Oncogene (2008) 27, 2686–2692 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Enhanced methyltransferase activity of SMYD3 by the cleavage of its N-terminal region in human cancer cells

F Pittella Silva1, R Hamamoto1, M Kunizaki1, M Tsuge1, Y Nakamura1 and Y Furukawa2

1Laboratory of Molecular Medicine, Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan and 2Promotion of Genome-Based Medicine Project, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Histone methylation is involved in the regulation of maintenance and cell division. Modifications include expression and DNA replication through alteration of methylation, phosphorylation, acetylation and ubiquiti- chromatin structure. We earlier showed that SMYD3, nation at various residues of histone tails (Peterson a histone H3- 4-specific methyltransferase, is and Laniel, 2004). The combination and degree of frequently upregulated in human colorectal, liver and the modifications lead to association or dissociation breast cancer compared to their matched non-cancerous of chromatin-interacting to the histone octa- cells, and that its activity is associated with the growth of mer, and thereby defines chromatin structure. Con- these tumors. In the present study, we found that human formational change of chromatin is a key factor for cancer cells express both the full-length and a cleaved transcription; untranscribed are compacted in form of SMYD3 . Amino acid sequence analysis heterochromatin, while transcribed genes are in euchro- uncovered that the cleaved form lacks the 34 amino acids matin, where transcriptional complexes are accessible to in the N-terminal region of the full-length protein. the target DNA (Jenuwein and Allis, 2001). Methylation Interestingly, the cleaved protein and mutant protein of H3 lysine 4 (H3-K4) has been shown to associate containing substitutions at glycines 15 and 17, two highly with transcriptional activation, while that of H3 lysine conserved amino acids in the N-terminal region, revealed a 9 or 27 (H3-K9 or H3-K27) with transcriptional repres- higher histone methyltransferase (HMTase) activity sion (Jenuwein and Allis, 2001; Cao et al., 2002; Lachner compared to the full-length protein. Furthermore, the and Jenuwein, 2002). Lysine histone methyltransferases N-terminal region is responsible for the association with (HMTases) have a conserved SET domain that provides heat shock protein 90a (HSP90a). These data indicate a place where methyl donor transfers H-residues to that the N-terminal region plays an important role for the their target substrate(s). The crystal structure of Set1, regulation of its methyltransferase activity and suggest a histone H3-K4 methyltransferase, demonstrated a that a structural change of the protein through the dimerization of the SET domain forming a donut-like cleavage of the region or interaction with HSP90a may structure, in which the histone tail associates with the be involved in the modulation. These findings may help for methyl donor (Tresaugues et al., 2006). Although a better understanding of the mechanisms that modulate studies on HMTases have accumulated a link between the HMTase activity of SMYD3, and contribute to the histone methylation and , the regulatory development of novel anticancer drugs targeting SMYD3 mechanism(s) of methyltransferases remains to be methyltransferase activity. elucidated. Oncogene (2008) 27, 2686–2692; doi:10.1038/sj.onc.1210929; Recent molecular studies have disclosed that altera- published online 12 November 2007 tions of global histone modification play a role in human tumorigenesis (Varambally et al., 2002). We Keywords: SMYD3; methyltransferase; cancer earlier reported that SMYD3, a histone H3-K4-specific methyltransferase, was frequently overexpressed in human colorectal, liver and breast cancers, and that its enhanced expression was essential for the growth Introduction of cancer cells (Hamamoto et al., 2004, 2006). SMYD3 encoded a 428-amino acid-protein containing a SET Modifications at histone tails play a crucial role in the domain (codons 117–246), a zf-MYND domain (codons regulation of transcription, DNA replication, telomere 49–87) and a SET-N region (codons 5–27). Mutation in the conserved sequence of the SET domain abolished the interaction of SMYD3 with methyl donor, showing Correspondence: Dr Y Furukawa, Promotion of Genome-Based that the SET domain is crucial for its methyltransferase Medicine Project, Institute of Medical Science, The University of activity. The role of SET-N region has not yet been Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. disclosed. SMYD3 promoted di- and tri-methylation in E-mail: [email protected] Received 22 January 2007; revised 13 September 2007; accepted 8 H3-K4, which was enhanced by the interaction with October 2007; published online 12 November 2007 heat shock protein 90a (HSP90a). We further clarified The histone methyltransferase activity of SMYD3 F Pittella Silva et al 2687 that SMYD3 interacted with an RNA helicase to form a of the same extract with anti-SMYD3 detected complex with RNA polymerase II, and that SMYD3 two bands. These data suggested that the full-length transactivated direct target genes including Nkx2.8 and protein was cleaved in the N-terminal region of SMYD3. WNT10B. Further investigations identified E2F1-bind- Since western blot analysis of HEK293 cells expressing ing elements in the 50 flanking region of SMYD3, and its N-terminal deletion mutants (FLAG-SMYD3-DN44, expression was enhanced by elevated E2F1 that is -DN99 and -DN249) with anti-SMYD3 antibody showed frequently activated in human cancer by the inactivation a single band, the cleavage site of SMYD3 was predicted of retinoblastoma protein 1 through its phosphorylation to localize between codons 1 and 45. by cyclin-dependent kinases or an interaction with viral proteins (Tsuge et al., 2005). Determination of cleavage site of SMYD3 protein We here report that cells express both full-length In an attempt to determine the exact cleavage site of SMYD3 and a cleaved form of the protein that lacks 34 SMYD3, we purified the 42-kDa protein from poly- N-terminal amino acids. The cleaved protein and vinylidene fluoride (PVDF) membrane transferred with mutant protein containing substitutions in the conserved immunoprecipitated FLAG-tagged SMYD3 protein amino acids in the SET-N region showed enhanced (Figure 1b), and determined its amino acid sequence. HMTase activities compared to the full-length protein. As a result, we identified a deleted form of SMYD3 Since HSP90a associates with the full-length protein but protein lacking 34 N-terminal amino acids, which not with the cleaved or mutant protein, conformational revealed a cleavage site between codon 34 (aspartic change by the cleavage or the binding with HSP90a may acid) and codon 35 (proline) (Figure 2a). SMYD3 play a role in the regulation of SMYD3 HMTase contains an amino acid sequence termed SET-N region activity. These findings will help to better understand between codons 5 and 27, which is conserved in SET the regulatory mechanisms of SMYD3 HMTase activ- domain proteins (Kouzarides, 2002; Lachner and ity, and contribute to the development of HMTase Jenuwein, 2002; Marmorstein, 2003). An alignment of inhibitors. amino acid sequences of SET-N region depicted the high similarity of the region between SMYD3 and other SET domain-containing methyltransferases (Figure 2b), indicating the importance of this region. Results

