Characterization of a Novel Histone H3K36 Methyltransferase Setd3 in Zebraﬁsh

Biosci. Biotechnol. Biochem., 75 (2), 289–294, 2011

Characterization of a Novel Histone H3K36 Methyltransferase setd3 in Zebraﬁsh

y Dong-Wook KIM,* Kee-Beom KIM,** Ji-Young KIM,** and Sang-Beom SEO

Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, South Korea

Received September 6, 2010; Accepted November 5, 2010; Online Publication, February 7, 2011 [doi:10.1271/bbb.100648]

Post-translational modifications of histones have been H3 and H4 have been identified in the target sites of demonstrated to play important roles in the regulation methylation: lysine 4, 9, 27, 36, 79 of histone H3, and of chromatin structure and transcriptional regulation. lysine 20 of histone H4.7) In histone modification, methylated lysine has an Modifications that are localized to active genes or important role in transcriptional regulation. The evolu- regions are acetylation of histone H3 and H4 and tionarily conserved SET domain was first identified methylation of H3K4, H3K36, and H3K79. Modifica- in Drosophila proteins: Suppressor of variegation tions that are associated with inactive transcription (Su(var)3-9), Enhancer of zeste (E(z)), and Trithorax. include H3K9, H3K27, and H4K20.1) SET domain-containing proteins have histone methyl- It has been found that several HMTases play critical transferase (HMTase) activity via the SET domain. Using roles in establishing and maintaining heritable programs a bioinformatics approach, we identified and cloned of gene expression during cellular differentiation and zebrafish setd3 containing SET and Rubis-subs-bind early embryonic development in zebrafish. In a previous domains. In this study, we report that setd3 had lysine study, a novel histone H3K4 HMTase, Smyd1, played a specificity toward histone H3K36. Methylation of his- critical role in zebrafish muscle differentiation.8) Also, tone H3K36 is known as one of the transcriptional setdb2 that methylated histone H3K9 participated in the activation markers. It transiently transfected setd3 regulation of dorsal organization in the zebrafish.9) activated general transcription in reporter assays. Surprisingly, a sequence alignment between SET Overexpression of setd3 decreased cell viability and domain of zebrafish HMTase setdb2 and setd3 indicated activated caspase-3, indicating possible roles in apop- significantly low amino acid sequence homology, totic cell death and cell cycle regulation. indicating somewhat wide amino acid composition in zebrafish. Key words: setd3; histone methyltransferase (HMTase); In this study, we identified, cloned, and characterized zebrafish; transcription; histone modification zebrafish HMTase setd3. Our data indicate that setd3 is a novel H3K36 HMTase that plays a role as an activator The fundamental unit of chromatin, the nucleosome, of general transcription and inhibits cell viability by is composed of 146 bp of DNA wrapped around a inducing caspase-3 activation overexpression. histone octamer consisting of two copies of each histone protein, H2A, H2B, H3, and H4. In eukaryotes, chromatin Materials and Methods structure is modulated by post-translational modifications of histones such as acetylation, phosphorylation, Plasmid constructs. The sequences of all constructs were confirmed 1) by DNA sequencing. The full-length open reading frame of zebrafish ubiquitination, SUMOylation, and methylation. setd3 cDNA was purchased from Openbiosystems (Huntsville, AL, The evolutionarily conserved SET domain was USA), which was used in PCR amplification as template. pGEX4T1- initially characterized as a common motif in the PEV setd3 was constructed by PCR amplification using the specific primer modifier SU(VAR)3-9, the polycomb group protein set against zebrafish setd3 (GeneBank accession no. BC055261). The E(Z), and the trithorax-group TRX.2,3) SET domain- PCR primer sequences were as follows: SalI site-introduced primer, 50-GTCGACGGATGGGCAAAAAGAGCAGAGTG-30 as forward containing proteins are potential histone methyltransfer- 0 ases (HMTase), and >50 putative SET domain-contain- primer and NotI site-introduced primer, 5 -GCGGCGGCCCCTA- TTTGCCAGCATCTTTTGG-50, as reverse primer. For eukayotic ing proteins have been identified in the past few years. expression, PCR products were subcloned into HA/myc/His-tagged Methylations of histone tails take place in the arginine pcDNA6 (Invitrogen, Carlsbad, CA). The coding sequence of setd3 (R) and lysine (K) residues. Lysine residues can be was amplified with specific primers. The PCR primer sequences methylated up to 3 times (mono-, di-, trimethylation), were as follows: HindIII site-introduced primer, 50-GCGAAGCTTAT- and arginine residues can be methylated once or twice, GGGCAAAAAGAGCAGAGTG-30 as forward primer, and NotI 0 dimethylation being either symmetric or asymmetric.4) site-introduced primer, 5 -GCGCTCGAGTTTGCCAGCATCTTTT- GGTTC-30, as reverse primer. Lysine methylation on histone H3 or H4 is involved in a variety of biological processes, including transcriptional Modeling of secondary structures. A molecular model of the setd3 regulation, DNA damage, and X-chromosome inactiva- protein was generated using the automated homology modeling server tion.5,6) Five lysine residues in the N-termini of histones SWISS-Model (http://swissmodel.expasy.org/).10) The generated setd3

