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Characterization of a Novel Histone H3K36 Methyltransferase Setd3 in Zebrafish

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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 modifica- tions 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 Æ 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.
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