A cleaved form of SMYD3 protein in human cancer cells Increased HMTase activity of the cleaved SMYD3 We showed in our earlier studies that the expression compared with the wild-type protein levels of SMYD3 protein are elevated in human To investigate the methyltransferase activity of the clea- hepatocellular carcinoma (HCC), colorectal carcinoma ved SMYD3 protein, we expressed 3 Â FLAG-tagged (CRC) and breast cancer (Hamamoto et al., 2004, 2006). wild-type, or a deleted form of SMYD3 (-DN34) Interestingly, western blot analysis with anti-SMYD3 exogenously in HEK293 cells, and immunoprecipitated antibody showed two bands of 45- and 42-kDa in all these proteins (Figure 3a). During the methylation breast cancer tissues examined, but it detected neither process, S-adenosyl methionine (SAM) is catalysed to of the two bands in normal mammary gland. Both the S-adenosyl homocysteine (SAH), whose accumulation is 45- and 42-kDa bands were observed in HCC, CRC supposed to compete with SAM leading to the inhibition and breast cancer cell lines (Figure 1a) and normal testis of methylation. We thus carried out a HMTase assay (data not shown). The predicted molecular weight of using wild-type and deleted SMYD3 proteins with SMYD3 was 45 kDa, and we did not find any other or without S-adenosyl homocysteine hydrolase (SAHH) alternative splicing forms of SMYD3 transcript in that hydrolyses SAH to homocysteine and adenosine. our reverse transcriptase (RT)–PCR analysis. Therefore, As we expected, addition of SAHH in the reaction we hypothesized that the 42-kDa band might result from mixture enhanced HMT activity (Figure 3b). This find- cleavage of the full-length SMYD3 protein. To examine ing should be useful for in vitro screening of methyl- the cleavage of SMYD3, we prepared plasmids expres- transferase inhibitor(s). To our surprise, we found that sing N-terminal HA-tagged SMYD3 or C-terminal the cleaved SMYD3 protein had significantly higher FLAG-tagged SMYD3. Extracts of HEK293 cells HMTase activity compared to the full-length protein expressing HA- or FLAG-tagged SMYD3 protein were (Figure 3b). Consistently, exogenous expression of cells used for immunoblot analysis with anti-HA or anti- with the cleaved form of SMYD3 revealed an increased FLAG , respectively. Although analysis of di- and tri-methylation of H3-K4 in comparison with extract from cells expressing FLAG-tagged SMYD3 that of wild-type SMYD3 (Supplementary Figure 1A). showed two bands with anti-FLAG antibody, analysis of Immunocytochemical staining indicated nuclear and cells expressing HA-tagged SMYD3 detected only one cytoplasmic localization of the cleaved protein in the band with anti-HA antibody (Figure 1b). We further cells, which is the same subcellular localization with the expressed N-terminal FLAG-tagged SMYD3 in full-length protein (Supplementary Figure 1B). These HEK293 cells that do not express endogenous SMYD3. data suggested that the N-terminal SET-N region may Western blot analysis with anti-FLAG antibody con- have a suppressive role for the HMTase activity, and sistently showed one band corresponding to the FLAG- that cleavage of this region may be involved in the tagged full-length protein (Figure 1c). However, analysis regulation of SMYD3 HMTase activity. It is of note