y To whom correspondence should be addressed. Tel: +82-2-820-5242; Fax: +82-2-822-3059; E-mail: [email protected] * Present address: Department of Biomedical Science, College of Life Science, CHA University, Seongnam 463-836, South Korea ** These authors contributed equally to this work. 290 D.-W. KIM et al. protein structures, including full-length (77–490 aa), SET domain, and (150 mL). The OD was determined with an ELISA reader (Biochrom, ssDNA binding motif, were visualized with the SWISS-Pdb Viewer, Cambridge, England) at a wavelength of 570 nm. A blank containing which showed chains, -helix, and -sheet in diﬀerent colors. DMSO alone was measured and subtracted from the values.

Cell culture. Cells were grown in Dulbecco’s Modified Eagle’s Statistical analysis. Data were expressed as means SD for three Medium (DMEM, Welgene, Daegu, Korea) containing 10% fetal or more independent experiments. Statistically significance effects bovine serum (FBS, Welgene) and 0.5% antibiotics at 37 Cina5% (p < 0:05) were evaluated with Microsoft EXCEL software. Dif- CO2 atmosphere. ferences between groups were evaluated by one-way analysis of variance (ANOVA), followed by Student’s t-test or Bonferroni’s test, Protein purification. For HMTase activity assay, the full-length as appropriate. open reading frame of the setd3 cDNA was inserted into pGEX4T1 vectors. The wild-type protein was expressed in Escherichia coli strain Results and Discussion BL21 (DE3) (Invitrogen) at 37 C for 4 h, and was purified with Glutathione-Sepharose 4B following the manufacturer’s protocol Characterization of the zebrafish setd3 gene (Amersham Bioscience, Orsay, France). The concentration of the wild type was determined by Coomassie Brilliant Blue R-250 staining SET domain-containing proteins were listed at of SDS–PAGE 12% gel. Bead-bound fusion proteins were used for EnsMart, a data mining tool contained in the Sanger in vitro HMTase activity assays. web site. Among the proteins, we selected setd3, which contains the SET domain. Setd3 was predicted to HMTase assay. In vitro HMTase assays were carried out at 30 C be present in the 14th chromosome of the zebrafish. for 2 h in 50-mL volumes of reaction buffer containing 50 mM Tris Data from blasting setd3 to the Conserved Domain (pH 8.5), 20 mM KCl, 10 mM MgCl2,10mM beta-mercaptoethanol, Database suggested the structure of setd3. The SET and 14 1.25 M sucrose, 100 nCi of S-adenosyl-[methyl- C]-L-methionine Rubis-subs-bind domains are located in the N-terminus (Amersham Bioscience), 1-mg/mL core histones from calf thymus (SET domain, 195–314 aa) and the C-terminus (the (Roche, Basel, Switzerland), and 0.5–2.5 mg of GST-setd3 or GST. Proteins were filtered using p81 filter paper (Millipore, Bedford, MA, Rubis-subs-bind domain, 343–475 aa) (Fig. 1A). The USA), and washed three times with pre-chilled 10% TCA and 95% Rubis-subs-bind domain permits binding of the protein ethanol for 5 min at room temperature. The filters were subjected to air to a substrate, such as the N-terminal tails of histones drying 2 mL of Gold Ultra (Perkin Elmer, Waltham, MA, USA) were H3 and H4.11) The SET domains of several proteins, added, and 14C-SAM was quantified using a scintillation counter including SUV39h1 and G9a, have been found to (Perkin Elmer). function as HMTase.12) The setd3 protein is composed of 17 -helices and eight -sheets in the secondary RT-PCR. Total RNA samples from each of the pcDNA6-setd3 structures (Fig. 1B). In secondary structure of the setd3, transfected HEK293 cells were extracted using Trizol reagent (Invitrogen), the manufacturer’s recommendations. Total RNA (2 mg) SET domain had three -helices and three -sheets was used to synthesize the cDNA. The cDNA synthesis was primed (Fig. 1B, gray box). Three-dimensional predictions of using oligo-dT primer (Fermentas, Vilnius, Lithuania) and the setd3 protein were performed using the SWISS-Model quantified cDNA was applied to setd3 mRNA expression pattern server. The setd3 protein secondary structure was analysis. The primer sequences forward, 50-GACGATATCAAAAGA- generated from 77 aa to 490 aa within the full length GAAGATTACTTCCCTG-30 and reverse, 50-GGATTCCACAAACT- 0 (Fig. 1C). In 3D protein modeling, setd3 had one CTGCATTTGACCG-3 . concave cleft in the center (Fig. 1C, red dotted line). The SET domain is located in cleft area, and this region Western blot. HEK293 cells were transfected with pcDNA6 vector or pcDNA6-setd3. The transfected cells were lysed and loaded onto likely contains the binding site for the substrate 14% SDS–PAGE and transferred to nitrocellulose membranes. The (Fig. 1D). Therefore, this cleft area in setd3 is consid- membranes were probed with anti-cleaved caspase-3 (Millipore), anti- ered an important area for HMTase activity. Several procaspase-9 (Santa Cruz Biotechnology), anti-H3K4-me2, anti- SET domain-containing HMTases, such as ALL-1, H3K9-me2, anti-H3K27-me2, anti-H3K36-me1, anti-H3K36-me2, TRX, and ASH-1, have a single-stranded DNA (ssDNA) anti-H3K36-me3, and anti-H4K20-me2 (Millipore) overnight at 4 C. binding motif located within the SET domain.13) The The blots were incubated with HRP-conjugated goat anti-rabbit 3D structure of the ssDNA-binding motif in setd3 is antibodies (Santa Cruz Biotechnology) and detected using ECL system (Santa Cruz Biotechnology). composed of two antiparallel -sheets (yellow ribbons) and a -helix (pink ribbon) (Fig. 1E). This antiparallel Transient transfection reporter assay. The transfection assay was -sheet structure was made by a flip-loop chain between conducted using pcDNA6-setd3, MH100-SV40-Luc, and MH100-TK- the -sheets (Fig. 1E, arrow). These structural features luc as internal controls where indicated. In 48-well plates, HEK293 of setd3 are very similar to those of ALL1, TRX, and the cells were co-transfected with Lipofectamine 2000 (Invitrogen) with ASH-1 ssDNA-binding motif.13) Predicted structures each reporter plasmid (100 ng) in the absence and the presence of such as pocket-like structure, SET domain, and ssDNA- pcDNA6-setd3 (50, 100, or 150 ng) and pCMV--gal plasmid (50 ng) binding motif in the setd3 protein strongly indicate for normalization of transfection efficiency. Cells were harvested 48 h post-transfection and lysed with cell culture lysis reagent (Promega, HMTase activity of setd3 in the zebrafish. Madison, WI). Luciferase activity was measured using a Glomax 20/ 20 luminometer (Promega). Each value is the mean of three replicates A novel HMTase activity of setd3 from a single assay and the results was repeated at least 3 times. The SET domain is evolutionarily conserved from Luciferase activities were normalized to that of -galactosidase. humans, to lower eukaryotes, suggesting the importance of HMTase activity in the SET domain.4) To character- MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) ize the HMTase activity of setd3, we performed an assay. HEK293, HeLa, and NIH-3T3 cells were seeded in 48-well in vitro HMTase assay with purified GST-setd3 fusion plates (2 103 cells/well) and transiently transfected with pcDNA6- setd3. Zero to 48 h post-transfection, MTT was added (20 mL, final protein by scintillation counting. Core histones and 14 concentration 0.5 mg/mL) to the cells and this was incubated for 4 h at S-adenosyl [methyl- C]-L-methionine (SAM) were 37 C, followed by aspiration of the medium and addition of DMSO utilized as substrate and methyl donor respectively. An A Novel HMTase setd3 in Zebrafish 291 A

