing buffer supplemented with EDTA-free protease inhibitor cock- senescence, we believe that SIRT1 and RBP1 proteins tail (Roche, Laval, QC, Canada). a-Flag M2-agarose (Sigma) may be important regulators in the equilibrium between was used to immunoprecipitate the complexes (as previously ageing and longevity. described by Binda et al., 2006), which were resolved by SDS– polyacrylamide gel electrophoresis (SDS–PAGE), transferred to PVDF membrane and analysed by immunoblotting using either a-HA monoclonal antibody (HA.11 from Covance Materials and methods (Emeryville, CA, USA)) or HRP-conjugated a-Flag M2 (Sigma). To overcome the masking of the R2 signal by the Cell lines and DNA transfections IgG light chain, the studies on the interaction of SIRT1 and H1299 cells (ATCC CRL-5803) and HeLa cells (ATCC CCL-2) R2 were conducted as outlined above, but HA-tagged R2 was were maintained in Dulbecco’s modified Eagle medium in vitro transcribed and translated from a pcDNA3 plasmid (DMEM; Invitrogen, Burlington, ON, Canada) supplemented using the TNT Coupled Reticulocyte Lysate System (Promega, with 10% fetal bovine serum (FBS; Cansera, Etobicoke, ON, Nepean, ON, Canada) and labelled with biotinylated lysine- 1 1 Canada), 100 U ml À Penicillin G, 100 mgmlÀ Streptomycin tRNA (Transcend from Promega) for detection by HRP- and 2 mM Glutamine (Invitrogen). DNA transfections were conjugated streptavidin (Promega). conducted using TranIT LT1 (Mirus, Madison, WI, USA) using a DNA:LT1 ratio of 1:3. Recombinant protein purification GST, GST-HDAC1 and GST-SIRT1 were purified essentially Plasmids as described previously (North et al., 2005; Binda et al., 2006). RBP1 and BCAA cDNA expression vectors were previously described (Binda et al., 2006). pcDNA3-SIRT1-Flag was In vitro binding assays obtained from Fuyuki Ishikawa (Takata and Ishikawa, In vitro translation (IVT) was performed using the TNT 2003). The Flag-tagged SIRT2-7 mammalian expression Coupled Reticulocyte Lysate System (Promega) and biotiny- vectors were obtained from Eric Verdin (North et al., 2005). lated lysine-tRNA (Transcend tRNA; Promega) according to The cDNAs of p33ING1b and p24ING1c were cloned from human the manufacturer’s guidelines. Equivalent amounts of IVT total RNA by reverse transcription (RT)–PCR into pCMV products were incubated with 2.0 mg GST recombinant proteins 3 Â Flag (Stratagene, La Jolla, CA, USA) using EcoRI and in nuclear lysis buffer (100 mM KCl), washed six times with 1mL XhoI restriction sites, while p33ING2 cDNA was subcloned from nuclear lysis buffer (100 mM KCl), resolved by Sodium dodecyl pFLAG-CMV-6c-ING2 (Pedeux et al., 2005) by PCR into sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), pCMV 3 Â Flag (Stratagene) using EcoRI and XhoI restriction transferred to a PVDF membrane and analysed by immuno- sites. The INGs cDNAs were also subcloned into pET-33b( þ ) blotting using HRP-conjugated streptavidin (Promega).

Oncogene SIRT1 regulates HDAC1-dependent repression O Binda et al 3391 Acknowledgements valuable discussions. This work was supported through grants from the Canadian Cancer Society, the National Cancer We thank Laurent Huck and Peter Moffett for critically Institute of Canada and the Canadian Institutes for Health reviewing this manuscript, and Jean-Se´ bastien Roy for Research.

References

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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