Oncogene The histone methyltransferase activity of SMYD3 F Pittella Silva et al 2688 Breast zf-MYND HCC CRC Carcinoma Breast Tissues 428 3 SMYD3 1 SET l 49 87117 246 T116 F7 SMYD3-wt 1-428 C mor ∆ SNU42 HepG2 SW480 HC T47D M Norma Tu Normal Tumor SMYD3- N44 45-428 SMYD3-∆N99 100-428 SMYD3 SMYD3-∆N249 250-428 * β-actin 9

t ∆N44 ∆N99 ∆N24 - -

SMYD3 SMYD3- G-SMYD3 AG- AG- FLAG-SMYD3-wFL FLA FL YD3 [kDa] YD3 M -S 50 * 37 IB: anti-SMYD3 25 ock -term-FLAG 20 Mock N-term-HA-SM M C 15

* 50 IB: anti-HA IB: anti-FLAG 37 IB: anti-FLAG 25 20 15

Figure 1 Cleavage of N-terminal region of SMYD3. (a) Expression of SMYD3 protein in human cancer cell lines and tissues. Western blot analysis was performed using anti-SMYD3 antibody. Expression of b-actin served as a quantitative control. (b) Western blot analysis of cells expressing N-terminal HA-tagged SMYD3 (left panel) or C-terminal Flag-tagged (right panel), with anti-HA antibody or anti-Flag antibody, respectively. (c) Schematic presentation of wild-type and deleted forms of SMYD3. (d) Plasmids expressing a series of N-terminal FLAG-tagged SMYD3 were transfected into HEK293 cells that do not express endogenous SMYD3. Western blot analysis of extracts from the HEK293 cells was performed using anti-SMYD3 antibody (upper panel) or anti-FLAG antibody (lower panel).