Fig. 1. Zebrafish setd3 Possesses a Conserved SET Domain. A, Schematic representation of the full-length setd3 and SET domain. B, Secondary structural elements are indicated above the amino acid sequence. Red rods represent -helices and blue arrows represent -sheets within the setd3 protein. The SET domain is indicated by a gray box. C, Three-dimensional modeling of setd3 (77–490 aa). Secondary structures are visualized in different colors. The red dotted line represents the cleft in setd3. D and E, Results of 3D modeling of the SET domain and the predicted ssDNA-binding motif of setd3. The structure of SET domain was generated from 195 aa to 314 aa (D), and the ssDNA-binding motif was used from 269 aa to 314 aa (E). The arrow indicates a flip-loop chain in the ssDNA-binding motif. The pink ribbons represent -helices, and -sheets are showed in yellow and green ribbons (D and E). All 3D models of setd3 were produced by SWISS-Model server, and were visualized with the SWISS-Pdb viewer. in vitro assay showed the HMTase activity of setd3 on ies. The histone H3K36-mono, -di, and -trimethylating core histones in a dose-dependent manner (Fig. 2A), but activities of setd3 were found to be increased compare to HMTase activity was not observed with GST protein the other sites investigated (Fig. 2D). Collectively, these (Fig. 2A). To investigate the lysine specificity of zebra- results indicate that setd3 has HMTase activity specif- fish setd3, an in vitro HMTase assay was performed with ically towards mono, di, and trimethylation of H3K36 purified GST-setd3 proteins. The results of western blot residues both in vitro and in vivo. analysis demonstrated that GST-setd3 methylated H3K36-me1/-me2/-me3 (Fig. 2B). Next we tried to Setd3 induced transcriptional activation confirm the HMTase activity of setd3 in vivo. Setd3 was In order to determine whether the methylation of cloned into eukaryotic expression vector. We transfected H3K36 of setd3 can be attributed to activation of general HEK293 cells with pcDNA6-setd3, and total RNA was transcription, we conducted transient transfection assays isolated and setd3 mRNA amplified using the specific using the MH100-SV40-Luc and MH100-Tk-Luc re- primer (Fig. 2C, upper panel). The pcDNA6-setd3 porter systems. Transactivation activities by H3K36 transiently transfected into eukaryotic cells was detected methylation have been reported in other HMTases, by western blot using anti-myc antibody (Fig. 2C, lower SETD2 and NSD1, through induction of H3K36 panel). To investigate further whether setd3 can meth- methylation.14,15) Transfection of pcDNA6-setd3 to ylate histones, we transfected HEK293 cells with HEK293 cells resulted in activation of luciferase pcDNA6-setd3 and carried out western blot using anti- activities in both reporter systems in a dose-dependent H3K4-me2, anti-H3K9-me2, anti-H3K27-me2, anti- manner. This indicates that relative luciferase activity H3K36-me1/-me2/-me3, and anti-H4K20-me2 antibod- ultimately increases to approximately 2.2-fold and 5- 292 D.-W. KIM et al. AB

Fig. 2. Zebrafish setd3 as a Novel HMTase. A, Core histones were used as substrates in the HMTase assay. Methylation levels were quantified by filter binding assay, and data are represented as raw counts per min incorporated. B, HMTase assay with GST-setd3 and core histones, followed by western blotting against -H3K4-me2, -H3K9-me2, -H3K27-me2, -H3K36-me1, -H3K36-me2, -H3K36-me3, and -H4K20-me2 antibodies. C, HEK293 cells were transfected with pcDNA6 or pcDNA6-setd3. cDNA were extracted from transfected cells. PCR products were amplified using specific primers to setd3 (upper panel). HEK293 cells were transfected with pcDNA6 or myc-tagged setd3. Total proteins were extracted from transfected cells using lysis buffer. Western blot analysis was done with -myc or --actin antibody (lower panel). D, Transiently pcDNA6- setd3-transfected cell extracts were used in western blot analysis, and then alterations of histone methylation levels were detected using -H3K4- me2, -H3K9-me2, -H3K27-me2, -H3K36-me1, -H3K36-me2, -H3K36-me3, and -H4K20-me2 antibodies. fold of basal transcription in both systems, when wild- formazan exocytosis, was significantly decreased in type setd3 was transfected, respectively (Fig. 3B and C). setd3 overexpressing cells compared with that of un- To investigate further the role of the SET domain in the treated cells at 48 h post-transfection. The relative transcriptional regulation of setd3, we also performed a viability in setd3 transfected cells gradually decreased reporter assay with setd3 deletion mutants (Fig. 3A). as compared to that of the control cells by approximately Similarly to the setd3 wild-type, transcriptional activity 40% in all the cell lines used (Fig. 4A, B, and C). These was increased only in SET domain intact setd3-N results demonstrate that setd3 inhibits cell proliferation. mutant transfected cells. However, SET domain deleted To further determine whether zebrafish setd3 over- setd3-C did not activate in transcription in either system expressing cells exhibit the expression level changes of (Fig. 3B and C). These results indicate that setd3 can apoptosis related proteins. We examined the expression activate transcription through H3K36 HMTase activity. of important regulators in apoptosis, such as caspase-9 and caspase-3, by western blot analysis. The inactivated A novel histone methyltransferase zebrafish setd3 form of procaspase-9 level decreased in zebrafish setd3 suppressed cell survival transfected cells compared to untreated and mock It has been found that several HMTases play (Fig. 4D). In contrast to the procaspase-9 level, cleaved important roles in development and cell growth.16) For caspase-3, which is an activated form of caspase-3 instance, G9a is essential for early embryogenesis and is (p17), was significantly increased in zebrafish setd3 involved in the transcriptional repression of develop- overexpression cells (Fig. 4D). These results indicate ment.17) To investigate the effects of setd3 on cell that zebrafish setd3 overexpression induces caspase- growth, we performed an MTT assay, and cell viability dependent apoptosis. was monitored every 12 h after transfection. Cell lines In this study, we predicted the structural features of from different human tissues (embryonic kidney and zebrafish setd3 and identified its H3K36 methylation cervical cancer) and different species (mouse fibroblast) activity. We report that setd3 acts as a transcriptional were used to show the general effects of setd3. The activator via induction of H3K36 methylation and has an formation of needle-like crystals, a marker of MTT inhibitory function in cell proliferation. Further studies A Novel HMTase setd3 in Zebrafish 293 A

Fig. 3. Zebraﬁsh setd3 Induced Transcriptional Activation. A, Schematic representation of the domain structure of the setd3 wild type and the deletion mutants. B and C, HEK293 cells were transiently co-transfected with pcDNA6-setd3 (50, 100, or 150 ng), pcDNA6-setd3-N (50, 100, or 150 ng), pcDNA6-setd3-C (50, 100, or 150 ng), MH100- SV40-Luc vector (100 ng) (B), and MH100-TK-Luc vector (100 ng) (C). The amounts of DNA in the transfection were kept constant using the pcDNA 6 empty vector. At 48 h post-transfection, cells were lysed with lysis buﬀer, and the lysate was assayed for luciferase assay. Luciferase activities were normalized to that of -galactosidase. Each value is the mean of three replicates from a single assay (SD). p < 0:001 compared with reporter plasmid alone.