SMYD3 amino acid sequence 134 Cleaved region MEPLKVEKFATANRGNGLRAVTPLRPGELLFRSDPLAYTVCKGSRGVVCDRCLLGKEKLMRCSQCRVAKYCSAKCQK KAWPDHKRECKCLKSCKPRYPPDSVRLLGRVVFKLMDGAPSESEKLYSFYDLESNINKLTEDKKEGLRQLVMTFQHFMRE EIQDASQLPPAFDLFEAFAKVICNSFTICNAEMQEVGVGLYPSISLLNHSCDPNCSIVFNGPHLLLRAVRDIEVGEELTICYLD MLMTSEERRKQLRDQYCFECDCFRCQTQDKDADMLTGDEQVWKEVQESLKKIEELKAHWKWEQVLAMCQAIISSNSER LPDINIYQLKVLDCAMDACINLGLLEEALFYGTRTMEPYRIFFPGSHPVRGVQVMKVGKLQLHQGMFPQAMKNLRLAFDI MRVTHGREHSLIEDLILLLEECDANIRAS

Specificity SMYD3 H3-K4 5 KVEKFATAN - -RG N G LRAVTPLRPG 27 DIM-5_Nc H3-K9 150 P L Q I F R T K D - - R GWG V KCPVN I KRG 172 SET7/9_Hs H3-K4 213RVYVAESL I SSAG E G LFSKVAVGPN 227 Clr4_Sp H3-K9 329 P L E I F K T K E - - K GWGVRSLRFAPAG 351 SET1_Sc H3-K4 938 P V M F A R S A I - - H N WG LYALDS I AAK 960 SET2_Sc H3-K36 121 P I A I F K T K H - - K G Y GVRAEQD I EAN 143 G9a_Hs H3-K9 / 27 830 R L Q L Y R T A K - - M GWG V RALQT I PQG 852 SUV39H1_Hs H3-K9 244 D L C I F R T D D G - R GWG V RTLEK I RKN 267 PR-SET7_At H3-K9 / 24, H4-K20 187 G M K I D L I D G - - K G R GV IATKQFSRG 187 EZH2_Hs H3-K9 / 27 611KHLLLAPSDV -AGWG IFIKDPVQKN 633 KRP_At H3-K9 447 N L E V F R S A K - - K GWA V RSWEY I PAG 469 ASH1_Dm H3-K9 / 24, H4-K20 1375 G V E R F M T A D - - K GWG V RTKLP I AKG 1397 SETDB1_Hs H3-K9 821 R L Q L F K T Q N - - K GWG IRCLDDI AKG 843 MLL2_Hs H3-K4 2576 A V G V Y R S A I - - H G R G LFCKRN I DAG 2598

Figure 2 (a) Determination of SMYD3 cleavage site by Edman amino acid sequence. (b) Alignment of amino acid sequences of SET-N in histone methyltransferases. Highly conserved amino acids are indicated in black boxes and moderately conserved amino acids are in shadowed boxes.

Oncogene The histone methyltransferase activity of SMYD3 F Pittella Silva et al 2689 10 9 ∆N34 ∆N44 8 FLAG-SMYD3 SMYD3-WT SMYD3- SMYD3- 7 6 IB: anti-FLAG 5 4 3