A B

Fig. 4. Zebrafish setd3 Suppresses Cell Viability. A, B, C, Cell viability was identified by MTT assay when setd3 was overexpressed in HEK293 (A), HeLa (B), and NIH-3T3 (C) cells. The cells were transfected with pcDNA6-setd3, and formazan was detected using an ELISA reader (570 nm) at each time point (24, 36, or 48 h). pcDNA6 empty vector used as control. D, Total proteins were extracted from pcDNA6-setd3-transfected HeLa cells and subjected to western blot using specific antibodies. -Actin was used as a loading control. All data are from three independent experiments as means SD ( p < 0:001). are required to understand of the mechanism of setd3 in Korea Grant, from the Ministry of Education, Science the transcriptional regulation of target genes through and Technology (R01-2008-000-20358-0). HMTase activity. The fact that setd3 inhibited cell growth indicates that it might be involved in the References regulation of apoptosis related-target genes, such as caspase-9 and caspase-3. 1) Li B, Carey M, and Workman JL, Cell, 128, 707–719 (2007). 2) Jones RS and Gelbart WM, Mol. Cell. Biol., 13, 6357–6366 (1993). Acknowledgment 3) Stassen MJ, Bailey D, Nelson S, Chinwalla V, and Harte PJ, Mech. Dev., 52, 209–223 (1995). This work was supported by the Mid-Career Re- 4) Lee DY, Teyssier C, Strahl BD, and Stallcup MR, Endocr. Rev., searcher Program, a National Research Foundation of 26, 147–170 (2005). 294 D.-W. KIM et al. 5) Lachner M, O’Sullivan RJ, and Jenuwein T, J. Cell Sci., 116, 12) Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schimid 2117–2124 (2003). M, Opravil S, Mechtler K, Ponting CP, Allis CD, and Jenuwein 6) Cao R and Zhang Y, Curr. Opin. Genet. Dev., 14, 155–164 T, Nature, 406, 593–599 (2000). (2004). 13) Krajewski WA, Nakamura T, Mazo A, and Canaani E, Mol. 7) Yin Y, Liu C, Tsai SN, Zhou B, Ngai SM, and Zhu G, J. Biol. Cell. Biol., 25, 1891–1899 (2005). Chem., 280, 30025–30031 (2005). 14) Sun XJ, Wei J, Wu XY, Hu M, Wang J, Wang HH, Zhang QH, 8) Tan X, Rotllant J, Li H, De Deyne P, and Du SJ, Proc. Natl. Chen SJ, Huang QH, and Chen Z, J. Biol. Chem., 280, 35261– Acad. Sci. USA, 103, 2713–2718 (2006). 35271 (2005). 9) Xu PF, Zhu KY, Jin Y, Chen Y, Sun XJ, Deng M, Chen SJ, 15) Wang GG, Cai L, Pasillas MP, and Kamps MP, Nat. Cell Biol., Chen Z, and Liu TX, Proc. Natl. Acad. Sci. USA, 107, 2521– 9, 804–812 (2007). 2526 (2010). 16) Margueron R, Trojer P, and Reinberg D, Curr. Opin. Genet. 10) Schwede T, Kopp J, Guex N, and Peitsch MC, Nucleic Acids Dev., 15, 163–176 (2005). Res., 31, 3381–3385 (2003). 17) Tachibana M, Ueda J, Fukuda M, Takeda N, Ohta T, Iwanari H, 11) Trievel RC, Flynn EM, Houtz RL, and Hurley JH, Nat. Struct. Sakihama T, Kodama T, Hamakubo T, and Shinkai Y, Genes Biol., 10, 545–552 (2003). Dev., 19, 815–826 (2005).