IB: anti-SMYD3 H-radioactivity [cpm] 3 2 1 IB: anti-β-actin 0

∆N34 ∆N44

SMYD3-WT SMYD3- SMYD3-

Enzyme 1µl Enzyme 4µl Enzyme 4µl + SAHH 0.5 µl

Figure 3 HMTase activity of wild-type and cleaved forms of SMYD3. (a) Western blot analysis of wild-type or deleted forms (DN34 and DN44) of SMYD3 proteins with anti-FLAG antibody (upper panel) and anti-SMYD3 antibody (middle panel). Proteins were extracted from cells expressing Flag-tagged SMYD3 proteins. Immunoprecipitated SMYD3 protein was used for in vitro HMTase assay. (b) HMTase activity of the wild-type and the mutant SMYD3 proteins. Addition of S-adenosyl homocysteine hydrolase (SAHH) increased the activity. 3H-radioactivity was measured by liquid scintillation counter. that the N-terminal region is important for the inter- SMYD3. Notably, we found that SetNm1, but not action with HSP90a, because wild-type SMYD3 but not SetNm2 or SetNm3 lost its interaction with HSP90a the cleaved protein was capable of binding with HSP90a (Supplementary Figure 2B). Since SMYD3-DN34 and (Supplementary Figure 2A). SetNm1 showed augmented HMTase activity, the N-terminal region may play a suppressive role for the HMTase activity. Considering that HSP90a increases Glycine 15 and 17 in the SET-N region is important for HMTase activity of wild-type SMYD3, conformational the HMT activity change of the N-terminal region by HSP90a may be To determine the importance of the conserved amino involved in the regulation. Substitution of glycine 15 acid sequence in the SET-N region for the suppressed or 27 alone may not be enough to alter the protein methyltransferase activity, we prepared additional plas- conformation into full active form. mids expressing mutant N-terminal FLAG-tagged SMYD3 proteins. Since glycines 15, 17 and 27 of SMYD3 are highly conserved in the SET-N region Deletion of nine N-terminal amino acids is critical for the (Figure 2b), we prepared SMYD3-SetNm1, -SetNm2 enhanced HMTase activity and -SetNm3 that contained substitutions of G15A/ The finding that the deletion or mutation of SMYD3 G17A, G15A or G27A, respectively (Figure 4a). Wes- N-terminal region enhanced its enzymatic activity tern blot analysis of the lysates from HEK293 cells tempted us to hypothesize two possible mechanisms; expressing these mutants showed that the substitu- the N-terminal region might associate with undeter- tions did not affect the cleavage of SMYD3 protein mined negative regulatory factor(s) for the enzyme (Figure 4b, upper panel). However, HMTase assay activity, or the deletion or substitution might confer using immunoprecipitated SMYD3 protein revealed conformational change of the protein leading to enhan- that SMYD3-SetNm1 containing substitutions of both ced enzyme activity. To determine whether additional G15A and G17A has significantly enhanced activity negative regulatory factor(s) may play a role in the compared to the wild-type protein, which was compar- enzyme activity, we prepared wild-type and N-terminal able to the cleaved SMYD3 protein (Figure 4c). On the deleted recombinant SMYD3 proteins, and investigated other hand, SMYD3-SetNm2 and -SetNm3 containing their HMTase activity in vitro. As shown in Figure 5, G15A or G27A, respectively, had similar HMTase all deletions mutants (SMYD3-DN9, -DN19, -DN29, activity to wild-type protein. These data suggest that -DN44, -DN74) exhibited four- to five-fold enhanced glycines 15 and 17 in the SET-N region may play an methyltransferase activity compared to the wild-type important role for the regulation of HMTase activity of protein in the absence of HSP90a (Figure 5). Notably,

Oncogene The histone methyltransferase activity of SMYD3 F Pittella Silva et al 2690 40000 zf-MYND 134 117 246 428 35000 SMYD3 SET 30000 SMYD3-wt MEPLKVEKFATANRGNGLRAVTPLRPGELLFRSD 25000 SMYD3-SETNm1 MEPLKVEKFATANRANALRAVTPLRPGELLFRSD SMYD3-SETNm2 MEPLKVEKFATANRANGLRAVTPLRPGELLFRSD 20000 SMYD3-SETNm3 MEPLKVEKFATANRGNGLRAVTPLRPAELLFRSD 15000 H-radioactivity [cpm] H-radioactivity

FLAG-SMYD3 3 10000

5000 N34 ∆ WT SETNm1 SETNm2 SETNm3 0 T 1 2 3 WT IB: anti-SMYD3 ∆N34 Control SETNm SETNm SETNm YD3- SMYD3 SM IB: anti-FLAG Enzyme 1l Enzyme 4l IB: anti-β-actin Enzyme 4l + SAHH 0.5l Background

Figure 4 Importance of the SET-N region for HMTase activity. (a) Schematic presentation of mutant SMYD3 protein containing substitution in the conserved amino acids of the SET-N region. (b) Immunoblot analysis of FLAG-tagged wild-type or mutant (DN34, SetNm1, SetNm2 and SetNm3) SMYD3 proteins with anti-SMYD3 (upper panel) or anti-Flag (middle panel) antibody. Immunoprecipitated protein with anti-Flag antibody from HEK293F cells expressing FLAG-tagged SMYD3 was used as enzyme source for HMTase assay. (c) HMTase activity of the wild-type, deleted and mutant forms of SMYD3. 3H-radioactivity was measured by liquid scintillation counter.

SMYD3-DN9 failed to associate with HSP90a, suggest- cleavage of ‘SDPL’, the amino acid sequence between ing that the nine amino acids in the N-terminal region codons 33 and 36 in SMYD3. It have been reported that are essential for the binding with HSP90a (data not caspase-3 recognizes the exact sequence ‘SDPL’ in the shown). These data suggest that additional factors microtubule-associated protein tau, and cuts the protein are not likely to play a role in the elevated activity of between ‘D’ and ‘P’ in neurodegenerative disorders the deleted proteins, and that the conformational change (Fasulo et al., 2000). Although further investigations of the SMYD3 protein by cleavage of its N-terminal are needed for the identification of the responsible region may be involved in the regulation of its protease, clarification of the regulatory mechanism of methyltransferase activity. the cleavage will contribute to a profound understanding of methyltransferase activity. SMYD3 has a growth-promoting effect in NIH3T3 Discussion cells, and its suppression leads to the induction of apoptosis in colorectal cancer, hepatocellular carcinoma The regulation of a protein function depends on its and breast cancer. Therefore, inhibitors of SMYD3 expression level, as well as post-translational modifica- should be a rational strategy to treat these types of tions including acetylation, phosphorylation, methyl- cancer. To confirm the transforming activity of the ation, glycosylation, sumoylation and ubiquitination cleaved SMYD3, we carried out colony formation assay (Yang, 2005). In addition to these modifications, the using the cleaved form or the full-length SMYD3 cleavage of proteins also plays a crucial role in the in NIH3T3 cells. Although both SMYD3-DN34 and regulation. For example, a number of enzymes such as full-length SMYD3 showed increased number of colo- pepsin, insulin, caspases, PARP and MMPs are nies compared with mock, the number of colonies regulated by cleavage. These proteins are expressed as was similar between the two forms (data not shown). proenzymes or inactive forms of enzymes, and the This result may imply that the HMTase activity of cleavage in a specific region of their amino acid chain full-length SMYD3 should be enough to induce the induces their enzyme activity. We uncovered in this growth-promoting effect. It is tempting to hypothesize report that the full-length SMYD3 protein is cleaved that HMTase activity of SMYD3 greater than a threshold between codons 34 and 35 in the N-terminal region, and may be needed to switch on the growth-promoting that the cleaved protein has higher methyltransferase pathway, and that undetermined factor(s) may regulate activity compared to the full-length protein. This finding the level of effect. Further investigation is necessary for additionally suggests that an undetermined mechanism of the clarification of mechanism(s). Although inhibitors of the cleavage may be involved in the modulation of SU(VAR)3–9, a histone H3-K9-specific HMTase have SMYD3 HMTase activity. A public peptidase database been identified (Greiner et al., 2005), those of histone H3- termed ‘MEROPS’ (Rawlings et al., 2006) predicted K4-specific HMTases have not been reported so far. Since Caspase-3, -7 and -8 as candidate proteases for the the cleaved form of SMYD3 has higher enzymatic

Oncogene The histone methyltransferase activity of SMYD3 F Pittella Silva et al 2691 zf-MYND 2002; Zhang et al., 2002). The SET domain consists of two non-contiguous region formed as SET-N and SET- SMYD3 SET 1 49 87117 246 428 C (Marmorstein, 2003) The SET-N region of SMYD3 contains three glycines that are highly conserved in SMYD3-wt 1-428 SMYD3-∆N9 10-428 several methyltransferases. The evidence that substitu- SMYD3-∆N19 20-428 tion of the conserved glycines (G15 and G17) dimin- SMYD3-∆N29 30-428 ished the interaction with HSP90a (Supplementary SMYD3-∆N44 45-428 SMYD3-∆N74 75-428 Figure 2B) and enhanced its methyltransferase activity corroborated the importance of the conserved glycines in the SET-N region. Since cleavage of the N-terminal t N9 N19 N29 N44 N74 ∆ ∆ ∆ ∆ ∆ region also increased its methyltransferase activity, this - - region should have a suppressive role for the enzyme activity. In our earlier study, we showed that addition -SMYD3- -SMYD3- of HSP90a enhanced the methyltransferase activity in vitro et al GST-SMYD3-wGST GST-SMYD3 GST GST-SMYD3 GST-SMYD3- of the full-length protein (Hamamoto ., 2004). These data are tempting us to speculate that the suppressive role in the N-terminal region may be IB: anti-GST regulated post-translationally through the cleavage or conformational change of the protein. This hypothesis needs to be investigated in future studies including CBB staining crystal structure of SMYD3. We have shown here that an N-terminal cleaved form 40000 of SMYD3 protein is expressed in cancer cells and that the cleaved protein has markedly higher HMTase 35000 activity than the full-length protein. These data implied that a post-translational regulatory system might play a 30000 role in the control of methyltransferase activity through 25000 a possible conformational change of the protein. In addition, we showed that the SET-N region of SMYD3 20000 is important for the regulation of the methyltransferase activity. We also found that addition of SAHH increases 15000 the methyltransferase activity what could be valuable

H-radioactivity [cpm] information for the screening of methyltransferase

3 10000 inhibitors. These findings will help for the better under- 5000 standing of the regulatory mechanisms of SMYD3 activity, and may contribute to the identification of 0 novel therapeutic strategies to inhibit the HMTase ControlSMYD3 SMYD3 SMYD3 SMYD3 SMYD3 SMYD3 wt −∆N9 −∆N19 −∆N29 −∆N44 −∆N74 activity. Figure 5 Enhanced HMTase activity by the deletion of N-terminal region in SMYD3. (a) Schematic presentation of deleted forms of SMYD3. Plasmids expressing a series of glutathione-S-transferase Materials and methods (GST)-fused SMYD3 protein were prepared. (b) Immunoblot analysis of recombinant SMYD3 proteins with anti-GST antibody. Cell lines and tissue specimens Wild-type and mutant recombinant SMYD3 protein fused with Human embryonic kidney 293 (HEK293), HEK293T and GST was expressed in bacterial cells, and was subsequently purified HEK293F cells were purchased from IWAKI. A human from the cells. (c) In vitro HMTase activity of the proteins. hepatoma cell line HepG2, and human colon cancer lines 3H-radioactivity was measured by liquid scintillation counter. HCT116 and SW480were obtained from the American Type Culture Collection. A human HCC cell line SNU423 was a gift from the Korea cell-line bank. T47D and MCF7 breast cancer activity compared to full-length protein, the cleaved cell lines were kindly provided from the cancer institute of the form may be more useful for the discovery of inhibitors. Japanese foundation for cancer research. All cell lines were In addition, we showed that the addition of SAHH grown in monolayers in appropriate media. Primary breast in the in vitro methyltransferase assay enhances the cancer tissues were obtained with informed consent from activity through hydrolysis of SAH, a catalysed product patients (Hamamoto et al., 2006). of SAM (Fontecave et al., 2004). These findings should contribute to the development of anti-cancer drug(s) Preparation of plasmids targeting SMYD3. Preparation of C-terminal FLAG-tagged SMYD3 was des- cribed previously (Hamamoto et al., 2004). We additionally SET domain containing proteins have been grouped prepared plasmids expressing N-terminal HA-tagged, or into several subfamilies according to the distribution of N-terminal 3 Â FLAG-tagged SMYD3 by cloning various their conserved domains such as pre-SET domain, post- PCR products containing either wild-type or deleted forms SET domain, post-SET AWS domain and N-terminal of SMYD3 cDNA into the appropriate site of pCMV-HA SET, C-Terminal Zn-finger domains (Jacobs et al., (Clontech, Palo Alto, CA, USA) or p3XFLAG-CMV10

Oncogene The histone methyltransferase activity of SMYD3 F Pittella Silva et al 2692 (Sigma, St Louis, MO, USA) vector. Primers used for wild- anti-FLAG antibody from the cells, separated on duplicated type plasmids were 50-AAGCTTGCGGCCGCGATGGAGC SDS–polyacrylamide gel electrophoresis gels, and transferred CGCTGAAGGTGGAAAAG-30, and 50-GGTACCTCTAGA to a nitrocellulose membrane and a sequence grade PVDF TTAGGATGCTCTGATGTTGGCGTC-30, and those used for membrane. The nitrocellulose membrane was used for mutants (FLAG-SMYD3-DN34, -DN44, -DN99 and -DN249) immunoblot analysis with anti-FLAG antibody to detect the were 50-GGGGTACCTTAGGATGCTCTGATGTTGGCGT two forms of SMYD3 protein. After staining of the PVDF C-30 and 50-CGGAATTCACCCTTGGCGTACACGGTGTGC membrane with CBB solution without acetic acid (0.025% AAGG-30,50-CGGAATTCTGGCGCGATGGAGCCGCTGA CBB in 40% methanol), we excised the band corresponding AGGTGGAAAAG-30,50-CGGAATTCTGACTCCGTTCGAC to the short form of SMYD3 and subjected to amino acid TTCTTGGCAG-30 or 50-CGGAATTCTCGGAAGCAGCTGA sequence. The amino acid sequence of the protein was GGGACCAGTACTGC-30, respectively. Mutant plasmids ex- determined by Edman amino acid sequence method (Shimadzu pressing substitution(s) at glycine 15, 17 or 27 were prepared using Biotechnologies, Tokyo, Japan). QuikChange II XL site-directed mutagenesis Kit according to the supplier’s protocol (Stratagene, Cedar Creek, TX, USA). In vitro histone methyltransferase assay FLAG-tagged SMYD3 was purified from 293T cells expres- Western blot analysis sing wild-type (p3XFLAG-CMV-SMYD3) or mutant SMYD3 A polyclonal antibody to SMYD3 was purified from sera of (p3 Â FLAG-DN34, -DN44, -SetNm1, -SetNm2 and -SetNm3) rabbits immunized with a recombinant His-tagged SMYD3 by immunoprecipitation with anti-FLAG antibody. GST-fused protein produced in Escherichia coli as described elsewhere. SMYD3 proteins were purified from bacterial cells expressing Proteins were separated by 10% SDS–polyacrylamide gel wild-type (GST-SMYD3-wt) or mutant SMYD3 constructs electrophoresis and immunoblotted with anti-SMYD3, anti- (GST-SMYD3-DN9, -DN19, -DN29, -DN44, -DN74). In vitro HA (Sigma), anti-FLAG (Sigma), anti-GST (BD-Pharmingen, HMTase assay was performed as described elsewhere (Hamamoto San Diego, CA, USA) or anti-b-actin (Sigma) antibody. HRP- et al., 2004). 3H-radioactivity was measured by liquid scintillation conjugated anti-rabbit immunoglobulin G (IgG), anti-mouse counter. IgG (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) or anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, Acknowledgements CA, USA) antibody served as the secondary antibody for the ECL Detection System (Amersham). We thank Dr Kiyoshi Yamguchi for helpful discussions and Ms Yuka Yamane for technical assistance. This work was Determination of cleavage site supported in part by a Grant-in-Aid for Scientific Research C-terminal-FLAG-tagged SMYD3 was expressed exogenously from the Ministry of Education, Culture, Sports, Science and in 293F cells. SMYD3 protein was immunoprecipitated with Technology, Japan.

References

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

Oncogene